Characterization of Lipases and Esterases from Metagenomes for Lipid Modification

Three hundred and fifty novel lipases and esterases discovered from environmental DNA samples were characterized for their fatty acid profile using GC-analysis. Enzymes were selected for further study based on activity and fatty acid chain length specificity. Additional characterization was based on enzyme activity towards tributyrin and 4-methylumbelliferyl butyrate, and enzyme heat stability. Several lipases were identified, which show high specificity towards short-chain fatty acids similar to pregastric lipases from kid and calf and a lipase from Mucor javanicus. Additionally, the metagenome-derived enzymes were thermostable. Selected metagenomic lipases were immobilized on Celite and used for the synthesis of structured triglycerides.     

Preparation of a promising whole cell biocatalyst of Geotrichum sp. lipase and its properties

BACKGROUND: As a new protein expression and self‐immobilization system, cell‐surface displayed enzymes have attracted increasing attention. In this study, Geotrichum sp. lipase (GSL), an important enzyme for the enrichment of polyunsaturated fatty acids (PUFAs), was first displayed on the cell surface of Saccharomyces cerevisiae. RESULTS: The activity of displayed GSL was higher (43.7 U g^?1 dry cell) than that of Candida antarctica lipase B (26.26 U g^?1 dry cell) and that of Rhizopus oryzae lipase (4.1 U g^?1 dry cell). It also exhibited higher thermostability than the free lipase, and retained 89% of the original activity after incubation at 40 °C for 3 h, compared with 48% at 35 °C for the free lipase at pH 8.5. Interestingly, the displayed lipase had a wider pH range and better pH stability. It had higher activity at all pH values than the free GSL, and retained 86% of the original activity in the pH range 9.5 to 10.5, whereas the activity of the free GSL could not be detected at pH 10. CONCLUSION: This work presented a method to prepare a whole‐cell biocatalyst with better stability and broader pH tolerance which will provide a useful strategy for other cost‐effective self‐immobilized industrial lipases. Copyright © 2011 Society of Chemical Industry     

Characterization of membrane-bound lipase from a thermophilic Rhizopus oryzae isolated from palm oil mill effluent

The characteristics of the membrane-bound lipase from a thermophilic Rhizopus oryzae were studied. The pH and temperature optima for lipase activity were at 7.0 and 37°C, respectively. The enzyme was stable and acidic conditions, retaining more than 80% of its initial activity at pH 4.0 after 30 min incubation. It was stable up to 50°C with 70% of initial activity retained after 3 h incubation. The enzyme is 1,3 specific and exhibits substrate preference. Monoacid triglyceride substrates were hydrolyzed better than methyl esters, polyoxysorbitan and sorbitan substrates.     

Burkholderia multivorans SB6 Lipase as a Detergent Ingredient: Characterization and Stabilization

In this study, 265 bacterial isolates were collected from kitchen wastewater samples using Rhodamine B agar medium. Of these, 115 isolates were found to respond positively to the addition of commercial detergents. Using 16S rRNA sequence analysis, the isolate demonstrating the high stability towards commercial detergents was identified as Burkholderia multivorans. An SB6 lipase with a molecular mass of 70 kDa was purified from B. multivorans. The purified enzyme showed optimal activity at pH 9.0 and 40 °C and remained stable in the presence of various metal ions, surfactants, and oxidizing agents. The addition of boron compounds improved the pH stability and thermostability of the enzyme, which displayed stability against some commercial detergents; moreover, this stability increased when boron compounds were added to the incubation medium as stabilizers. These properties make SB6 lipase an ideal choice as an additive in detergent formulations.     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

The Use of Boron Compounds for Stabilization of Lipase from Pseudomonas aeruginosa ES3 for the Detergent Industry

This study aimed to characterize a lipase that is highly active and stable under typical washing conditions for use as a detergent ingredient by investigating the effects of various boron compounds on lipase stabilization under different conditions. In addition, the antimicrobial activity of the boron compounds used in enzyme stabilization was examined in order to obtain an effective antimicrobial detergent. A lipase‐producing bacterium was isolated from kitchen wastewater samples using Rhodamine‐B Agar medium and identified as Pseudomonas aeruginosa based on 16S rDNA sequence analysis. The ES3 lipase obtained from P. aeruginosa was purified, and the purified enzyme was found to have a molecular mass of 40 kDa. The enzyme showed optimal activity at pH 9.0–10.0 and 40 °C and remained stable in the presence of various metal ions, surfactants and oxidizing agents. Moreover, the pH stability and thermostability of the enzyme was improved by the addition of boron compounds, which, when used as stabilizers in the incubation media, also increased the stability of the enzyme towards commercial detergents. Furthermore, the enzyme displayed properties comparable with the commercial product Lipolase^®, which has shown excellent stability towards various commercial detergents. Finally, boron compounds used to stabilize the lipase were found to possess antimicrobial properties, suggesting that detergents incorporating these compounds will also exhibit antimicrobial activity when washing clothes and dishes.     

丁酸梭杆菌VPI 3266甘油脱水酶和1,3-丙二醇氧化还原酶的克隆、表达

对丁酸梭杆菌VPI 3266的1,3-丙二醇代谢途径关键酶甘油脱水酶(DhaB)与1,3-丙二醇氧化还原酶(DhaT)进行了研究。甘油脱水酶基因dhaB全长3315bp,编码两个蛋白亚基,分别为甘油脱水酶的核心酶和脱水酶的再激活酶。前者全长2367bp,编码788个氨基酸,后者全长918bp,编码305个氨基酸。通过构建重组质粒,使其在大肠杆菌中实现了活性表达。1,3-丙二醇氧化还原酶基因dhaT全长1166bp,编码388个氨基酸,属于NADP依赖的离子激活的醇脱氢酶家族III。通过IPTG诱导,在大肠杆菌中实现了高效表达,酶活达到5.2U/mL。并通过Ni亲和柱获得了电泳纯的蛋白。蛋白相对分子质量为4.19×10⁴。该酶的最适反应温度为50℃,最适反应pH值为10.0,在pH值8.5~10.0范围内比较稳定,在45℃保温2h,酶活还残存50%。该酶以1,3-丙二醇为底物,在生理条件下的V_max和K_m分别为29.2U/mg和19.8mmol/L。     

Electron Transfer and Stability of a Quinohaemoprotein Alcohol Dehydrogenase Electrode

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni (QH-EDH) was immobilised in a redox polymer network of a polyvinylpyridine, partially N-complexed with osmiumbis(bipyridine)chloride. Substrate-dependent electron transfer occurred, indicating that the enzyme was active and that effective electron transport was achieved. It was shown that the enzyme molecular weight is of importance with respect to the enzyme electrode stability. Long term stability and current density of the QH-EDH electrodes were highest when the enzyme was immobilised at pH 10·0 and 4°C, followed by an additional cross-linking step with glutaraldehyde (1%) at pH 7·0. With such an electrode current densities of 40 μA cm⁻² were obtained for several primary alcohols. The affinity of the immobilised enzyme for these substrates (K_m(app) values) was similar to that of the enzyme in solution. The half-life time of the electrodes was between 50 h and 200 h depending on the time the enzyme was in contact with the substrate. When the immobilised enzyme electrode was operated at temperatures above 37°C the stability decreased. © 1997 SCI.     

Tailoring a stabilized variant of hydroxynitrile lyase from Arabidopsis thaliana

The R-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface-modified variant incorporating 11 changes in the amino acids on the protein surface. Comparative characterization of the variant and the wild-type enzyme showed a broadened pH optimum towards the acidic range, along with enhancement of activity by up to twofold and significantly increased pH- and thermostabilities. The effect can most probably be explained by a shift of the isoelectic point from pH 5.1 to 4.8. Application of the variant for the synthesis of (R)-cyanohydrins in an aqueous/organic two-phase system at pH 4.5 demonstrated the high stereoselectivity and robustness of the variant relative to the wild-type enzyme, which is immediately inactivated under these conditions.     

The stability of lactic acid production in resting suspensions of Lactobacillus delbrueckii

The stability of resting glycolytic activity (RGA) i.e. lactic acid production from glucose, in non-growing suspensions of Lactobacillus delbrueckii was examined. Non-growing conditions were imposed by removal of essential nutrients or by the addition of antibiotics. The stability of RGA was reduced in fast-grown chemostat cells. Mg²⁺, reducing agents, the rigorous exclusion of oxygen and a constant supply of energy source gave a five-fold increase in RGA and a 25-fold increase in stability in cells deprived of a nitrogen source. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity severely limited RGA after about 8 h of incubation. No evidence of excessive ATP accumulation or ADP depletion was found. Addition of chloramphenicol, actinomycin D or mitomycin C decreased RGA stability over a 24-h period. Addition of yeast extract and tryptone to a mixture containing phosphate, Mg²⁺, thioglycollate and chloramphenicol gave a three-fold increase in RGA which lasted for about 1 h. Mg²⁺ is believed to stimulate the glycolytic sequence directly and oxygen probably inhibits GAPDH by interaction with the -SH groups of the enzyme. Tryptone and yeast extract probably stimulate pyruvate kinase activity, and this enzyme is also absolutely dependent on fructose-1, 6-diphosphate for activity.     

Immobilization of a thermostable inorganic pyrophosphatase from the archaeon Pyrococcus furiosus onto amino‐functionalized silica beads

This study focuses on the preparation and application of a recombinant thermophilic inorganic pyrophosphatase from the archaeon Pyrococcus furiosus on amino‐functionalized silica beads. The amino‐functionalized silica beads were prepared by coating with 3‐aminopropyltriethoxysilane by silanization. The thermostable inorganic pyrophosphatase was rapidly and successfully immobilized onto the amino‐functionalized silica beads with glutaraldehyde as a coupling agent (within 12 min, >95.4% protein was immobilized onto the support). The results show that the protein could be immobilized efficiently, with up to 1 mg of protein/g of support with 92.9% activity. Compared with the free enzyme, the immobilized enzyme displayed a high activity toward inorganic pyrophosphate, less sensitivity toward the pH, and increased thermal stability. The immobilized enzyme retained 56.9% of its initial activity after hydrolysis of the inorganic pyrophosphate after 12 consecutive cycles (total = 330 min) at high temperature; this indicated a high protein stability suitable for practical applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40700.     

Optimization of nitrilase production from a new thermophilic isolate

A new thermostable nitrilase‐producing isolate identified as Streptomyces sp. MTCC 7546 has been studied extensively for the optimization of enzyme production operating in batch mode. The benzonitrile was observed as inducer of nitrilase production. The isolate showed maximum nitrilase production after 24 h of incubation at optimal conditions. The strain grows well on a variety of carbon sources and produces the nitrilase that catalyses the hydrolysis of nitriles to acids without formation of amides. The enzyme is mostly active against mono‐ and di‐aliphatic nitriles (10 mmol L^?1) at pH of 7.4 and at a temperature of 50 °C. Copyright © 2007 Society of Chemical Industry     

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-l-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba²⁺. This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。     

Improvement of an Acid Phosphatase/DHAP‐Dependent Aldolase Cascade Reaction by Using Directed Evolution

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild‐type enzyme at pH 6.0. This variant is 17 times more active than the wild‐type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.     

磁性壳聚糖微球的制备及其用于甲酸脱氢酶的固定化

分别采用乳化交联法和共沉淀法制备磁性壳聚糖微球载体,并对形貌结构进行比较,结果表明,采用共沉淀法制备的磁性壳聚糖微球负载Fe3O4的效果好,故将其作为载体固定甲酸脱氢酶。最佳固定化条件:添加酶量9 U.g-1,pH=7.0,固定化时间5 h。游离酶和固定化酶的最适宜反应温度分别为50℃和30℃;游离酶的最适宜pH=7.0,固定化酶的最适宜pH=6.0;将游离酶和固定化酶分别置于60℃恒温水浴放置180 min后,游离酶和固定化酶的相对酶活力分别为0.78%和40.39%;将游离酶和固定化酶置于不同pH的缓冲液中保存1 h后,在强酸(pH=2.0)和强碱(pH=10.0)条件下,固定化酶的相对酶活力分别为11.03%和38.43%,游离酶已全部失活;固定化酶重复使用6次后,相对酶活力为73.53%,表明固定化酶具有较好的热稳定性、酸碱稳定性和操作稳定性。     

Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability

Baeyer–Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo‐, regio‐, and enantioselectivity in many cases. Large‐scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface‐exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H₂O₂ (0.2 M ), whereas the wild‐type enzyme's activity was completely abolished at concentrations as low as 5 mM H₂O₂. We propose that oxidation‐stable mutants might well be a “prerequisite” for thermostabilization, because laboratory‐evolved thermostability in CHMO might be masked by a high degree of oxidation instability.     

Chemoselective hydrolysis of methyl 2‐acetoxybenzoate using free and entrapped esterase in K‐carrageenan beads

Porcine liver esterase was entrapped in natural polysaccharides K‐carrageenan and retention of its activity was determined using p‐nitrophenyl acetate as the substrate. The optimum pH for esterase activity of entrapped enzyme showed a little shift towards acidic side. Immobilized enzyme showed improved thermal and storage stability. The entrapped esterase retained 50% of its activity after eight repetitive cycles. Michaelis constant K_m for the free and entrapped enzymes was almost same indicting no conformational change during immobilization. Maximum velocity V_max was observed to decrease on immobilization. The free and entrapped esterase was used for selective hydrolysis of methyl 2‐acetoxybenzoate to methyl 2‐hydroxybenzoate in batch process as well as in a fixed bed reactor. The hydrolysis was observed to be 99% within 2 h for free as well as immobilized enzyme in batch process. The rate of hydrolysis was found to depend on pH. The turn over number of selective hydrolysis in batch and fixed bed reactor was 3.08 × 10⁶ and 1.19 × 10⁷, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Isolation and characterization of a nitrile hydratase from a Rhodococcus sp.

A new nitrile hydratase producing strain of Rhodococcus has been found. A method for purification of the nitrile hydratase and a characterization of the enzyme is described. The hydratase is a 52-kdal protein consisting of two subunits of molecular weights, 26 and 23 kdal, respectively. The hydratase exhibits a broad substrate specificity. Aliphatic saturated or unsaturated nitriles, as well as aromatic nitriles, are substrates for the enzyme. Inhibition of the enzyme activity by amides and carboxylic acids was observed. The optimum pH of the hydratase is 7.5. The enzyme is rather unstable, even at room temperature. The enzyme may be applied for the production of acrylamide. For this application of the enzyme, the optimal temperature is about 4°C, where the enzyme exhibits a satisfactory activity and stability.     

A simple method for biocatalyst immobilization using PVA‐based hydrogel particles

BACKGROUND: The aim of this study was to evaluate the feasibility of enzyme immobilization in PVA particles through extrusion of LentiKat^®Liquid in polyethylene glycol. Inulinase, with invertase activity for sucrose hydrolysis, was used as model system. RESULTS: Inulinase was effectively immobilized in PVA particles. The pH optimum of the enzyme activity was broadened for lower pH values. Mechanical instability of the PVA under prolonged incubation above 55 °C was observed. A 1.8‐fold increase in the apparent K_M (Michaelis constant) suggests diffusion limitations as a result of immobilization. The immobilized biocatalyst exhibited considerable operational stability, since a decrease of roughly 10% in the product yield after 24 h biotransformation runs was observed in trials performed at 50 °C, following 20 repeated, consecutive batches. CONCLUSION: The results obtained highlight the potential of PVA‐based particles obtained through extrusion into PEG for the production of suitable biocatalysts for application in large‐scale processes. Copyright © 2008 Society of Chemical Industry