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Organic solvent tolerant lipase by Pseudomonas sp. strain S5: Stability of enzyme in organic solvent and physical factors affecting its production

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An organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5 and shown to be BTEX (Benzene, Toluene, Ethyl-Benzene and Xylene) degrader. The strain produces a lipase that is stable in the presence of organic solvents such as n-hexane, cyclohexane, toluene and 1-octanol. The production of lipase by Pseudomonas sp. strain S5 was optimum at 37°C, pH 7.0 and 6% starting inoculum.
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... It is widely accepted that abiotic factors such as pH and temperature can have a strong influence on enzymatic processes, as well as cell membrane permeability [23]. Lipases can metabolise well in a wide range of pH and temperature conditions, and bacterial lipases are most commonly effective in an alkaline medium [24]. ...
... The ability of strain LSK25 to hydrolyse long chain fatty acids mirrors that reported in Pseudomonas sp. S5 [23]. ...
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In recent years, studies on psychrophilic lipases have been an emerging area of research in the field of enzymology. This study focuses on bacterial strains isolated from anthropogenically-influenced soil samples collected around Signy Island Research Station (South Orkney Islands, maritime Antarctic). Limited information on lipase activities from bacteria isolated from Signy station is currently available. The presence of lipase genes was determined using real time quantification PCR (qPCR) in samples obtained from three different locations on Signy Island. Twenty strains from the location with highest lipase gene detection were screened for lipolytic activities at a temperature of 4 °C, and from this one strain was selected for further examination based on the highest enzymatic activities obtained. Analysis of 16S rRNA sequence data of this strain showed the highest level of sequence similarity (98%) to a Pseudomonas sp. strain also isolated from Antarctica. In order to increase lipase production of this psychrophilic strain, optimisation of different parameters of physical and nutritional factors were investigated. Optimal production was obtained at 10 °C and pH 7.0, at 150 rev/min shaking rate over 36 h incubation.
... One of the abilities of rhizobacteria is to induce plant systemic resistance. Biocontrol agent rhizobacteria have been reported to induce plant systemic resistance to disease-causing pathogens, which in turn increases the yield resulting from long-term disease control [17,44]. ...
Article
Rhizobacteria play a positive role as biocontrol agents as well as Plant Growth Promoting Rhizobacteria (PGPR) agents. The research objective was to obtain indigenous rhizobacteria isolates on cocoa plants that have the potential to inhibit the attack of P. palmivora fungal pathogens, and act as PGPR in vitro and in vivo. The results of the study concluded that isolates TRI 7/1, TRI 8/8, GM 7/9 and GM 7/10 had the highest ability to inhibit the growth of pathogen. The lowest disease severity (20%) was obtained in the seedlings treated using isolates TRI 7/1 and TRI 8/8. Rhizobacterial isolates GM 3/6, GM 5/6, GM 7/9 and GM 8/8 produce high amounts of IAA. Rhizobacteria isolates GM 5/6, GM 7/9 and GM 8/8 has very high peroxidase enzyme activity. High production of HCN compounds was obtained in rhizobacteria isolates TRI 3/3, TRI 4/10 and TRI GM 8/11. All rhizobacterial isolates gave an increase in the value of maximum growth potential, germination and vigor values for growth strength compared with the control. The rizobacteria treatments using isolates TRI 7/1, TRI 8/8, GM 7/9 and GM 7/10 were able to increase plant height, stem diameter and number of leaves at 30, 40, 50, 60, and 70 DAP compared to control treatment.
... Hexane is known has a log P value (3.5) higher than toluene (2.5). [26] To optimize the enzymatic hydrolysis of anchovy oil by lipase, in this study was evaluated the effect of incubation time and enzyme concentration to Omega-3 content. The results was presented in Figure 3. Reaction time is one of parameters for an enzyme to catalyze and generate the product. ...
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Anchovy (Stolephorus sp.) is an economically important fish in Indonesia. Anchovy contains Omega-3 that important to maintain the health of the heart and brain. This study aimed to enrich the Omega 3 content of anchovy oil from the North Sea of West Java. The extraction of anchovy oil was carried out by the soxhlet method. Enrichment of omega 3 from anchovy fish oil is carried out by hydrolysis with a commercial lipase enzyme at concentration 500, 1000, 1500 and 2000 unit/600g fish oil, for 5, 10, 15 and 20 hour. Before hydrolysis, fish oil was added with solvent water, ethanol, toluene, and n-hexane. Omega 3 content of fish oil products were analyzed by using Gas Chromatography (GC) with FID detector with retention time 14.068 min and 15.506 min for α-Linolenat (ALA) and eicosapentaenoic (EPA), respectively. The results showed the highest omega 3 content (ALA 0.54% and EPA 1.103% ) was produced by addition n-hexane with a ratio 1:6 with the concentration of lipase was 1000 units for 20 hours.
... However it was noted that the lipase production was declined with increase in pH 7.0 to pH 10.0 but was able to produce lipase towards alkalotolerant nature. Factors like pH and temperature have a high influence on processes such as enzymatic reactions and permeability of cell membrane [20]. Bacterial lipases show maximum activity in alkaline pH [1]. ...
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Lipases are an important hydrolyzing enzyme with various applications and industrial potential. The maintenance of microorganisms in high oxygen concentrated liquid nutrient medium is possible by submerged fermentation. The present study focuses on the optimum production of lipase enzyme from Bacillus Substilis KUBT4 through submerged fermentation. The presence of 1% sucrose and 1% tryptone in the medium enhanced lipase production when compared with other carbon and nitrogen sources. The enzyme production by this strain expressed slightly alkaline. It has optimal activity at pH 7.5 and temperature at 36ºC where it is stable at pH 6.5-8.5 and temperature at 32ºC to 38ºC. Furthermore, 10% of inoculum size for 48 hrs of incubation period with the agitation of 150rpm increased the lipase production.
... The majority of lipases can hydrolyze fatty acids on the outer parts of the glycerol backbone (i.e., sn-1 and sn-3 positions) and are therefore sn-1,3 regiospecific. Very few lipases can hydrolyze fatty acids in the middle (sn-2) position Baharum et al., 2003). Van Pouderoyen, G., Eggert, T., Jaeger, K.-E., & Dijkstra, B. W., The crystal structure of Bacillus subtilis lipase: a minimal α/β hydrolase fold enzyme, 215-226, Copyright (2001), with permission from Elsevier. ...
... A few solvent stable lipases have been reported from solvent tolerant Pseudomonas and Bacillus sp. (Isken and de Bont, 1998;Baharum et al., 2003). Madayanti et al. (2008) cultivated and collected 10 isolates from thermogenic phases (50-70°C) during composting process which showed lipolytic activity. ...
... In contrast to our results, Gilbert et al. (1991) indicated that maximum lipase activity by Pseudomonas spp. was detected at 30°C, while the results reported by Qamsari et al. (2011) showed that maximum lipase activity produced by P. aeruginosa KM110 was at temperature range (35 to 45°C). Also, Baharum et al. (2003) found that the maximum production of lipase by Pseudomonas spp. strain S5 was at 37°C. ...
Chapter
A search for lipase in the NCBI PubMed returns 35,767 articles spanning across a century with the earliest article dating back to the year 1903. The NCBI protein database on lipases gives a total of 10,95,718 submissions of protein sequences. A quick analysis of the period between the years 2000 and 2015 shows that 9,15,412 lipase sequences have been submitted to the NCBI database, of which 8,35,372 releases were made between 2010 and 2015 alone. This span of 5 years has been very prolific in terms of numbers of sequence submissions, accounting for a whopping 75% of total sequence submissions. Starting from the beginning of the year 2015 until the time this book was written, over 15% of the total number of lipase sequence releases have already been accomplished. Lipases, a group of triacylglycerol acyl hydrolases, catalyze the hydrolysis of fats and oils with the release of free fatty acids, diglycerides, monoglycerides, and glycerol and are undoubtedly crucial enzymes from the research as well as the industrial point of view as suggested by the extent of their applications in the various industrial sectors. Lipases occur widely in the living world. Even viruses, considered to be at the borderline of the living and the nonliving, have lipase-encoding genes. For instance, vLIP is a secreted glycoprotein found in the genome of Marek disease virus (MDV). MDV causes a fatal infection of poultry. The vLIP protein bears significant homology to the a/b-hydrolase fold of vertebrate triacylglycerol lipase [1]. This protein, however, lacks the phospholipid or triglyceride hydrolysis activity typical of lipases. Instead, it has been implicated in enhancing the virulence of MDV [2]. The role of lipases as virulence factors is not restricted to viruses but is also widely seen in pathogenic bacteria [3]. These extracellular lipases wreak havoc on the membrane of host cells, such as macrophages. Over 70 lipases have also been implicated in plant pathogenicity [4]. In higher organisms, lipases catalyze the digestion of dietary lipids. In some insects and migratory birds, they are also important for the mobilization of lipid reserves, used during flight [7,8].
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Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
Chapter
Lipolytic enzymes are a diverse group of industrially important hydrolases. There are currently 15 families of lipases, classified based on their amino acid sequence and function. This chapter focuses on the updated classification of these lipolytic enzymes based on their structure-function relationships. Further, a whole new group of extremophilic enzymes has now emerged. Because such extremophilic lipases, spanning the organic-solvent-tolerant, thermophilic, psychrophilic, halophilic, and alkalophilic enzymes, are important industrially, they are covered as well. Metagenomics has led to the discovery of many enzymes; thus metagenomically discovered lipases are also briefly treated. Over the years, with advances in detection technology, methods of lipase activity estimation have also witnessed a phenomenal increase in terms of the available techniques. Hence, a dedicated section on the assay of lipolytic enzymes is also encompassed. Last, because the industrial applications have been a constant driving force in enzyme research, industrially relevant lipases and their applications are also covered to a certain extent.
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Newly isolated Pseudomonas sp. KWI-56 produced an extracellular thermostable lipase. The enzyme was purified 13.9-fold by acetone precipitation and gel filtration by HPLC with 2.9% recovery. The purified enzyme, which showed a single band on SDS-PAGE, had a molecular weight of 33, 000. The thermostability was very high, such that more than 96% of initial activity remained after incubation at 60°C for 24hr. The optimum temperature was 60°C and the maximum hydrolysis of beef tallow reached 95% at the reaction temperature of 50°C.
Article
A lipolytic Pseudomonas sp. has been successfully immobilized in strontium alginate gel bead for use in the production of lipase. This paper reports on the various conditions required for its optimum production. A 3% alginate gel concentration with a 20% (w/v) cell loading gives the highest production of lipase. The production of lipase can be enhanced by aeration and shaking but mass transfer effect may be dependent on the ratio of cell mass and bead size. Prolonged recycling with aeration accelerates the rupturing of the beads compared to non-aerated system. A maximum production of lipase is given by 1.5 g of immobilized bacteria in 50 ml of culture broth. The immobilized bacteria can withstand recycling up to 24 days, (72 h cycle), after which time the beads ruptured. The production, however, remains at 70%.
Article
Lipases from Rhizomucor Miehei, Candida antarctica, and Fusarium solani pisi were chemically modified with the aim to improve their catalytic properties in organic solvents. The chemical modifiers, two activated polyethylene glycol derivatives and activated n-octanol, were covalently linked to lysine residues at the surface of the enzyme leading to varying surface hydrophobicities. The modified lipases were tested for hydrolytic activity in water and for transesterification activity in the organic solvents o-xylene, tert-butyl methyl ether, tert-butanol, and 2-butanone. Whereas the hydrolytic activity was only slightly affected by the modifications, the transesterification activities were influenced strongly even though the modified lipases were still not soluble in organic solvents. The most effective modifier is tryesyl-activated polyethylene glycol 2000 monomethyl ether, activating lipases up to 27-fold in organic solvents while it is the least hydrophobic. The more hydrophobic modifiers, tresyl-activated polyethylene glycol 400 mono-octyl ethyl (tOPEG) and tresyl-activated octanol (tOCT), may lead to inactivation. Co-lyophilization of unmodified Candida antartica lipase B (CALB) with additives such as polyethylene glycol dimethyl ether and crown ether also positively affects the activity of CALB in organic solvents. However, we found that covalent linking of MPEG to CALB is more effective because the activation by additives is partially lost during washing of the enzyme for reuse. The thermostability of CALB in o-xylene is not affected by modification, whereas in 2-butanone the thermostability is decreased by MPEG modification and increased by OPEG or OCT modification. Our results suggest that MPEG positively influences the porosity of the lipase aggregates in organic media, whereas OPEG and OCT induce tighter aggregates.
Article
A simple and rapid colorimetric method was developed to determine the lipase activity for fat splitting. Free fatty acids produced by lipase from triacylglycerols were determined by observing the color developed using cupric acetate-pyridine as a color developing reagent. This modified method requires only a few minutes to determine the free fatty acids, whereas it takes over 20 min by the conventional methods which require solvent evaporation and centrifugation steps. The sensitivity and reproducibility of the method were good for caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids.
Article
An extracellular, alkali-tolerant, thermostable lipase was from a Pseudomonas sp. It had optimal activity at 65C and retained 75% of its activity at 65C for 90min. The pH optimum was 9.6 and it retained more than 70% activity between pH5 and 9 for 2h. The culture broth was free of protease and, at 30C, the culture filtrate retained all the activity for at least 7 days, without any stabilizer. In shake flask culture, addition of groundnut oil (3gl–1) towards the end of growth phase increased the activity from 4Uml–1 to 8 ml–1.
Article
The influence of the concentration of oxygen on lipase production by the fungus Rhizopus delemar was studied in different fermenters. The effect of oxygen limitation (≤ 47 μmol/l) on lipase production by R. delemar is large as could be demonstrated in pellet and filamentous cultures. A model is proposed to describe the extent of oxygen limitation in pellet cultures. Model estimates indicate that oxygen is the limiting substrate in shake flask cultures and that an optimal inoculum size for oxygen-dependent processes can occur. Low oxygen concentrations greatly negatively affect the metabolism of R. delemar, which could be shown by cultivation in continuous cultures in filamentous growth form (Doptimal=0.086 h-1). Continuous cultivations of R. delemar at constant, low-oxygen concentrations are a useful tool to scale down fermentation processes in cases where a transient or local oxygen limitation occurs.
Article
Enzymes from extremophiles (extremozymes) show activity and stability at extremes of temperature, low water activity, and high hydrostatic pressure. Aqueous/organic and nonaqueous media allow the modification of reaction equilibria and enzyme specificity, creating pathways for synthesizing novel compounds. Used in combination with such media, extremozymes show great potential as shown by their unique properties in aqueous media. This review introduces organic media biocatalysis before addressing the state of the art of recent fundamental and applied aspects of extremozyme biocatalysis. The aim is to encourage further exploitation of this technology, drawing on the limited work published in this field and important methods developed using mesophilic enzymes. Enzymes from three classes of extremophile will be considered: psychrophiles, halophiles, and thermophiles. Low temperature processes using psychrophilic (cold-active) enzymes may enhance yields of heat-sensitive products and reduce energy consumption. Halophilic enzymes require KCl/NaCl from 1 M to saturation, i.e. low water activity media, a feature in common with organic solvent systems. Thermophilic enzymes can be active and stable at up to 130°C and are highly resistant to proteases, detergents, and chaotropic agents. These features may afford resistance to the effects of organic solvents. Enhancing extremozyme performance via chemical modification, complexation, immobilization, and protein engineering is also discussed.
Article
Mucor hiemalis f. hiemalis is a major contaminant of cameroonian palm fruit and produces an inducible extracellular lipase in batch fermentation. Rape oil was the best inducer for enzyme production, with the highest activity being achieved after 6 days of incubation. The enzyme was purified 2200-fold by ultrafiltration, ammonium sulfate fractionation, Sephadex G75 chromatography, Q-Sepharose chromatography, and Sephacryl S-200 chromatography. The purified enzyme showed a prominent polypeptide band in polyacrylamide gel electrophoresis, associated with esterase activity according to activity staining. Molecular weight of the lipase was estimated to be 49 kDa using gel filtration on Sephadex G75, and 49 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was identified as a glycoprotein with pI of 4.6. The N-terminal amino acid sequence data (19 residues) and the amino acid composition were determined. The optimum pH and temperature for activity of the enzyme were 7.0 and 40°C, respectively. The lipase was stable in the pH range of 4–9 and at 45°C for 15 min. It hydrolyzed both synthetic and natural triglycerides with optimal activities recorded on tricaprylin and rape oil, respectively. Ca2+, Mg2+, Co2+, Mn2+, and Na+-enhanced lipase activity, whereas Fe2+, Cu2+, Ba2+, and surfactants—such as taurocholic acid, triton X-100, and Tween 20—strongly reduced lipase activity. The enzyme activity was not affected by EDTA (ethylenediaminetetraacetic acid disodium dihydrate), PMSF (phenylmethylsulfonylfluoride), (p-chloromercuribenzoic), and Benzamidine.
Article
The bioremediation of hydrocarbon in contaminated soils by mixed cultures of hydrocarbon-degrading bacteria was investigated. The mixtures or consortia of bacteria, denoted as Consortium 1 and Consortium 2 consisted of 3 and 6 bacterial strains, respectively. Bacterial strains used in this study were from the Center for Research in Enzymes and Microbiology (CREAM) collection of strains, at Universiti Putra Malaysia, and were isolated from hydrocarbon-contaminated soil samples by enrichments on either crude oil or individual hydrocarbons as the sole carbon source. The strains were selected based on the criteria that they were able to display good growth in crude oil, individual hydrocarbon compounds or both. Their ability to degrade hydrocarbon contamination in the environment was investigated using soil samples that were contaminated with diesel, crude oil or engine oil. Consortium 2, which consisted of 6 bacterial strains, was more efficient at removing the medium- and long-chain alkanes in the diesel-contaminated soil compared to Consortium 1. Further, Consortium 2 could effectively remove the medium- and long-chain alkanes in the engine oil such that the alkanes were undetectable after a 30-day incubation period. Consortium 2 consisted predominantly of Bacillus and Pseudomonas spp.