Environmental Biocatalysis. From remediation with enzymes to novel green processes
... Nowadays concern for the environment is becoming increasingly important. Intensive industrialization, large-scale heavy metals and xenobiotics use, such as: oil, polycyclic aromatic hydrocarbons (PAH), BTEX (benzene, toluene, ethylbenzene and xylene), chlorinated hydrocarbons, eg polychlorinated biphenyls (PCB), trichlorethylene (TCE) and perchlorethylene, nitroaromatic compounds, organophosphorus compounds and solvents, pesticides[1,2]can cause a number of environmental problems, including the possibility of harmful effects on the biogeochemical circulation of different elements and they can be toxic to organisms, including humans. Therefore, the risk associated with the accumulation of degradation-resistant contaminants in the environment is increasing and it is necessary to search for new, safe and often unconventional methods of dealing with pollution[3,4]. ...
... Natural bioremediation is especially important in the case of spills of petroleum products, as their distribution is monitored, and the migration of contamination, growth rate, microbial activity and the presence of biogenic elements are tracked[2,9]. The process of spontaneous purification of contaminated environment may be prolonged, which is associated with the spontaneous course of the physico-chemical reactions during the degradation of pollutants[1,4]and with the specific enzymatic activity of the indigenous microorganisms living in this environment (eg enzyme inhibition by substrates or competition for degrading enzyme active sites, toxicity of biodegradation products of pollutants, competition for oxygen etc.). The ability of microorganisms to bioremediation of pollutants is based on their oxidation and decomposition (biodegradation), assimilation or transformation into non-toxic compounds such as CO 2 and H 2 O (biotransformation)[4,7]. ...
... Moreover, in bioremediation one can use enzymes secreted outside the cell, as well as intracellular ones. The most studied enzymes in bioremediation are bacterial mono-or dioxygenases, reductases, dehalogenases, cytochrome P450 monooxygenase, phosphotriesterases, and enzymes involved in lignin metabolism (such as laccases, lignin-and manganese peroxidases) from white-rot fungi[1,3,42]. According to Arora et al[43], oxygenases play a key role in the metabolism of organic compounds by increasing their reactivity or water solubility or bringing about cleavage of the aromatic ring. ...
The aim of this paper was to present possibilities of using different substrates to assist the bioremediation of soils contaminated with heavy metals, pesticides and other substances. Today's bioengineering offers many solutions that enable the effective conduct of biological remediation, including both biostimulation and bioaugmentation. For this purpose, they are used to enrich various organic substances, sorbents, microbiological and enzymatic preparations, chemical substances of natural origin or nanoparticles. The use of genetic engineering as a tool to obtain microorganisms and plants capable of efficient degradation of pollutants may cause the risks that entails the introduction of transgenic plants and microorganisms into the environment. In order to determine the efficacy and possible effects of the various bioremediation techniques, it is required to conduct many studies and projects on a larger scale than only in the laboratory. Furthermore, it should be emphasized that bioremediation involves interdisciplinary issues and therefore, there is a need to combine knowledge from different disciplines, such as: microbiology, biochemistry, ecology, environmental engineering and process engineering.
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... These approaches must be carefully studied and planned for each type of contaminant and environmental condition, as both present advantages and disadvantages. For instance, bioaugmentation success depends on the competitiveness of the inoculated strains in different environments [20]. Genetically modified organisms (GMOs) can also be used to improve petroleum degradation efficiency, but other limitations may complicate the procedure, such as problems with international legislation [20, 21]. ...
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... These approaches must be carefully studied and planned for each type of contaminant and environmental condition, as both present advantages and disadvantages. For instance, bioaugmentation success depends on the competitiveness of the inoculated strains in different environments [20]. Genetically modified organisms (GMOs) can also be used to improve petroleum degradation efficiency, but other limitations may complicate the procedure, such as problems with international legislation [20, 21]. ...
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... The characterisation of petroleum-degrading strains and their metabolic pathways serves to improve bioremediation approaches. Bioremediation can occur either naturally or by the use of bioaugmentation (whole cell introduction) or biostimulation approaches (use of nutrients or conditions to stimulate the native microbial community) [5, 20]; isolated enzymes may also be used to transform the contaminant into less-toxic or nontoxic compounds [3, 5, 20]. Many authors have described bioaugmentation and biostimulation approaches to restore different petroleumcontaminated sites; both are accepted options for minimizing the impact of petroleum spills [5]. ...
... These approaches must be carefully studied and planned for each type of contaminant and environmental condition, as both present advantages and disadvantages. For instance, bioaugmentation success depends on the competitiveness of the inoculated strains in different environments [20]. Genetically modified organisms (GMOs) can also be used to improve petroleum degradation efficiency, but other limitations may complicate the procedure, such as problems with international legislation [20, 21]. ...
... Microbial bioremediation, however, suffers from some limitations . Besides the difficult proliferation (Alcalde et al., 2006), often oxidation products derived from bacterial metabolism display enhanced toxicity compared to the PAH precursors themselves (Bamforth and Singleton, 2005). In addition, microorganisms require appropriate environmental growth conditions such as suitable nutrients, temperature, pH, redox potential and oxygen content which, overall, may strongly affect their proliferation (Adams et al., 2015). ...
... As a consequence, the whole process is generally slow (Pilon-Smits, 2005; Salt et al., 1998; Sharma and Pandey, 2014; Kathi and Khan, 2011) or even totally inhibited, in this case too. In order to overcome these problems, we reasoned that cell-free (Alcalde et al., 2006 ) plant enzymes could be capable of transforming PAHs into degraded/oxidized products. In particular, it is well known that peroxidase (POD) and polyphenol oxidase (PPO) are involved into the oxidation of phenols (Aniszewski et al., 2008; Hiraga et al., 2001) and also play an important role in the resistance of plants to biotic and abiotic stress (Foyer and Noctor, 2005; Sharma et al., 2012). ...
... Nonetheless, the application of this knowledge into effective microbial bioremediation protocols is still on a preliminary phase [147]. Because enzymes are simpler systems than the whole microorganism, in the past years enzymatic bioremediation has been seen as a possible alternative148149. Some advantages in using enzymes instead of microorganisms or chemicals have been pointed out. ...
... Some advantages in using enzymes instead of microorganisms or chemicals have been pointed out. The degradation of pollutants does not generate toxic or bio-hazardous products, the enzymes are themselves biodegradable by the indigenous microorganisms and the efficiency of the process can be improved by recombinant-DNA technology [148, 150]. The role of bacterial extracellular enzymes in the degradation of organic matter and their broad range of substrates makes them suitable candidates for remediation of pollutants from contaminated environments [151]. ...
Heterotrophic bacteria are key players in the processes of organic matter recycling, decomposition and mineralization in aquatic environments. Although only small and chemically simple compounds can be passively transported through bacterial membranes, substrates for bacterial utilization in aquatic environments are dominated by particulate or high-molecular-weight dissolved organic matter. Complex substrates must first be hydrolyzed outside the cell into smaller size molecules by extracellular enzymes and this process represents a limiting step in nutrient cycling. Bacterial extracellular enzymatic activity is regulated at the ecosystem level, by environmental factors and at the micro-environment level by enzyme-substrate interactions. Over the last century, changes in the atmosphere concentration of CO 2 and other green-house gases caused changes in climate patterns that have repercussions in ecosystem function and biodiversity. Microorganisms are generally able to respond very quickly to environmental changes because of their close contact with the surrounding environment and rapid growth. As mediators in important biogeochemical processes, namely decomposition and transformation of organic matter, release of inorganic nutrients for higher trophic levels and detoxification of xenobiotics, bacterial enzyme activities have the potential to be used as descriptors of biological responses to changing environmental conditions. The present paper reviews the currently available information on environmental regulation of bacterial extracellular enzymatic activity in aquatic environments and discusses the potential implications of direct and indirect effects global changes on heterotrophic bacterial communities and on the processes of organic matter recycling.
... The traditional treatment methods for phenolic pollutants mainly include physical adsorption and chemical catalysis [6,7], but these methods usually have the disadvantages of low efficiency and easy to cause secondary pollution. The enzymatic method is considered to be an effective method for degrading phenolic pollutants due to its high specificity, environmental friendliness and high catalytic efficiency [8,9]. Laccase is a kind of oxidoreductase with copper as the catalytic center. ...
In this study, polyethyleneimine was combined with magnetic Fe3O4 nanoparticles through the bridging of carboxyl-functionalized ionic liquid, and laccase was loaded onto the carrier by Cu2+ chelation to achieve laccase immobilization (MCIL–PEI–Cu–lac). The carrier was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, thermogravimetric analysis, X-ray diffraction analysis, magnetic hysteresis loop and so on. MCIL–PEI–Cu–lac has good immobilization ability; its loading and activity retention could reach 52.19 mg/g and 91.65%, respectively. Compared with free laccase, its thermal stability and storage stability have been significantly improved, as well. After 6 h of storage at 60 °C, 51.45% of the laccase activity could still be retained, and 81.13% of the laccase activity remained after 1 month of storage at 3 °C. In the pollutants removal test, the removal rate of 2,4-dichlorophenol (10 mg/L) by MCIL–PEI–Cu–lac could reach 100% within 10 h, and the removal efficiency could still be maintained 60.21% after repeated use for 8 times. In addition, MCIL–PEI–Cu–lac also has a good removal effect on other phenolic pollutants (such as bisphenol A, phenol, 4-chlorophenol, etc.). Research results indicated that an efficient strategy for laccase immobilization to biodegrade phenolic pollutants was developed.
... Glycosidic enzymes require mild reaction conditions (moderate temperature and slightly acidic pH), display high efficiency, and allow for control of the composition of the final product on the basis of enzyme specificity [4,[18][19][20][21][22], which can also be altered by protein engineering techniques [23,24]. The enzymatic strategies are more environmentally friendly and generate less waste than the chemical methods [3,25,26]. The physicochemical properties of the starting chitosan also influence the composition of the resulting oligosaccharides [27,28]. ...
By controlled hydrolysis of chitosan or chitin with different enzymes, three types of chitooligosaccharides (COS) with MW between 0.2 and 1.2 kDa were obtained: fully deacetylated (fdCOS), partially acetylated (paCOS), and fully acetylated (faCOS). The chemical composition of the samples was analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry. The synthesized fdCOS was basically formed by GlcN, (GlcN)2, (GlcN)3, and (GlcN)4. On the contrary, faCOS contained mostly GlcNAc, (GlcNAc)2 and (GlcNAc)3, while paCOS corresponded to a mixture of at least 11 oligosaccharides with different proportions of GlcNAc and GlcN. The anti-inflammatory activity of the three COS mixtures was studied by measuring their ability to reduce the level of TNF-α (tumor necrosis factor) in murine macrophages (RAW 264.7) after stimulation with a mixture of lipopolysaccharides (LPS). Only fdCOS and faCOS were able to significantly reduce the production of tumor necrosis factor (TNF)-α at 6 h after stimulation with lipopolysaccharides.
... Among the various nanomaterials magnetic nanoparticles that showed promising possibilities of industrial application had large magnetic behavior, controlled size, more chemical reactivity [196] and less toxicity [197]. Magnetic nanoparticle-enzyme conjugates (MNP-Es) are one class of bio-NP conjugates used for biotechnological purposes where high catalytic specificity, prolonged reaction time, and also the ability to recycle an expensive biocatalyst are required [189,198] . Another main advantage of magnetic nanoparticle is easy separation by an external magnetic device [199,200], thus allowing immobilized cellulase to be used several times thus reducing the loss of enzyme and production cost. ...
... There are several aspects, in which the study about dehalogenation couples the approaches including metagenomics, which is a culture-independent technique for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants (Nishino et al., 2013). In addition to this, the proteomics has also having significant contribution to understand the individual organisms at the molecular level thereby finding various protein functions with respect to the responses (Alcalde et al., 2006). During the last few years, the field of dehalogenation has been seen in a rapid progress in the identification of novel fungi as well as gene, sequencing of whole genomes, and the genetic adaptability potential. ...
... The present work contributes to the ever expanding literature on enzyme applications in the chemical industry and for polysaccharides modifications(for reviews, see: Alcalde, Ferrer, Plou, & Ballestreros, 2006; Bornscheuer et al., 2012; Cheng & Gu, 2012; Elboutachfaiti, Delattre, Petit, & Michaud, 2011; Li, Yang, Yang, Zhu, & Wang, 2012; Nestl, Nebel, & Hauer, 2011). We have applied the laccase/TEMPO chemo-oxidation reaction to fenugreek gum in aqueous solution, followed by lyophilization , and have obtained a water-insoluble aerogel (EOLFG), with high water and DMSO uptake capacity. ...
We describe a new aerogel obtained from laccase-oxidized galactomannans of the leguminous plant fenugreek (Trigonella foenum-graecum) and suggest its potential practical use. Laccase/TEMPO oxidation of fenugreek in aqueous solution caused a viscosity increase of over 15-fold. A structured, elastic, stable hydrogel was generated, due to formation of carbonyl groups from primary OH of galactose side units and subsequent establishment of hemiacetalic bonds with available free hydroxyl groups. Upon lyophilization of this hydrogel, a water-insoluble aerogel was obtained (EOLFG), capable of uptaking aqueous or organic solvents over 20 times its own weight. The material was characterized by scanning electron microscopy, FT-IR, elemental analysis and 13C CP-MAS NMR spectroscopy and its mechanical properties were investigated. To test the EOLFG as a delivery system, the anti-microbial enzyme lysozyme was used as model active principle. Lysozyme was added before or after formation of the aerogel, entrapped or absorbed in the gel, retained and released in active form, as proven by its hydrolytic glycosidase activity on lyophilized Micrococcus lysodeikticus cells wall peptidoglycans. This new biomaterial, composed of a chemo-enzymatically modified plant polysaccharide, might represent a versatile, biocompatible "delivery system" of active principles in food and non-food products.
... Recent advancement in biotechnology and molecular tools has enhanced the production and recovery of enzymes. Many authors have suggested the use of enzyme (Biocatalysis) in bioremediation instead of microorganism [52] [53]. The use of enzyme in bioaugmentation can result in avoiding the competition between indigenous bacteria and the new strains. ...
This study discusses factors affecting various processes involved in bioremediation coupled with electrokinetics. The study presents innovative solutions, and proposes new directions. Environmental conditions that have an influence on the characteristics, behavior, and metabolism of indigenous microorganisms are presented. The discussion focuses on overcoming the unfavorable conditions created by electrolysis reactions, prolongation the survival of the microbes at contaminated sites, increase of microbial enzyme secretion, improvement of the indigenous bacteria metabolic pathways, and exploration of metagenomics resources from soil biota. The challenge facing the implementation of conventional bioremediation techniques in precisely and effectively delivering nutrients to indigenous bacteria, particularly in soils with tortuous paths and low hydraulic conductivity is discussed. Current knowledge in application of enhanced biostimulation using electrokinetics is reviewed. The implementation of bioaugmentation in bioremediation coupled with electrokinetics to enhance the outcome of bioremediation is presented. Effects of phenomena associated with electrokinetics in the hybrid remediation approach are discussed.
... Extreme environments represent a great microbial resource for novel enzymes, the majority of which remains to be discovered. Metagenomics, the technique to access the genome content resource of noncultivated microbes, is a powerful tool used in the discovery of novel industrial enzymes for biotechnological and pharmaceutical applications12345. More than 99 % of the microorganisms cannot be cultivated [6] but discovered , using an alternative metagenomics approach to the traditional microbial screening methods to isolate enzyme from extreme environments789. ...
The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.
Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.
The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.
... The biotechnological applicability of laccase may therefore be extended by the use of laccase-mediator system (LMS). Thus, laccase and LMS find potential application in delignification (Virk et al. 2012), and biobleaching of pulp (Ibarra et al. 2006; Weirick et al. 2014); enzymatic modification of dye-bleaching and fibres in the textile and dye industries (Kunamneni et al. 2008); enzymatic removal of phenolic compounds in beverages— wine and beer stabilization, fruit juice processing (Minussi et al. 2002); enzymatic cross linking of lignin-based materials to produce medium density fibreboards (Widsten et al. 2004); bioremediation and detoxification of aromatic pollutants (Alcalde et al. 2006; Khambhaty et al. 2015); detoxification of lignocellulose hydrolysates for ethanol production by yeast (Larsson et al. 1999); treatment of industrial wastewater (Berrio et al. 2007; Viswanath et al. 2014) and construction of biofuel cells and biosensors (Ghindilis 2000; Shraddha et al. 2011). Due to its catalytical properties, laccase has gained considerable interest for potential biotechnological applicability (Bourbonnais et al. 1995; Abdel-Hamid et al. 2013). ...
Laccase belongs to a small group of enzymes called the blue multicopper oxidases, having the potential ability of oxidation. It belongs to enzymes, which have innate properties of reactive radical production, but its utilization in many fields has been ignored because of its unavailability in the commercial field. There are diverse sources of laccase producing organisms like bacteria, fungi and plants. In fungi, laccase is present in Ascomycetes, Deuteromycetes, Basidiomycetes and is particularly abundant in many white-rot fungi that degrade lignin. Laccases can degrade both phenolic and non-phenolic compounds. They also have the ability to detoxify a range of environmental pollutants. Due to their property to detoxify a range of pollutants, they have been used for several purposes in many industries including paper, pulp, textile and petrochemical industries. Some other application of laccase includes in food processing industry, medical and health care. Recently, laccase has found applications in other fields such as in the design of biosensors and nanotechnology. The present review provides an overview of biological functions of laccase, its mechanism of action, laccase mediator system, and various biotechnological applications of laccase obtained from endophytic fungi.
... However, historical information [30,31] suggests the opposite has actually been the case. Considering the relative low cost and low energy requirements of bioremediation technologies [6,[58][59][60], the gulf between aspiration (as shown here) and practice might be due to various factors involving the risk-averse nature of the contaminated-land industry, or difficulties in project design. The latter include identifying appropriate organisms for removing specified contaminants, optimizing environmental conditions for their action, ascertaining extents of eventual clean-up, and the incomplete understanding of all the mechanisms and processes involved. ...
A global survey examining the use of bioremediation technologies for addressing environmental pollution problems has been carried out. There were respondents from all continents (except Antarctica), though North America was comparatively over-represented. Despite a high aspiration to apply bioremediation techniques, this was not borne out in current practice. Air pollution was the lowest priority. Otherwise, a clear association was seen between the per capita income of a region and the concerns, remediation techniques and research practice adopted. For example, contamination of groundwater had higher priority in developed countries/regions. Toxic metals and aromatic hydrocarbons were the most common concern, while alkyl halides were of greater concern in North temperate (comparatively economically developed) countries than elsewhere. Only 15-35% of respondents used online databases to guide the design of their experiments, and these were largely restricted to North America and Europe, three quarters of US respondents used modelling software compared with about a third elsewhere. Consequently, while the developed economies made higher use of low-cost in situ bioremediation technologies (e.g. Monitored Natural Attenuation), their developing counterparts appeared to focus on the more expensive, sometimes ex situ, technologies. Despite the significant investment in and widespread availability of online resources, their limited use emphasizes the need to explore avenues for improved training and the development of more user-friendly resources. In this regard, this survey has produced a bioremediation research wish list to guide such developments. The data from this survey may also contribute to policy-decision making worldwide.
... Industrial and agriculture practices results the release high level of xenobiotic compounds, such as polycyclic aromatic hydrocarbon (PAH), pentachlorophenol (PCP), polychlorinated biphenyls (PCB), DDT, pesticides, benzene, toluene, ethylbenzene, xylene and TNT in the environment (Riva 2006). These are highly toxic and persistent in nature, many of them exert mutagenic and carcinogenic effects on living systems (Samanta 2002; Alcalde et al. 2006). Hence, in view of public health, it becomes necessary to put serious efforts in order to remove them from the environment. ...
Present study reports the validation (oxidation) of computationally predicted oxidation of xenobiotic contaminants by commercially available pure laccase from Trametes versicolor. Selected contaminants were predicted as potential targets for laccase oxidation by using in-silico docking tool. The oxidation by laccase was measured by change in absorbance at specific λ max of each compound. Sinapic acid and tyrosine were taken as positive and negative controls, respectively. Oxidation was observed in m-chlorophenol, 2,4 di-chlorophenol, 2,4,6 trichlorophenol, captan, atrazine and thiodicarb, except malathion, which showed no activity. It could be speculated that the predicted substrates showing oxidation shared homology at structural and chemical level with positive control compounds. In case of malathion, structural non-homology with sinapic acid could be attributed to its inactivity towards laccase that required further structural analysis. Thus, a remediation tool proposing an advanced remediation approach combining the application of theoretical in-silico method and subsequent experimental validation using pure laccase could be proposed. As number and type of xenobiotics increase, the unfeasibility to screen them experimentally for bioremediation also rise. This approach would be useful to reduce the time and cost required in other screening methods.
... Fermentation, with special reference to the production of alcoholic beverages, bioethanol, dairy products, organic acids and drugs which also comprise antibiotics, is the most important example of microbiological processes. The enzyme industry, as it is currently known, is the result of a rapid development of biotechnology, especially during the past four decades (Alcalde et al., 2006). Since ancient times, enzymes found in nature have been used in the production of food products such as cheese, beer, wine and vinegar (Kirk et al., 2002). ...
Trichoderma spp. are fungi that are present in nearly all soils and other diverse habitats. These are highly efficient producers of many extracellular enzymes. They are used commercially for production of cellulases and other enzymes that degrade complex polysaccharides. They are frequently used in the food and textile industries for these purposes. For example, cellulases from these fungi are used in "biostoning" of denim fabrics to give rise to the soft, whitened fabric-stone-washed denim. The enzymes are also used in poultry feed to increase the digestibility of hemicelluloses from barley or other crops. This book chapter provides the properties, production, and applications of Trichoderma enzymes within food industry.
... Plant oxidative enzymes (oxidoreductases), such as peroxidases (PODs) and polyphenol oxidases (PPOs), have been used in several applications, pertaining to biosensors, biocatalysis, immunoassays, organic synthesis, etc. (Hamid and Rehman, 2009; Ryan et al., 2006; Xu, 2005). However, a vast number of studies have been conducted on the use of oxidative enzymes for bioremediation processes, based on concrete evidence that their deployment might be advantageous over conventional treatments, which aim at detoxifying recalcitrant organic pollutants (Alcalde et al., 2006; Demarche et al., 2012; Durán and Esposito, 2000; Karam and Nicell, 1997). The potential advantages of enzymic treatment as compared with conventional treatment include: Another significant advantage over conventional chemical treatments is enzyme specificity, which limits undesired side reactions that could increase reactant consumption and raise the cost of treatment. ...
Food processing by-products' exploitation includes the recovery of fine chemicals and production of precious metabolites, such as natural antioxidants, enzymes, starch, pigments, etc., which are value-added products of high significance to the pharmaceutical, cosmetics, and food industries. Contemporary biocatalysis is based upon new tools and methodologies to improve a wide range of production processes. The development of modern biocatalytic procedures promotes advances in the environment (bioremediation), food (bio-based functional ingredients), and also renewable and clean energies (biofuels) sectors. However, despite the obvious benefits of biocatalysis, the major drawbacks associated with the exploitation of enzymes are, in many cases, the high production costs and the low yields obtained. In recent years there has been a substantial amount of knowledge gained on the richness of food wastes and by-products in valuable enzymes, with high prospects in several environmental and food processes. This chapter deals with the recovery and utilization of enzymes from food wastes, by describing the major aspects of practical importance.
... Recent advancement in biotechnology and molecular tools has enhanced the production and recovery of enzymes. Many authors have suggested the use of enzyme (Biocatalysis) in bioremediation instead of microorganism [52] [53]. The use of enzyme in bioaugmentation can result in avoiding the competition between indigenous bacteria and the new strains. ...
Petroleum hydrocarbons are hydrophobic and tend to adhere to soil when released into the environment. Desorption of the contaminant from soil is necessary for most remediation technologies. In subsurface and for in-situ remediation, the dominant desorption mechanism is back diffusion. In this study, three tests were conducted to establish sorption and desorption kinetics for phenanthrene as a contaminant and kaolinite as a soil matrix. Sorption isotherm tests were conducted at temperatures of 14, 22, and 30 °C using six phenanthrene solution containing concentrations from 300 to 800 μg/L. Freundlich equation constant, Kf, values of 0.147, 0.133, and 0.109 μgLn/g(1+n) were determined for the three temperatures. Desorption tests were conducted using phenanthrene-free solution to determine desorption parameters at room temperature (∼22 °C). In addition, two test series were performed to compare phenanthrene desorption by hydraulic and electroosmotic flows at room temperature. A fixed wall hydraulic permeability apparatus was used to generate a hydraulic flow rate of 1.4 × 10-3 mL/s at a pressure of 260 kPa, while a low level direct current density (0.3-0.43 mA/cm2) was applied to generate electroosmotic flow rate equivalent to the hydraulic flow. The phenanthrene concentration in effluent samples after desorption by electroosmotic flow was found to be three to four times the concentration after desorption by hydraulic flow. Moreover, the power required in the hydraulic flow test was three orders of magnitude higher than the consumed power in the electrokinetic flow test. These results show that phenanthrene desorption by electroosmotic flow is more efficient than by hydraulic flow.
... However, the advent of PCR and methods based on recombination, such as DNA shuffling (Stemmer, 1994), which imitate in vitro natural evolutionary processes, have revolutionized this approach, producing some notable successes in the field of biocatalysis (Percival Zhang et al. , 2006). Whatever the in vitro method employed to generate artificial diversity, the key to random engineering is the availability of a robust, high throughput screen that will accurately pinpoint enzyme variants in large libraries that display the targeted property (Alcalde et al. , 2006, Brustad and Arnold, 2010, Dougherty and Arnold, 2009). In the field of biomass-modifying enzymes this is particularly important, because many attempts to create better enzymes have produced only very modest improvements, basically because the substrates used for screening, generally isolated polysaccharides (e.g. ...
White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'Omics' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose.
Copyright © 2015. Published by Elsevier Inc.
... Lipids are one of the major pollutants in domestic and industrial effluents. The use of lipases in the treatment of these effluents as well as in the bioremediation of contaminated environments represents an environmentally safe alternative to chemical methods [1]. Lipases (EC 3.1.1.3) ...
... Plant oxidative enzymes (oxidoreductases), such as peroxidases (PODs) and polyphenol oxidases (PPOs), have been used in several applications, pertaining to biosensors, biocatalysis, immunoassays, organic synthesis, etc. (Hamid and Rehman, 2009; Ryan et al., 2006; Xu, 2005). However, a vast number of studies have been conducted on the use of oxidative enzymes for bioremediation processes, based on concrete evidence that their deployment might be advantageous over conventional treatments, which aim at detoxifying recalcitrant organic pollutants (Alcalde et al., 2006; Demarche et al., 2012; Durán and Esposito, 2000; Karam and Nicell, 1997). The potential advantages of enzymic treatment as compared with conventional treatment include: Another significant advantage over conventional chemical treatments is enzyme specificity, which limits undesired side reactions that could increase reactant consumption and raise the cost of treatment. ...
Food Waste Recovery: Processing Technologies and Techniques acts as a guide to add more value to by-products from food waste in an economic and sustainable way. The book investigates recovery issues and compares different techniques to help you advance your research and to develop new applications. Strong coverage of the different technologies is included keeping a balance between the characteristics of current conventional technologies and emerging technologies making this is an essential reference for research outcomes.
... because of their high specificity, lack of acclimatization period, less energy requirement as well as operability over a wide temperature , pH and salinity at low or high concentrations of pollutants which exhibit attractive replacement for conventional wastewater treatment15161718. The use of oxygen by enzyme laccase as a non-limited electron acceptor and its extensive substrate specificity can be attributed to fact that laccase possesses substantial concern for the degradation of pollutants192021 . Enzyme laccase, (benzenediol:oxygen oxidoreductase , EC 1.10.3.2) which is found in the fungi, few bacteria and in higher plants, belongs to the polyphenol oxidases, a group of enzymes. ...
... The production can be enhanced, although to a limited extent, in laboratory conditions through optimization of growth parameters. On the otherhand, the recombinant DNA technology offers a cost effective process for large scale production of enzymes with enhanced stability and activity (Alcalde et al., 2006). In fact, the production of enzymes in industrial scale from the suitable microbial strain is of paramount importance for wide spread practical applications of enzymatic bioremediation process. ...
... The idea of using biotechnological approaches, and more specifically biocatalysis, for dealing with recalcitrant pollutants has been gaining acceptance in recent years. Most xenobiotics can be subjected to enzymatic bioremediation, including polycyclic aromatic hydrocarbons, polynitrated aromatic compounds, pesticides (e.g., organochlorine insecticides), bleach plant effluents, synthetic dyes, and polymers (Alcalde et al., 2006). Further, enzymes from various plant and microbial sources have several advantages over conventional physical and chemical treatment processes. ...
Plant food wastes and by-products might contain a range of enzymes capable of transforming bio-organic molecules, and thus they may have potential uses in bioremediation processes. Potato peels are an abundant plant food waste and might be considered a promising means of bioremediation since they contain the oxidative enzyme polyphenol oxidase (PPO), able to oxidize a range of pollutants. With this in view, this study aimed at investigating the influence of the side-chain structure of several o-diphenolic acids on their oxidizability by a crude potato peel PPO. After establishing optimal conditions concerning pH and temperature, the examinations carried out showed that the crude enzyme preparation used exhibited the highest catalytic efficiency with the physiological substrate, L -DOPA, which is an amino group-bearing O-diphenolic acid. Critical comparison of the data that emerged from testing chlorogenic acid (CGA), hydrocaffeic acid (HCA), and caffeic acid (CA) revealed that the unsaturation on the side chain might be a factor of instability for substrate binding, as judged by the particularly high K M for CA, but lack of double bond (HCA) or conjugation of the carboxyl group (CGA) restores stability. Based on the K M values determined, it was also shown that the higher the side-chain length, the lower the affinity between the substrate and the enzyme.
... The efficiency, selectivity, and specificity of proteins as molecular workers in life are related to the complexity of their structures. These features and the non-toxicity of proteins are the reasons for our need to introduce proteins to all our life aspects, such as the production of fine chemicals (Panke et al., 2004; Matsumura et al., 2006), pharmaceuticals (Margolin, 1993; Panke and Wubbolts, 2005), food processing (James and Simpson, 1996), remediation (Alcalde et al., 2006), and mining (Rawlings et al., 2003; Valenzuela et al., 2006). The biggest limitation of using natural proteins is that most of proteins have been perfectly created to work efficiently only under physiological conditions. ...
... Over the last few decades, a variety of sustainable " green chemistry " approaches in chemical industries have been developed (Alcalde et al. 2007). Biocatalysis is one such green technology that has been suggested as a significant alternative to conventional chemical synthesis of high value products. ...
A stilbene-based membrane spanning conjugated oligoelectrolyte 4,4'-bis(4'-N,N-bis(6″-(N,N,N-trimethyl ammonium) hexyl) amino)-styryl) stilbene tetraiodide (DSSN+) has been reported to be able to interact with bacterial cells and enhance their bioelectricity generation in bioelectrochemical devices, although the mechanism remains elusive. The goal of this study was to elucidate the impacts of DSSN+ on extracellular bioactivity and the underlying mechanism. Specifically, extracellular ferrihydrite reduction by Shewanella oneidensis was used to evaluate the influence of cell-DSSN+ interaction. Our results show that DSSN+ enhanced ferrihydrite reduction by S. oneidensis in a growth-dependent manner. The incorporation of DSSN+ into S. oneidensis cell membrane increased the extracellular concentration of redox shuttles, i.e., flavins, and extracellular enzyme activities without significantly decreasing cell viability. The findings suggested that membrane permeabilization is the dominant mechanism for the enhancement of extracellular bioactivity in S. oneidensis by DSSN+. We further demonstrated that the interaction between DSSN+ and S. oneidensis cells enhanced biofilm formation and stability without compromising the overall biofilm activity. Taken together, our results suggest that membrane spanning conjugated oligoelectrolytes, of which DSSN+ is one of many possible molecular structures, may be applied to enhance extracellular bioactivity in bacteria toward more efficient biofilm-based biocatalysis.
... In this context, immobilized biocatalysts fully participate in the ''green chemistry'' concept that was introduced in the 1990s (Pollard & Woodley, 2007) and its effect on a sustainable environment is now established beyond question. Although the benefits of biocatalyst for environment friendly finechemical transformations have been accepted, but issues, such as the use of enzymes for industrial processes involving clean energy production and bioremediation are still unsettled (Alcalde et al., 2006). To use the biocatalysts in an integrated sustainable way it is necessary to minimize the cost and the use of chemicals and at the same time, increase the yields and efficiency of the whole process. ...
Abstract Biocatalytic membrane reactors have been widely used in different industries including food, fine chemicals, biological, biomedical, pharmaceuticals, environmental treatment and so on. This article gives an overview of the different immobilized enzymatic processes and their advantages over the conventional chemical catalysts. The application of a membrane bioreactor (MBR) reduces the energy consumption, and system size, in line with process intensification. The performances of MBR are considerably influenced by substrate concentration, immobilized matrix material, types of immobilization and the type of reactor. Advantages of a membrane associated bioreactor over a free-enzyme biochemical reaction, and a packed bed reactor are, large surface area of immobilization matrix, reuse of enzymes, better product recovery along with heterogeneous reactions, and continuous operation of the reactor. The present research work highlights immobilization techniques, reactor setup, enzyme stability under immobilized conditions, the hydrodynamics of MBR, and its application, particularly, in the field of sugar, starch, drinks, milk, pharmaceutical industries and energy generation.
... A study performed by Liu et al. [116] using genetically modified bacteria showed that its use is a promising strategy in the bioremediation process of environments contaminated by arsenic. Other technologies of bioremediation in development include the use of protein engineering, metabolic engineering, transcriptome and proteomics [117, 118]. For the development of tests in field using genetically modified bacteria, the major obstacle is the environmental concern and political restrictions for the use of these organisms [113]. ...
... Furthermore, in the presence of redox mediators from natural or synthetic sources, laccases can expand their substrate specifi city to oxidize higher redox potential compounds (including non-phenolic substrates such as lignin derivatives, synthetic organic dyes and PAHs) (Ca ñ as & Camarero 2010). Thus, fungal laccases may have many potential applications in the front line of green chemistry, in the bioremediation of pollutants, the production of second generation biofuels (bioethanol, biobutanol), the engineering of biosensors and biofuel cells, in the paper, textile and food industries and in the synthesis of antibiotics, drugs, cosmetics, polymers and other compounds (Alcalde et al. 2006a; Kunamneni et al. 2007 Kunamneni et al. , 2008a Xu 2005). Typically, laccases contain four copper atoms arranged in two highly conserved regions. ...
During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs610T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs610T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs610T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.
... In recent years, the use of biocatalysis in industrial processes has become more common as it offers efficient tools for transformation of natural or synthetic materials under mild reaction conditions, reduced energy and raw material consumption. Furthermore, biocatalysis enables the synthesis of new chemicals and decreases the generation of waste and toxic side-products, as compared to the equivalent chemical catalyst3456. One group of biocatalysts with special interest for the chemical industry is the oxygenases. ...
Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.
Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD+ ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (FdhSNO ) from the soil bacterium Starkeya novella strictly specific for NAD+ . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of FdhSNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+ ) with better catalytic efficiency than NAD+ . The single mutation D221Q enabled the reduction of NADP+ with a catalytic efficiency kCAT /KM of 0.4 s-1 mM-1 at 200 mM formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a 5-fold increase in catalytic efficiency for NADP+ compared to the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP+ . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of FdhSNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.
In the past few decades, remediation and management of water resources has been a major challenge. Global increase in population and industrialization has burdened the water resources. The organic pollutants discharged in wastewater from industries and anthropogenic activities have been major driving force that causes a threat to water resources. By 2050, around 5.7 billion people are expected to have a residence in the area with water scarcity for more than a month in a year. The urge is to achieve environmental sustainability by adopting sustainable alternatives for treating and managing organic pollutants using clean and eco-friendly alternatives. Enzymatic bioremediation is a green bioprocess that provides efficient detoxification of organic waste using myriad of enzymes. In this context, chapter gives an overview of organic pollutants present in wastewater and its impact on the environment and human health. This encompasses enzymatic bioremediation strategies to detoxify organic pollutants with an attempt to highlight the role of diverse range of enzymes, and the key challenges of enzymatic bioremediation. The emerging role of nanotechnology integrated with conventional bioprocess is amalgamated to provide sustainable, green, and energy-efficient bioremediation.
Enzymes are biological catalysts capable of recognizing a substrate and catalyze reactions of hydrolysis and synthesis. The most significant property of enzymes is their high specificity toward their substrates since they are able to recognize and act upon a molecule from a pool of similar compounds. Enzymes are labile catalysts at certain operative conditions that may severely affect their stability. However, the attachment of enzymes to solid supports has proven to be a good solution to stabilize them and, thus, to preserve their catalytic performances. The fundamentals of enzyme biocatalysis in sustainable processes are summarized in this chapter. The advantages of immobilized enzymes in environmental applications and sustainable processes will be addressed considering the most suitable materials and the most common immobilization methods. The use of biocatalysts in bioremediation, biofuel production, and in the valorization of waste streams is reviewed.
The consistent discharge of xenobiotic compounds including hydrocarbon-containing substances, pesticides, plastics, fertilizers, etc., in the surrounding is the main source of pollution in our ecosystem and must be eradicated and controlled. Numerous conventional remediation strategies such as biological, chemical and physical techniques have been adopted and are presently in use. However, several limitations attached to the utilization of the aforementioned techniques had led to the fervent search for more cost-effective and efficient methods. Enzyme-based remediation is an eco-friendly, quick, easy and acceptable strategy utilized for the degradation of these contaminants in the ecosystem. Numerous enzymes with potent biodegradation potentials have been identified and distinguished from their innate sources; conversely, less extraction of these enzymes has limited further exploitation of the enzymes. More recently, research has shown that genetic engineering strategy has the capacity to produce numerous types of enzymes. In addition, enzyme immobilization techniques can be used to enhance the stability, half-life and overall catalytic rate of enzymes. In this current appraisal, we have presented a concise overview of the enzymes used for the remediation of hazardous and toxic contaminants. The comprehension of the mode of action of enzymatic bioremediation of enzymes like tyrosinase, laccase, peroxidase, oxygenase has also been extensively revealed in this current review. Diverse enzyme methods (enzyme immobilization and engineering) are used to overcome those limitations. Hopefully, this appraisal will expose the world to considerable description about enzymes and numerous enzymatic technologies which can be utilized for the remediation of pollutants in the ecosystem.
Application of pesticides to enhance productivity is a common practice in agricultural field. Pesticides no doubt play an important role in protecting the majority of crops from the pests and enhancing yield but extensive use of pesticides is a major concern today due to its presence at various trophic levels as well as in drinking water. They pose detrimental effects on the survival of wildlife, plants, animals, aquatic ecosystem as well as other non‐target species. They are considered as chemical mutagens in animals are resulting in mutations, damage of genetic material and chromosomal aberrations which are life threatening. Necrosis, chlorosis, stunted growth, etc. are common phytotoxic symptoms of pesticides in plants. Detoxification of pesticides is possible by various physico‐chemical as well as biological techniques. Physico‐chemical methods include detoxification systems which are very costly, laborious, and time consuming. Biological techniques use living biota which is eco‐friendly technique. The present review makes an approach to emphasize on physico‐chemical methods as well as biological methods including use of microbes and plants.
Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV, as well as an increase in the H/T ratio, was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6–3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. Binding of hydroquinone was further investigated by soaking in absence of fructose, showing a flexible site that likely allows productive motion of the intermediates. Therefore, the acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein, this flexibility being essential for the transfructosylation reaction to proceed. Finally, the binding affinity of the phenolic compounds was explained based on the two sites previously reported for pXd-INV.
To date, commercial laccase preparations are used in the food, textile, and paper and pulp industries (mild pH). Laccases are attractive in the synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (≥8.0). So far, one fungal laccase has been reported to be active at alkaline pH. Herein, engineering of the fungal laccase from Melanocarpus albomyces (MaL) for increased activity toward the substrate 2,6‐dimethoxyphenol at pH (≥9.0) is reported. Through a knowledge‐gaining directed evolution (KnowVolution) campaign, the key positions Leu365 and Leu513 were identified to increase alkaline tolerance. Both positions are located in close proximity of the T1Cu site. Molecular docking and simulations studies reveal that both substitutions act in a synergic way to stabilize and improve laccase activity at higher pH. Kinetic characterization of the final variant MaL‐M1 (L365E/L513M) revealed at pH 9.8 a threefold improved kcat (kcat=(6.0±0.2) s⁻¹) compared with that of wild‐type M. albomyces laccase (kcat=(2.11±0.07) s⁻¹).
This year, the Nobel Prize in Chemistry was awarded to three pioneering scientists who applied laboratory evolution for protein engineering: Frances Arnold, George P. Smith, and Sir Gregory P. Winter. This approach has had major impact in various applications and inspires the search for the general principles of design through evolution.
The glycosylation of plant polyphenols may modulate their solubility and bioavailability, and protect these molecules from oxygen, light degradation and during gastrointestinal transit. In this work, the synthesis of various α-glucosyl derivatives of (‒)-epigallocatechin gallate (EGCG), the predominant catechin in green tea, was performed in water at 50 °C by a transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. The molecular weight of reaction products was determined by HPLC-MS. Using hydrolyzed potato starch as glucosyl donor, two main monoglucosides were obtained with conversion yields of 58% and 13%, respectively. The products were isolated and chemically characterized by combining 2D-NMR methods. The major derivative was epigallocatechin gallate 3’-O-α-D-glucopyranoside (1) and the minor epigallocatechin gallate 7-O-α-D-glucopyranoside (2).
Microorganisms are found throughout every corner of nature, and vast number of microorganisms is difficult to cultivate by classical microbiological techniques. The advent of metagenomics has revolutionized the field of microbial biotechnology. Metagenomics allow the recovery of genetic material directly from environmental niches without any cultivation techniques. Currently, metagenomic tools are widely employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the uncultivable component of microbial communities. The employment of next-generation sequencing techniques for metagenomics resulted in the generation of large sequence data sets derived from various environments, such as soil, the human body and ocean water. This review article describes the state-of-the-art techniques and tools in metagenomics and discusses the potential of metagenomic approaches for the bioprospecting of industrial enzymes from various environmental samples. We also describe the unusual novel enzymes discovered via metagenomic approaches and discuss the future prospects for metagenome technologies.
The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 μg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.
Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is widely distributed among various organisms. Since GABA has several well-known physiological functions, such as mediating neurotransmission and hypotensive activity, as well as having tranquilizer effects, it is commonly used as a bioactive compound in the food, pharmaceutical and feed industries. The major pathway of GABA biosynthesis is the irreversible decarboxylation of l-glutamate catalyzed by glutamate decarboxylase (GAD), which develops a safe, sustainable and environmentally friendly alternative in comparison with traditional chemical synthesis methods. To date, several microorganisms have been successfully engineered for high-level GABA biosynthesis by overexpressing exogenous GADs. However, the activity of almost all reported microbial GADs sharply decreases at physiological near-neutral pH, which in turn provokes negative effects on the application of these GADs in the recombinant strains for GABA production. Therefore, ongoing efforts in the molecular evolution of GADs, in combination with high-throughput screening and metabolic engineering of particular producer strains, offer fascinating new prospects for effective, environmentally friendly and economically viable GABA biosynthesis. In this review, we briefly introduce the applications in which GABA is used, and summarize the most important methods associated with GABA production. The major achievements and present challenges in the biotechnological synthesis of GABA, focusing on screening and enzyme engineering of GADs, as well as metabolic engineering strategy for one-step GABA biosynthesis, will be extensively discussed.
Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas.
A gram-positive bacterium, denominated CFA-06, was isolated from Brazilian petroleum in the Campos Basin and is responsible for the degradation of aromatic compounds and petroleum aromatic fractions. The CFA-06 strain was identified as Bacillus safensis using the 16S rRNA and gyrase B sequence. Enzymatic assays revealed the presence of two oxidoreductases: a catalase and a new oxidoreductase. The oxidoreductases were enzymatically digested and analysed via ESI-LTQ-Orbitrap mass spectrometry. The mass data revealed a novel oxidoreductase (named BsPMO) containing 224 amino acids and 89% homology with a hypothetic protein from B. safensis (CFA-06) and a catalase (named BsCat) with 491 amino acids and 60% similarity with the catalase from Bacillus pumilus (SAFR-032). The new protein BsPMO contains iron atom(s) and shows catalytic activity toward a monooxygenase fluorogenic probe in the presence of cofactors (NADH, NADPH and NAD). This study enhances our knowledge of the biodegradation process of petroleum by B. safensis.
Environmental microbiology and biotechnology are crucial in the modern society. They influence human health, environment and serve as a source of energy and provider of novel bioproducts. The key areas of interest are bioremediation, microbial communities, pathogen detection, wastewater treatment, energy generation, bioprocesses, and natural product discovery. Environmental microbiology must interact with hydrology, biogeochemistry, limnology, oceanography and numerical modeling. This subject will continue to engage future generations of scientists, engineers, entrepreneurs and policy makers.
While many human activities rely heavily on the use of petrogenic hydrocarbons, contamination of the environment by petrogenic hydrocarbons during extraction, refining, and transport of petroleum remains a global issue. The aim of this chapter is to assess the potential for phytoremediation (plant-assisted bioremediation) and necrophytoremediation (dead plant biomass-assisted bioremediation) for the remediation of hydrocarbon-contaminated soils. Phytoremediation is defined as the use of plants and their associated microorganisms to remove or degrade organic or inorganic contaminants. In terms of hydrocarbon degradation, microorganisms present in the plant rhizosphere (root zone) have been shown to be effective at remediating the contaminants; this is called rhizoremediation (rhizodegradation). Plant roots increase microbial activities (including hydrocarbon degrading activities) through the release of root exudates such as nutrients, oxygen, and hydrocarbon analogues, which may contribute to the acceleration of the degradation of petrogenic hydrocarbons. However, toxicity associated with many hydrocarbon products towards the plants coupled with undesirable soil conditions such as salinity may limit the effectiveness of phytoremediation (rhizoremediation). In this case, necrophytoremediation can be used as an alternative method; this is defined as the use of dead plant biomass (e.g. straw) and its associated microflora to degrade the contaminant. Necrophytoremediation is a toxic-independent process, one which is less affected by soil conditions unlike phytoremediation. In summary, both these technologies are promising tools which can be used to remediate hydrocarbon-contaminated soils as they are cost-effective, sustainable, and environmental friendly.
Lenardao, EJ Freitag, RA Dabdoub, MJ Batista, ACF Silveira, CD
Our aim is to show, that the enzymes as electrocatalysts are able to improve the performance characteristics of the fuel cells. The hydrogen fuel electrode based on hydrogenase from Thiocapsa roseopersicina immobilized directly on carbon filament material has been made. The enzyme electrode has operated according to electron tunneling between the enzyme active site and the electrode support; this mechanism is called direct bioelectrocatalysis. Under pure hydrogen the efficiencies in energy conversion of the enzyme electrode and the noble metal based commercial fuel electrode are similar concerning both the hydrogen equilibrium potential achieved and the current densities in H 2 oxidation. However, the use of the enzyme electrodes completely avoids the problem of poisoning the anode by the impurities of carbon monoxide present in reforming gas, which limits the use of cheap hydrogen containing fuel. The stability of the biological catalysts can be drastically improved by their immobilization on electrode supports, which provide the development of commercially competitive biofuel cells. Ó 2002 Elsevier Science B.V. All rights reserved.
Whether one considers the "light side" or the "dark side" of hydrogen production, significant progress is being made.
Nature provides a fantastic array of catalysts extremely well suited to
supporting life, but usually not so well suited for technology. Whether biocatalysis
will have a significant technological impact depends on our finding robust
routes for tailoring nature's catalysts or redesigning them anew. Laboratory
evolution methods are now used widely to fine-tune the selectivity and activity
of enzymes. The current rapid development of these combinatorial methods promises
solutions to more complex problems, including the creation of new biosynthetic
pathways. Computational methods are also developing quickly. The marriage
of these approaches will allow us to generate the efficient, effective catalysts
needed by the pharmaceutical, food and chemicals industries and should open
up new opportunities for producing energy and chemicals from renewable resources.
We examine the life cycles of gasoline, diesel, compressed natural gas (CNG), and ethanol (C2H5OH)-fueled internal combustion engine (ICE) automobiles. Port and direct injection and spark and compression ignition engines are examined. We investigate diesel fuel from both petroleum and biosources as well as C2H5OH from corn, herbaceous bio-mass, and woody biomass. The baseline vehicle is a gasoline-fueled 1998 Ford Taurus. We optimize the other fuel/powertrain combinations for each specific fuel as a part of making the vehicles comparable to the baseline in terms of range, emissions level, and vehicle lifetime. Life-cycle calculations are done using the economic input-output life-cycle analysis (EIO-LCA) software; fuel cycles and vehicle end-of-life stages are based on published model results. We find that recent advances in gasoline vehicles, the low petroleum price, and the extensive gasoline infrastructure make it difficult for any alternative fuel to become commercially viable. The most attractive alternative fuel is compressed natural gas because it is less expensive than gasoline, has lower regulated pollutant and toxics emissions, produces less greenhouse gas (GHG) emissions, and is available in North America in large quantities. However, the bulk and weight of gas storage cylinders required for the vehicle to attain a range comparable to that of gasoline vehicles necessitates a redesign of the engine and chassis. Additional natural gas transportation and distribution infrastructure is required for large-scale use of natural gas for transportation. Diesel engines are extremely attractive in terms of energy efficiency, but expert judgment is divided on whether these engines will be able to meet strict emissions standards, even with reformulated fuel. The attractiveness of direct injection engines depends on their being able to meet strict emissions standards without losing their greater efficiency. Biofuels offer lower GHG emissions, are sustainable, and reduce the demand for imported fuels. Fuels from food sources, such as biodiesel from soybeans and C2H5OH from corn, can be attractive only if the co-products are in high demand and if the fuel production does not diminish the food supply. C2H5OH from herbaceous or woody biomass could replace the gasoline burned in the light-duty fleet while supplying electricity as a co-product. While it costs more than gasoline, bioethanol would be attractive if the price of gasoline doubled, if significant reductions in GHG emissions were required, or if fuel economy regulations for gasoline vehicles were tightened.
With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors-that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.
Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate
nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to
relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance
the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell
membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities.
A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion.
Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular,
22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed
evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating
other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents
such as sarin and soman.
Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together
with a 22-fold increase in kcat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective
mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site
of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus
is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations
accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability
in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular
mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible
for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.
Organophosphate-degrading enzyme from Agrobacterium radiobacter P230 (OPDA) is a recently discovered enzyme that degrades a broad range of organophosphates. It is very similar to OPH first
isolated from Pseudomonas diminuta MG. Despite a high level of sequence identity, OPH and OPDA exhibit different substrate specificities. We report here the
structure of OPDA and identify regions of the protein that are likely to give it a preference for substrates that have shorter
alkyl substituents. Directed evolution was used to evolve a series of OPH mutants that had activities similar to those of
OPDA. Mutants were selected for on the basis of their ability to degrade a number of substrates. The mutations tended to cluster
in particular regions of the protein and in most cases, these regions were where OPH and OPDA had significant differences
in their sequences.
The realization that fossil fuel reserves are limited and their adverse effect on the environment has forced us to look into alternative sources of energy. Hydrogen is a strong contender as a future fuel. Biological hydrogen production ranges from 0.37 to 3.3 moles H(2) per mole of glucose and, considering the high theoretical values of production (4.0 moles H(2) per mole of glucose), it is worth exploring approaches to increase hydrogen yields. Screening the untapped microbial population is a promising possibility. Sequence analysis and pathway alignment of hydrogen metabolism in complete and incomplete genomes has led to the identification of potential hydrogen producers.
Enzymes are powerful tools that help sustain a clean environment in several ways. They are utilized for environmental purposes in a number of industries including agro-food, oil, animal feed, detergent, pulp and paper, textile, leather, petroleum, and specialty chemical and biochemical industry. Enzymes also help to maintain an unpolluted environment through their use in waste management. Recombinant DNA technology, protein engineering, and rational enzyme design are the emerging areas of research pertaining to environmental applications of enzymes. The future will also see the employment of various technologies including gene shuffling, high throughput screening, and nanotechnology. This article presents an overview of the enzymatic applications in pollution control and the promising research avenues in this area.
Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.
Brazil is one of the world's largest producers of alcohol from biomass at low cost and is responsible for more than 1 million direct jobs. In 1973, the Brazilian Program of Alcohol (Proalcool) stimulated the creation of a bioethanol industry that has led to large economic, social, and scientific improvements. In the year 1984, 94.5% of Brazil's cars used bioethanol as fuel. In 2003/2004, 350.3 million of sugarcane produced 24.2 million t of sugar and 14.4 billion L of ethanol for an average 4.3 million cars using ethanol. Since its inception, cumulative investment in Proalcool totals US$11 billion, and Brazil has saved US$27 billion in oil imports. The ethanol production industry from sugarcane gene-rates 152 times more jobs than would have been the case if the same amount of fuel was produced from petroleum, and the use of ethanol as a fuel is advantageous for environmental reasons. In 2003, one of the biggest Brazilian ethanol industries started consuming 50% of the residual sugarcane bagasse to produce electrical energy (60 MW), a new alternative use of bioenergy for the Brazilian market. Other technologies for commercial uses of bagasse are in development, such as in the production of natural fibers, sweeteners (glucose and xylitol), single-cell proteins, lactic acid, microbial enzymes, and many other products based on fermentations (submerged and semisolid). Furthermore, studies aimed at the increase in the biosynthesis of sucrose and, consequently, ethanol productivity are being conducted to understand the genetics of sugarcane. Although, at present, there remain technical obstacles to the economic use of some ethanol industry residues, several research projects have been carried out and useful data generated. Efficient utilization of ethanol industry residues has created new opportunities for new value-added products, especially in Brazil, where they are produced in high quantities.
Despite its long-term use in bioremediation, bioaugmentation of contaminated sites with microbial cells continues to be a source of controversy within environmental microbiology. This largely results from its notoriously unreliable performance record. In this article, we argue that the unpredictable nature of the approach comes from the initial strain selection step. Up until now, this has been dictated by the search for catabolically competent microorganisms, with little or no consideration given to other essential features that are required to be functionally active and persistent in target habitats. We describe how technical advances in molecular biology and analytical chemistry, now enable assessments of the functional diversity and spatial distribution of microbial communities to be made in situ. These advances now enable microbial populations, targeted for exploitation, to be differentiated to the cell level, an advance that is bound to improve microbial selection and exploitation. We argue that this information-based approach is already proving to be more effective than the traditional 'black-box' approach of strain selection. The future perspectives and opportunities for improving selection of effective microbial strains for bioaugmentation are also discussed.
An area of intense scientific and practical interest is the biogeochemical and microbial processes determining the success of natural attenuation, biostimulation and/or bioaugmentation treatments for organic contaminants in groundwater. Recent studies in this area have focused on the reductive dechlorination of chlorinated solvents, the degradation of the fuel additive methyl tert-butyl ether, and the removal of long-term hydrocarbon contamination. These studies have been facilitated by the use of stable isotope analysis to demonstrate in situ bioremediation and push-pull tests, in which isotopes are injected into aquifers and then quickly retrieved and analyzed, to measure in situ activity. Molecular tools such as quantitative PCR, the detection of mRNA expression, and numerous DNA fingerprinting methods have also proved valuable, being employed to identify and sometimes quantify environmentally important organisms or changes in communities. Methods to track bacteria and tools to characterize bacterial attachment properties have also offered insight into bacterial transport in situ.
Many research groups successfully rely on whole-gene random mutagenesis and recombination approaches for the directed evolution of enzymes. Recent advances in enzyme engineering have used a combination of these random methods of directed evolution with elements of rational enzyme modification to successfully by-pass certain limitations of both directed evolution and rational design. Semi-rational approaches that target multiple, specific residues to mutate on the basis of prior structural or functional knowledge create 'smart' libraries that are more likely to yield positive results. Efficient sampling of mutations likely to affect enzyme function has been conducted both experimentally and, on a much greater scale, computationally, with remarkable improvements in substrate selectivity and specificity and in the de novo design of enzyme activities within scaffolds of known structure.
Oleic acid alkyl esters (biodiesel) were synthesised by biocatalysis in solvent-free conditions. Different commercial immo-bilised lipases, namely Candida antarctica B, Rizhomucor miehei, and Pseudomonas cepacia, were tested towards the reaction between triolein and butanol to produce butyl oleate. Pseudomonas cepacia lipase resulted to be the most active enzyme reaching 100% of conversion after 6 h. Different operative conditions such as reaction temperature, water activity, and reagent stoichio-metric ratio were investigated and optimised. These conditions were then used to investigate the effect of linear and branched short chain alcohols. Methanol and 2-butanol were the worst alcohols: the former, probably, due to its low miscibility with the oil and the latter because secondary alcohols usually are less reactive than primary alcohols. Conversely, linear and branched primary alcohols with short alkyl chains (C 2 –C 4) showed high reaction rate and conversion. A mixture of linear and branched short chain alcohols that mimics the residual of ethanol distillation (fusel oil) was successfully used for oleic acid ester synthesis. These compounds are important in biodiesel mixtures since they improve low temperature properties.
Biocatalysis offers mild reaction conditions, an environmentally attractive catalyst–solvent system, high activities, and chemo-, regio-, and stereoselectivities, while the use of enzymes generally circumvents the need for functional group activation and avoids protection/deprotection steps required in traditional organic syntheses. This review, using β-lactam antibiotics as an example, discusses recent advances in biocatalysis research towards the goal of ‘green’ methodologies for the manufacture of (fine) chemicals and the emulation of a cell's enzymatic cascade processes.
Under aerobic conditions, the enzyme γ-hexachlorocyclohexane dechlorinase (LinA) from Sphingomonas paucimobilis UT26 catalyses the elimination of chlorine atoms from the molecule of γ-hexachlorocyclohexane (γ-HCH) or lindane, a recalcitrant pesticide that is still widely used. In its native metabolic context, LinA starts the biodegradation process of lindane by transforming γ-HCH to 1,2,4 trichlorobenzene (TCB), a less persistent chemical. In an attempt to generate an improved version of this enzyme to be used in lindane bioremediation schemes, we have run an experimental evolution procedure on LinA, using Escherichia coli as the surrogate host. One round of random mutagenesis and subsequent screening for improved dechlorination in vivo sufficed to yield one mutant enzyme (LinAT10), bearing a single substitution C132R, that displayed a two-fold enhanced expression and three-fold enhanced solubility of the enzyme compared to the wild type protein. This resulted in a biological product with a six-fold increase in dechlorination ability when expressed in E. coli. The potential of this protein and its expression system for in situ bioremediation is discussed.
The ability of peroxidases and laccases enzymes to treat organic pollutants is reviewed. Enzymatic methods generally have low energy requirements, are easy to control, can operate over a wide range of conditions and have a minimal environmental impact. Peroxidases and laccases have broad substrate specificities and can catalyze the oxidation of a wide range of toxic organic compounds. The results show that an enzymatic oxidation can diminish the toxicity of some polycyclic aromatic hydrocarbons (PAHs), phenols, organophosphorus pesticides and azo dyes in laboratory and some field conditions. Due to the hydrophobicity and low aqueous solubility of these substrates, reactions are usually performed in the presence of organic solvents. However, it was detected that organic solvents can provoke enzyme denaturation, unfavorable substrate partition, inhibition or stabilization of enzyme–substrate complexes, depending on the enzyme, substrate and organic solvent used. Strategies to overcome these problems are proposed. Additionally, the low stability of heme-containing peroxidases to hydrogen peroxide, the low reaction rates of laccases, the mediators toxicity, the limited availability and high costs of these enzymes are other limitations detected for commercial applications. Due to field reaction conditions are more complex than laboratory conditions efforts have to be made to achieve the cheap overproduction of these biocatalysts in heterologous hosts and also their modification by chemical means or protein engineering to obtain more robust and active enzymes.
This topical review and analysis seeks to provide others with some insight to strategies for the development of new process technologies in the 21st century which align with the traditional desires of green and/or environmental chemistry. Rather then dwelling on existing approaches or step-wise developments, guidance is offered about those targets and approaches which offer the greatest reward and green benefits. Catalysis and catalysts are a key parts of multiple approaches, but those approaches must be taken with some anticipation about the vitality of the technological alternative that is considered in view of overall issues of economics, politics, societal demands, and total energy requirements. The importance of considering energy usage in designing and modifying new chemical processes will be emphasized. Examples will be given to illustrate the importance of considering the total energy input and output of any new alternative process. When this is done, it becomes apparent that several popular approaches are not really so green. Opportunities for improvement to existing products are discussed with a focus on seeking dramatic, breakthrough process improvements with minimal energy and materials costs.
A field release of a genetically engineered microorganism was performed at the Field Lysimeter Site on the Oak Ridge Reservation. Six large lysimeters were filled with soil that had been contaminated with a mixture of naphthalene, phenanthrene, and anthracene. A genetically engineered bacterial strain, Pseudomonas fluorescens HK44, was sprayed onto the surface of the soil during soil loading. This strain contains a fusion between the lux genes of Vibrio fischeri and the promoter for the lower pathway of naphthalene degradation, enabling the strain to become bioluminescent when it is degrading naphthalene. Release of the bacteria outside the lysimeters was monitored, using selective agar plates and one-stage Anderson air samplers. Although approximately 10(14) bacteria were sprayed during the loading process, escape was only detected sporadically; the highest incidence of bacterial escape was found when the relative humidity and wind speed were low.
Biocatalysis inherently offers the prospect of clean industrial processing and has become an accepted technology throughout most sectors. The convergence of biology and chemistry has enabled a plethora of industrial opportunities to be targeted, while discoveries in biodiversity and the impact of molecular biology and computational science are extending the range of natural and engineered biocatalysts that can be customised for clean industrial requirements.
The use of biocatalysis for industrial synthetic chemistry is on the verge
of significant growth. Biocatalytic processes can now be carried out in organic
solvents as well as aqueous environments, so that apolar organic compounds
as well as water-soluble compounds can be modified selectively and efficiently
with enzymes and biocatalytically active cells. As the use of biocatalysis
for industrial chemical synthesis becomes easier, several chemical companies
have begun to increase significantly the number and sophistication of the
biocatalytic processes used in their synthesis operations.
Enzymes are the subset of proteins that catalyse the chemistry of life,
transforming both macromolecular substrates and small molecules. The precise
three-dimensional architecture of enzymes permits almost unerring selectivity
in physical and chemical steps to impose remarkable rate accelerations and
specificity in product-determining reactions. Many enzymes are members of
families that carry out related chemical transformations and offer opportunities
for directed in vitro evolution, to tailor catalytic properties to
particular functions.
Directed evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space.
Both parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme.
Permutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. Although various physicochemical methods have been used to remove these compounds from our environment, they have many limitations. Xenobiotic-degrading microorganisms have tremendous potential for bioremediation but new modifications are required to make such microorganisms effective and efficient in removing these compounds, which were once thought to be recalcitrant. Metabolic engineering might help to improve the efficiency of degradation of toxic compounds by microorganisms. However, efficiency of naturally occurring microorganisms for field bioremediation could be significantly improved by optimizing certain factors such as bioavailability, adsorption and mass transfer. Chemotaxis could also have an important role in enhancing biodegradation of pollutants. Here, we discuss the problems of PAH pollution and PAH degradation, and relevant bioremediation efforts.
The chemical industry plays a key role in sustaining the world economy and underpinning future technologies, yet is under
unprecedented pressure from the effects of globalization and change in many of its traditional markets. Against this background,
what will be needed for the industry to embrace efforts to make it “greener”? We explore some of the issues raised by the
development of “green chemistry” techniques and identify potential barriers to their implementation by industry.
Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
Directed evolution has now been used for over two decades as an alternative to rational design for protein engineering. Protein function, however, is complex, and modifying enzyme activity is a tall order. We can now improve existing enzyme activity, change enzyme selectivity and evolve function de novo using directed evolution. Although directed evolution is now used routinely to improve existing enzyme activity, there are still only a handful of examples where substrate selectivity has been modified sufficiently for practical application, and the de novo evolution of function largely eludes us.
Water flows were analysed for the filling phase and the first 4 years after closure of two types of full-scale landfill cells: 'special cells' containing mostly fly ash from municipal solid waste (MSW) incineration disposed with other special/hazardous waste, and 'biocells' (biological cells) containing co-disposed MSW and food industry sludge. The landfill cells were constructed about -1.5 m above sea level (masl) at Lomma Bay, southern Sweden. The hydrological effects of water intrusion into the special cells from surroundings and sludge moisture within the biocells were studied. HELP modelling of hydrological processes predicted delay in peaks of leachate generation from uncovered special cells following rain, which was not confirmed. Faster leachate production as a response to rainfall from special cells than from biocells was observed. It was inferred that special waste has more intensive channelling, lower water absorption and higher hydraulic conductivity than mixtures of sludge/MSW. To avoid convergence problems in modelling uncovered special cells, the use of a 5 cm deep top layer with saturated hydraulic conductivity 1.7 x 10(-3) cm s(-1), porosity 0.437, and field capacity 0.105, is suggested.
A custom-made enzyme: The gene and protein sequence of a new (R)-hydroxynitrile lyase from Prunus amygdalus was cloned and engineered for recombinant production on a large scale. The enzyme is stable and active at low pH values. A rationally designed active-site mutant now permits the rapid and stereoselective synthesis of sterically hindered cyanohydrins such as ortho-chloromandelonitrile (see picture).
Research conducted over the past seven years by the biotechnology byproducts consortium (BBC) addresses its mission to investigate the opportunities to add value to agricultural products, byproducts and coproducts and to manage the wastewater arising from agribusinesses in an environmentally favorable way. Since a wide variety of research approaches have been taken, the results are collected in five topic groups: (1) bioremediation that includes anaerobic fermentations of wastes to produce methane and hydrogen, the genetics of methanogenesis and in situ remediation of contaminated aquifer systems, landfill leachates and industrial effluents; (2) land application of fermentation byproducts and their use in animal feeds; (3) biocatalytic studies of transformations of components of corn and soybean oils, peroxidases present in plant products, such as soybean hulls; (4) biochemical reactions for the production of de-icers from industrial water streams, biodiesel production from fats and greases, biodegradable plastics from polymerizable sugar derivatives, single cell foods derived from fungal growth on waste streams, and bacterial polysaccharides from Erwinia species; (5) separation and recovery of components by membrane technologies.
A limited number of bacteria, yeast and fungi can convert hemicellulose or its monomers (xylose, arabinose, mannose and galactose) into ethanol with a satisfactory yield and productivity. In the present study we tested a number of thermophilic enrichment cultures, and new isolates of thermophilic anaerobic bacterial strains growing optimally at 70-80 degrees C for their ethanol production from D-xylose. The new isolates came from different natural and man-made systems such as hot springs, paper pulp mills and brewery waste water. The test was composed of three different steps; (i) test for conversion of D-xylose into ethanol; (ii) test for viability and ethanol production in pretreated wheat straw hemicellulose hydrolysate; (iii) test for tolerance against high D-xylose concentrations. A total of 86 enrichment cultures and 58 pure cultures were tested and five candidates were selected which successfully fulfilled the criteria defined for the screening test.
Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.
Use of biotechnology in pulp bleaching has attracted considerable attention and achieved interesting results in recent years. Enzymes of the hemicellulolytic type, particularly xylan-attacking enzymes, xylanases are now used commercially in the mills for pulp treatment and subsequent incorporation into bleach sequences. The aims of the enzymatic treatment depend on the actual mill conditions and may be related to environmental demands, reduction of chemical costs or maintenance or even improvement of product quality. The use of oxidative enzymes from white-rot fungi, that can directly attack lignin, is a second-generation approach, which could produce larger chemical savings than xylanase but has not yet been developed to the full scale. It is being studied in several laboratories in Canada, Japan, the U.S.A. and Europe. Certain white-rot fungi can delignify kraft pulps increasing their brightness and their responsiveness to brightening with chemicals. The fungal treatments are too slow but the enzyme manganese peroxidase and laccase can also delignify pulps and enzymatic processes are likely to be easier to optimize and apply than the fungal treatments. Development work on laccase and manganese peroxidase continues. This article presents an overview of developments in the application of hemicellulase enzymes, lignin-oxidizing enzymes and white-rot fungi in bleaching of chemical pulps. The basic enzymology involved and the present knowledge of the mechanisms of the action of enzymes as well as the practical results and advantages obtained on the laboratory and industrial scale are discussed.
Hydrogenases have found use in a variety of biotechnological applications, including biohydrogen production, wastewater treatment, the prevention of microbial-induced corrosion and the generation and regeneration of NADP cofactors. In the future, advances in genome mining and screening techniques are likely to identify new hydrogenases for novel applications.
Biocatalysis has expanded rapidly in the last decades with the discoveries of highly stereoselective enzymes with broad substrate specificity. A new frontier for biocatalysis is broad reaction specificity, where enzymes catalyze alternate reactions. Although often under-appreciated, catalytic promiscuity has a natural role in evolution and occasionally in the biosynthesis of secondary metabolites. Examples of catalytic promiscuity with current or potential applications in synthesis are reviewed here. Combined with protein engineering, the catalytic promiscuity of enzymes may broadly extend their usefulness in organic synthesis.
1. Enzymatic bioremediation is potentially a rapid method of removing environmental pesticide residues. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables and spent dip liquors.
2. A specific application for some organophosphate-degrading enzymes involves detoxification of nerve agent stockpiles. Effective and affordable remediation requires highly specialized enzymes, so protein engineering techniques are being used to improve properties of various source enzymes to enhance catalytic rates, stability and substrate range.
3. Trials with an optimized organophosphate-degrading enzyme have shown the feasibility of such technology in various applications.
4. The enzymes developed for environmental remediation for specific pesticide classes also have applications as antidotes for high-dose pesticide poisonings and as prophylaxis for people at risk of high pesticide doses.
Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.
Wheat bran (WB), produced worldwide in large quantities as a by-product of the wheat milling industry, constitutes a significant underutilized source of sugars. This paper describes various methods of hydrolyzing the abundant polysaccharides in bran to yield a sugar feedstock suitable for fermentation into bioethanol. Firstly, the starch in the bran was released using amylolytic enzymes. The fibrous material remaining was further hydrolyzed. Acid hydrolysis, heat pretreatment followed by enzymatic hydrolysis and direct enzymatic hydrolysis were compared in terms of total sugar yield and pentose sugar yield. The maximum total sugar yield was achieved when small amounts of acid were added at the pretreatment step prior to enzymatic hydrolysis. This form of pretreatment released most pentosans and significantly enhanced the hydrolysis of cellulose. The overall sugar yield of this combined hydrolysis method reached 80% of the theoretical and it consisted of 13.5 g arabinose, 22.8 g xylose and 16.7 g glucose per 100 g starch-free bran.
Biological methods for decontamination promise an improved substitute for ineffective and costly physico-chemical remediation methods, although so far only a fraction of the total microbial diversity (i.e. the culturable fraction with metabolic potential) has been harnessed for this purpose. Exploring and exploiting the "overlooked" genetic resource might ameliorate concerns associated with the degradation of recalcitrant and xenobiotic pollutants that are not degraded or only poorly degraded by known culturable bacteria. Recent advances in the molecular genetics of biodegradation and in knowledge-based methods of rational protein modification provide insight into the development of "designer biocatalysts" for environmental restoration. The application of such genetically engineered microorganisms (GEMs) in the environment has been limited, however, owing to the risks associated with uncontrolled growth and proliferation of the introduced biocatalyst and horizontal gene transfer. Programming rapid death of the biocatalyst soon after the depletion of the pollutant could minimize the risks in developing these technologies for successful bioremediation.
Enzyme immobilisation is experiencing an important transition. Combinatorial approaches are increasingly applied in the design of robust immobilised enzymes by rational combination of fundamental immobilisation techniques (i.e. non-covalent adsorption, covalent binding, entrapment and encapsulation) or with other relevant technologies. The objective is to solve specific problems that cannot be solved by one of these basic immobilisation techniques.
It is now widely accepted that the application of standard microbiological methods for the recovery of microorganisms from the environment has had limited success in providing access to the true extent of microbial biodiversity. It follows that much of the extant microbial genetic diversity (collectively termed the metagenome) remains unexploited, an issue of considerable relevance to a wider understanding of microbial communities and of considerable importance to the biotechnology industry. The recent development of technologies designed to access this wealth of genetic information through environmental nucleic acid extraction has provided a means of avoiding the limitations of culture-dependent genetic exploitation.
Various high-throughput experimental techniques are routinely used for generating large amounts of omics data. In parallel, in silico modelling and simulation approaches are being developed for quantitatively analyzing cellular metabolism at the systems level. Thus informative high-throughput analysis and predictive computational modelling or simulation can be combined to generate new knowledge through iterative modification of an in silico model and experimental design. On the basis of such global cellular information we can design cells that have improved metabolic properties for industrial applications. This article highlights the recent developments in these systems approaches, which we call systems biotechnology, and discusses future prospects.
Microorganisms can degrade numerous organic pollutants owing to their metabolic machinery and to their capacity to adapt to inhospitable environments. Thus, microorganisms are major players in site remediation. However, their efficiency depends on many factors, including the chemical nature and the concentration of pollutants, their availability to microorganisms, and the physicochemical characteristics of the environment. The capacity of a microbial population to degrade pollutants within an environmental matrix (e.g. soil, sediment, sludge or wastewater) can be enhanced either by stimulation of the indigenous microorganisms by addition of nutrients or electron acceptors (biostimulation) or by the introduction of specific microorganisms to the local population (bioaugmentation). Although it has been practiced in agriculture and in wastewater treatment for years, bioaugmentation is still experimental. Many factors (e.g. predation, competition or sorption) conspire against it. However, several strategies are currently being explored to make bioaugmentation a successful technology in sites that lack significant populations of biodegrading microorganisms. Under optimal local conditions, the rate of pollutant degradation might increase upon addition of an inoculant to remediate a chemical spill; however, the most successful cases of bioaugmentation occur in confined systems, such as bioreactors in which the conditions can be controlled to favour survival and prolonged activity of the exogenous microbial population.
Industrialization and the quest for a more comfortable lifestyle have led to increasing amounts of pollution in the environment. To address this problem, several biotechnological applications aimed at removing this pollution have been investigated. Among these pollutants are xenobiotic compounds such as polynitroaromatic compounds--recalcitrant chemicals that are degraded slowly. Whereas 2,4,6-trinitrophenol (TNP) can be mineralized and converted into carbon dioxide, nitrite and water, 2,4,6-trinitrotoluene (TNT) is more recalcitrant--although several microbes can use it as a nitrogen source. The most effective in situ biotreatments for TNT are the use of bioslurry (which can be preceded by an abiotic step) and phytoremediation. Phytoremediation can be enhanced by using transgenic plants alone or together with microbes.