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Literature Review
Industrial enzyme applications. Curr Opin Biotechnol
Abstract
The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.
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- ... Previous studies of Sang-Mok and Koo, (2001); Kirk et al., (2002) pointed that cellulases are inducible enzymes produced by several microorganisms, mainly by bacteria and fungi. Moreover, Immanuel et al., (2006); Kasana et al., (2008) added that cellulases have various potential industrial and biotechnological applications, and hence are in high demand. ...
- ... Previous studies of Sang-Mok and Koo, (2001); Kirk et al., (2002) pointed that cellulases are inducible enzymes produced by several microorganisms, mainly by bacteria and fungi. Moreover, Immanuel et al., (2006); Kasana et al., (2008) added that cellulases have various potential industrial and biotechnological applications, and hence are in high demand. ...
- The use of biomolecules toward environmental analysis provides impressive advantages in terms of selectivity and efficiency. Proteins have served as the classical choice of biomolecules in this regard partly due to their natural function as strong and specific binding agents. Nevertheless, biomolecules are usually considered unsuitable for large-scale environmental applications due to their fragility and cost. In this chapter, we first examine the emergence of nucleic acid based nanotechnology in the context of environmental analysis. Notably, the development of nucleic acid aptamers, aptazymes, and nano-architectures has facilitated application as both a receptor in biosensors as well as versatile scaffolds for engineering functional constructs. Further, we present a proof-of-concept of nucleic acid based nanoconstructs as a reusable adsorbing agent. We have developed nucleic acid three-way junction-based matrices that are capable of retrieval and reuse of a commonly used staining agent. Immobilization of the nucleic acid architectures on magnetic nanoparticles enables their reuse across samples. Inherent sophistication of biomolecules in general and nucleic acid based constructs, in particular, supports their deployment in specialized applications at a smaller scale pertinent to individual human activity. The perspective presented in this chapter is expected to encourage environmental engineering in distinctive and atypical contexts.
- Aspergillus terreus was chosen for production of alkaline protease using solid-state fermentation (SSF). The maximum enzyme yield reached about 34.87 U/mg protein after optimization of fermentation parameters. The produced alkaline protease was purified by precipitation with iso-propanol and then purified through gel filtration and ion exchange column chromatography with a yield of 53.58% and 5.09- fold purification. The enzyme has shown to have a molecular weight of 35 kDa. Optimal pH and temperature for the enzyme activity were 9.5 and 50°C respectively. The highest activity was reported towards casein, with an apparent Km value of 6.66 mg/mL and Vmax was 30 U/mL. The enzyme activity was greatly repressed by phenylmethylsulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) caused activation in enzyme activity. The enzyme retained about 83.8, 70.6, 74.5, 76.4 and 66.4% of its original activity after incubation with Aerial, Leader, Oxi, Persil and Tide, respectively for 8 h at 60°C. Adding of the enzyme in detergents improved the cleansing performance to the blood stains and suggested to be used as a detergent additive. Our outcomes showed that protease could be used as environment green-approach in dehairing process.
- Les canaux biologiques sont des protéines situées sur la membrane cellulaire. Ils permettent de maintenir l'équilibre des électrolytes et le transport des molécules. Ces canaux protéiques peuvent répondre à de multiples stimuli tels que la lumière, le pH, la force ionique et le potentiel transmembranaire afin de réguler leur perméabilité et leur sélectivité. Pour imiter ces pores biologiques, des nanopores artificiels ont été fabriqués des matériaux inorganiques ou polymères. Leurs avantages sont la simplicité de fabrication et leur stabilité mécanique et chimique. Cependant, ils sont généralement moins sélectifs et ne répondent pas aux stimuli externes. Pour améliorer leurs propriétés, leur fonctionnalisation est requise pour améliorer leurs propriétés de perméabilité, de sélectivité ou leur donner la capacité de détecter des biomolécules spécifiques.Dans cette thèse, nous avons cherché à fonctionnaliser des nanopores obtenus par la méthode des traces attaquée avec des polymères pour trois applications: mimer canal ionique sensible aux stimuli, la récupération de l’énergie osmotique et les biocapteurs. Pour mimer les canaux ioniques stimuli répondant, nous avons construit deux types de nanopore : l’un fonctionnalisé par auto-assemblage couche par couche de différent polyélectrolytes est sensible au pH. Le second est sensible à la lumière et au pH est obtenu par greffage chimique de Polyethylène glycol-spiropyran. L'isomérisation induites par la lumière et la protonation / déprotonation causées par le changement de pH peuvent modifier la charge et la conformation de molécules fonctionnelles modulant ainsi l’ouverture et la perm-sélectivité du pore. Pour la récupération d'énergie osmotique, nous avons suivi deux stratégies pour améliorer la sélectivité ionique de la membrane afin d’améliorer le rendement énergétique. La première approche consiste au dépôt couche par couche de polyélectrolytes sur la surface des pores pour augmenter la densité de charge de surface. La seconde stratégie est basée sur le remplissage sur pore par un hydrogel hautement chargé synthétisé in-situ. Les résultats ont montré une production d'énergie de 20 pW par pore pour les deux stratégies. La fonctionnalisation avec l’hydrogel permet d'utiliser une géométrie cylindrique permettant de fabriquer des membranes avec une haute densité de pores. Pour l’application biocapteur, , nous avons développé un nanopore fonctionnalisé par PLL afin de détecter la contamination de l’héparine par chodroitine sulfate oversulfatée (OSCS) en utilisant le principe de la diode ionique. Après avoir été traitée aux héparinases, la concentration en héparine dans les résidus peut refléter quantitativement la concentration en OSCS par la mesure de la rectification du courant ionique (ICR). Ces expériences ont aussi permis de confirmé la possibilité de caractériser la cinétique de dégradations enzymatiques par une nanopore unique. Pour aller plus loin, nous avons immobilisé des hyaluronidases dans le nanopore afin de caractériser les réactions enzymatiques au niveau d'une molécule à l'aide de la technique de pulse de résistance. Les résultats ont montré que la durée du complexe entre une hyaluronidase et une molécule d'acide hyaluronique pour une réaction est d'environ 1 seconde.Dans cette thèse, nous démontrons l’intérêt des nanopores obtenus par la technique des traces attaquée et fonctionnalisés par polyélectrolytes. Ces nanopore uniques peuvent être facilement reproduit et adapter pour concevoir d'autres systèmes pour des applications dans santé et d'énergie.
- This research reports the transport behaviors of long flexible polymers (hyaluronic acids) through long conical track-etched nanochannels with and without grafted enzymes. The impacts of the channel diameter as well as the polymer regimes in solution (dilute and semi-dilute) have been investigated. Without enzymes, the experimental results can be well explained by the analytical models of the scaling law of de Gennes. Then, the corresponding enzymes (hyaluronidase) are grafted inside the channel. When enzymes are located in the base side, polymers get degraded at the entrance and degraded products were detected. When enzymes are grafted at the tip side, the extension of translocation duration due to the binding of substrate-enzyme is observed. This is for the first time that the enzymatic degradation reactions are characterized in situ at single molecule level by nanopore technology.
Literature Review
- Effects of starch (bacterial α-amylase and NMYL) and non-starch (xylanase PTP, lipase NVZ and glucose-oxidase GLZ) degrading enzymes on dough quality parameters (rheological, fermentative, texrural and thermal characteristics) were determined by means of the response surface methodology to a central composite design of white wheat dough samples. PTP showed linear, interactive and/or quadratic significant effects on most functional properties. Higher dosages of PTP yielded softer and less adhesive doughs with higher gas retention capability but with weakened gluten and poorer machinability (stickier). Negative linear and positive quadratic effects of PTP were observed on dough hardness and derived mechanical properties gumminess and chewiness. Optimized dosage of 30 mg/100 g flour led to improved handling and fermentative dough characteristics and avoided excessive dough hardness and stickiness. The simultaneous presence of PTP and NVZ decreased fermentation time and increased dough extensibility. The combination of both PTP and NMYL and/or incorporation of NVZ decreased dough resilience, which corresponded to a greater extensibility and a lower dough consistency. Although GLZ addition induced some desirable effects on the final dough volume and the amylose-lipid complexation, the incorporation of the pair PTP/ GLZ into dough formulas is not recommended because of the reduction of the induced softening effect of PTP. A significant negative and quadratic effect of NMYL addition was observed for the enthalpy of dissociation of the amylose-lipid complex. Incorporation of this enzyme up to 30 mg/100 g flour did not significantly decrease values for this thermal property. No criteria for optimum dosage of NVZ were established, and it is recommended that doses higher than those recommended by the manufacturer (5 mg/100 g of flour) be used.
- Phytate is a molecule rich in phosphorus (P). However, the P in phytate is of low availability to monogastric animals because they lack the proper enzyme system to hydrolyse phytate. Consequently, there is a high P concentration in the manure from animals fed on diets containing phytate, and this can lead to pollution of the environment. Because phytate can complex with minerals, starch, proteins and digestive enzymes, it also has anti-nutritional properties. Limiting the P output by monogastric animals, by increasing the digestibility (availability) of the P in the diet by hydrolysing phosphate from phytate, was the original reason for developing the microbial phytase, Natuphos®. It has been shown in many studies that P excretion by pigs and poultry can be reduced by 30% by including phytase in their diets. The digestibility of other nutrients bound to phytate can also be increased considerably by hydrolysis of the phytate molecule by phytase. A number of studies have been performed in poultry and pigs to determine the effect on amino acid digestibility of adding microbial phytase to the feed. In general, an increase of 1-3% has been reported. It was shown by meta-analysis that these improvements were significant for most amino acids at a phytase supplementation rate of 500 FTU/kg diet. In piglets and broilers an improvement in performance of 1.5-3.0% was often observed when phytase was included in the diet, even if the diet met the digestible/available P requirement. This improvement in performance cannot be explained by improvements in amino acid digestibility alone. It has been suggested that there is an effect on energy utilisation as well, and this has now been confirmed in studies with poultry. To apply this information in feed compounding, matrix values are proposed for use by the industry in linear programming. Depending on many factors, feed costs can be decreased by up to EUR 3.50 (US$ 3.00)/tonne by the addition of phytase to diets that are not limiting in P. Limiting the total P content in the diet to a lower concentration can increase the economic advantage of adding phytase. Processing of feed, especially pelleting, can reduce the activity of phytase. It is therefore necessary to protect the enzyme and this can be very difficult, especially when it is important that the enzyme should also become rapidly available to the animal. The product Natuphos® 10000 G fulfils these requirements. The broadness of the impact of this enzyme on the nutritional value of feed makes it a really remarkable enzyme.
- Bacillus licheniformis α-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme’s half-life at 80 °C (pH 5.6, 0.1 mM CaCl2).
- Industrial-scale starch liquefaction is currently constrained to operating at pH 6.0 and above, as the enzyme used in the process, Bacillus licheniformis α-amylase, is unstable at lower pH under the conditions used. There is a need develop an enzyme that can operate at lower pH. Recent progress has been made in engineering the B. licheniformis enzyme for improved industrial performance. The availability of crystal structures and subsequent analysis of improved variants, in a structural context, is revealing common factors and a rationale to make further improvements.
- Effects of starch (bacterial α-amylase and NMYL) and non-starch (xylanase PTP, lipase NVZ and glucose-oxidase GLZ) degrading enzymes on dough quality parameters (rheological, fermentative, textural and thermal characteristics) were determined by means of the response surface methodology to a central composite design of white wheat dough samples. PTP showed linear, interactive and/or quadratic significant effects on most functional properties. Higher dosages of PTP yielded softer and less adhesive doughs with higher gas retention capability but with weakened gluten and poorer machinability (stickier). Negative linear and positive quadratic effects of PTP were observed on dough hardness and derived mechanical properties gumminess and chewiness. Optimized dosage of 30 mg/100g flour led to improved handling and fermentative dough characteristics and avoided excessive dough hardness and stickiness. The simultaneous presence of PTP and NVZ decreased fermentation time and increased dough extensibility. The combination of both PTP and NMYL and / or incorporation of NVZ decreased dough resilience, which corresponded to a greater extensibility and a lower dough consistency. Although GLZ addition induced some desirable effects on the final dough volume and the amylose-lipid complexation, the incorporation of the pair PTP/GLZ into dough formulas is not recommended because of the reduction of the induced softening effect of PTP. A significant negative and quadratic effect of NMYL addition was observed for the enthalpy of dissociation of the amylose-lipid complex. Incorporation of this enzyme up to 30 mg/100 g flour did not significantly decrease values for this thermal property. No criteria for optimum dosage of NVZ were established, and it is recommended that doses higher than those recommended by the manufacturer (5 mg/100 g of flour) be used.
- This study attempted to introduce the bio-processes in the conventional scouring and bleaching preparation of cotton. The scouring with two types of pectinases, acting under acidic and alkaline conditions respectively, was as efficient as the chemical process in terms of obtained adequate water absorbency of the fabrics. The necessity of surfactants application in scouring was outlined. Bleaching of the fabrics was performed with hydrogen peroxide, which was enzymatically produced by glucose oxidase during oxidation of glucose. The aeration plays an important role in the enhancement of the enzyme reaction, so that the quantity of generated peroxide is sufficient to overcome the stabilizing effect of the glucose and protein in the subsequent bleaching. A closed-loop process reusing starch containing desizing baths in a single step scouring/bleaching operation with enzyme-generated peroxide was performed.
