Stability Of Lipase Research Articles

Pseudomonas sp. BCNU 106이 생산하는 유기용매 내성 리파아제의 특성

유기용매 내성 세균 Pseudomonas sp. BCNU 106으로부터 생산된 리파아제 조효소액은 pH 4-10의 넓은 범위의 pH와 37℃에서 매우 안정적이었다. BCNU 106의 리파아제 안정성은 25% xylene, hexane, octane, toluene, chloroform 및 dodecane에서 증가하였으며, 상업적인 고정화 효소와 비교해도 우수한 안정성을 보이고 있다. 그리고 Cu²⁺, Hg²⁺, Zn²⁺ 및 Mn²⁺ 존재 하에서 110% 이상의 상대활성을 나타낸 반면에, Fe²⁺에서는 효소활성이 억제되었다. 게다가 계면활성제인 tween 80과 triton X-100 및 SDS에서도 높은 안정성을 유지됨이 확인되었다. 본 연구에서 유기용매 내성 Pseudomonas sp. BCNU 106의 리파아제는 고정화 효소에 못지않은 효소 활성 및 안정성을 유지함이 밝혀져 다양한 산업공정에서 잠재적인 생물촉매로 적용될 수 있는 가능성을 확인할 수 있었다. A crude extracellular lipase from solvent-tolerant bacterium Pseudomonas sp. BCNU 106 was highly stable in the broad pH range of 4-10 and at temperature of 37℃. Crude lipase of BCNU 106 exhibited enhanced stability in 25% organic solvents such as xylene (121.85%), hexane (120.35%), octane (120.41 %), toluene (118.14%), chloroform (103.66%) and dodecane (102.94%) and showed excellent stability comparable with the commercial immobilized enzyme. In addition, the stability of BCNU 106 lipase retained above 110% of its enzyme activity in the presence of Cu²⁺, Hg²⁺, Zn²⁺ and Mn²⁺, whereas Fe²⁺ strongly inhibited its stability. The detergents including tween 80, triton X-100 and SDS were positive signals for lipase stability. Because of its stability in multiple organic solvents, cations and surfactants, the Pseudomonas sp. BCNU 106 lipase could be considered as a potential biocatalyst in the industrial chemical processes without using immobilization.

Stabilization of lipase from Thermomyces lanuginosus by crosslinking in PEGylated polyurethane particles by polymerization: Application on fish oil ethanolysis

The adsorption of Thermomyces lanuginosus lipase (TLL) on PEGylated polyurethane particles as support permitted the development of several strategies to improve the properties of this commercial low-cost enzyme. The supports were synthesized by miniemulsion technique using isophoronediisocyanate (IPDI) and poly(ε-caprolactone) diol (PCL530) as monomers. The aqueous phase was composed of distilled water, surfactant sodium dodecyl sulfate (SDS), and poly(ethylene glycol) with different molar mass (PEG 400, 4000 or 6000). Polyethyleneimine (PEI) and trehalose were used to coat the PU-PEG polyurethane particles in order to increase the stability. In general, the coating with PEI (20%) allowed a greater stability of the derivatives. (100% of relative activity at 50°C during 8h). TLL immobilized on PEGylated polyurethane particles was efficiently used in the production of ethyl esters from fish oil compared to the free TLL (data not shown). The values of ethyl esters production of EPA and DHA were dependent on the support used for immobilization, which proved to be a determining factor in the activity. The highest selectivity obtained value was 45.8 for the PU-PEG4000-PEI20 derivative.

Improved catalytic performance of porcine pancreas lipase immobilized onto nanoporous gold via covalent coupling

Nanoporous gold (NPG) is a promising candidate material for lipase immobilization. However, the immobilization approach greatly influences the usability of immobilized lipase composites. In the present study, a type of lipase–NPG biocomposites is constructed by the covalent coupling method. The effects of immobilizing method as well as the NPG pore size on the lipase loading, catalytic activity, and stability of lipase–NPG biocomposites are investigated. The results show that the lipase–NPG biocomposite with a pore size of 25 nm obtained by using covalent coupling is an optimal strategy due to its excellent catalytic properties and remarkable stability. This particular lipase–NPG biocomposite not only retains over 85 % of its initial catalytic activity after 10 recycles but also exhibits good resistance to denaturation over a broader range of pH values and temperatures. Therefore, good activity and stability render this strategy promising in the biocatalyst field.

Easy stabilization of interfacially activated lipases using heterofunctional divinyl sulfone activated-octyl agarose beads. Modulation of the immobilized enzymes by altering their nanoenvironment

Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of lipases. However, the enzyme may be released from the support under drastic conditions. This paper describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions without risking enzyme desorption. The three enzymes failed in immobilization on glyoxyl-octyl supports for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose. Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and much more stable (e.g., 20 using CRL). The end of the enzyme-support reaction was achieved via blocking the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only determined the final enzyme stability, but also the activity, selectivity and even specificity of the different immobilized preparations.

Effect of deep eutectic solvent mixtures on lipase activity and stability

DESs (deep eutectic solvents) have many potential applications as cosolvents or anhydrous reaction media for biocatalytic reactions, owing to their non-volatility, non-flammability, non-toxicity, biocompatibility, biodegradability, and low cost. In this work, choline chloride ([Ch]Cl)-based DESs and DES mixtures containing two hydrogen bond donors were used as cosolvents to enhance the activity and stability of Candida rugosa lipase in aqueous reactions. The activity of lipase in an aqueous solution of [Ch]Cl:urea:glycerol was 155% higher than that in buffer. The half-life time of lipase at 40°C in an aqueous solution of [Ch]Cl:glycerol was enhanced by 9.2 times. The lipase showed the highest acid stability and base stability in the aqueous solutions of [Ch]Cl:glycerol:thiourea and [Ch]Cl:ethylene glycol:formamide, respectively. In general, glycerol-containing DES mixtures were very useful in enhancing the activity and stability of lipase, while formamide-containing DES mixtures could not efficiently enhance the activity and stability of lipase. To understand the effect of DES mixtures on the activity and stability of lipase in aqueous solution, four solvatochromic parameters of DES mixtures were determined. When the solvatochromic parameters of DES mixtures were correlated with the stability of lipase in aqueous solutions of DES mixtures, it was found that thermal stability and storage stability of lipase were associated with the hydrogen bond acidity of DES mixtures. Acid stability and base stability of lipase were correlated with polarity based on Reichardt’s dye and the dipolarity/polarizability of DES mixtures, respectively.

Development and in vitro evaluation of an oral SEDDS for desmopressin

Context: Self-emulsifying drug delivery systems (SEDDS) are among most promising tools for improving oral peptide bioavailability.Objective: In this study, in vitro protective effect of SEDDS containing desmopressin against presystemic inactivation by glutathione and α-chymotrypsin was evaluated.Materials and methods: The partitioning coefficient (log P) of desmopressin was increased via hydrophobic ion pairing using anionic surfactants. Solubility studies were performed to select the appropriate solvents for SEDDS preparation. Subsequently, droplet size and emulsification properties of 22 SEDDS formulations were evaluated. Moreover, the peptide-surfactant complex was dissolved in two chosen SEDDS formulations. Finally, SEDDS containing desmopressin were characterized regarding lipase stability, toxicity, and in vitro protective effect toward glutathione and α-chymotrypsin.Results: Desmopressin log P was increased from initial −6.13 to 0.33 using sodium docusate. The resulting desmopressin docusate complex (DES/AOT) was incorporated in two different SEDDS formulations, containing Capmul 907 P as main solvent. DES/AOT-SEDDS-F4 (containing 0.07% w/w DES/AOT) was composed of 50% Capmul 907P, 40% Cremophor RH40, and 10% Transcutol. The comparatively more hydrophilic formulation DES/AOT-SEDDS-F15 (containing 0.25% w/w DES/AOT) consisted of 20% Capmul 907P, 40% Acconon MC8-2, and 40% Tween 20. Both formulations were stable toward digestion by lipase and protected desmopressin toward α-chymotrypsin degradation. Moreover, DES/AOT-SEDDS-F4 also protected the peptide from thiol/disulfide exchange reactions with glutathione and was not cytotoxic at a concentration of 0.375% (w/w).Conclusion: DES/AOT-SEDDS-F4 protected desmopressin from in vitro glutathione and α-chymotrypsin degradation. DES/AOT-SEDDS-F4 was metabolically stable and nontoxic. Therefore, it could be considered as a potential delivery system for oral desmopressin administration.

Design of biocompatible immobilized Candida rugosa lipase with potential application in food industry.

Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity. Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively. This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.

Synthesis, characterization and kinetic analysis of chitosan coated magnetic nanobiocatalyst and its application on glucose oleate ester synthesis

In this work, we report the covalent immobilization of lipase enzyme on the surface of chitosan coated magnetic nanoparticles (MNPs) using N-(3-dimethylaminoproyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. Surface functional groups of chitosan coated MNPs, lipase enzyme, and immobilized lipase enzyme were characterized by Fourier transform infrared spectroscopic (FT-IR) analysis. The structural characteristics of the chitosan coated magnetic nanoparticles were analyzed by X-ray Diffraction (XRD) studies. The statistical methodology with central composite design (CCD) was applied to evaluate the effects of immobilization, including the magnetic nanoparticles, lipase enzyme, pH and immobilization time on the enzyme activity. Based on the statistical analysis, the optimum immobilization conditions of magnetic nanoparticles (40mg), lipase enzyme (40mg), pH (9), and immobilization time (6h), the maximum enzyme activity obtained was 16.94U/mL. The optimum pH and temperature for maximum enzyme activity of immobilized lipase are found to be pH 9 and 40°C respectively. The immobilized lipase exhibited excellent catalytic activity over eight successive cycles and retain 64% original stability. The Michaelis–Menten enzyme kinetic studies of immobilized lipase on chitosan coated MNPs showed maximum activity (Vmax) and Michaelis–Menten constant (Km) of 33.7 (μmol/mLmin) and 0.89mM respectively. The thermal stability of lipase was significantly improved after immobilization. The thermal deactivation rate of immobilized lipase was studied to follow the Arrhenius law with deactivation energy of 73kJ/mol. Glucose ester yield of 80.8% was achieved in this study revealed that immobilized lipase on MNPs have promising application in industrial scale for sugar ester production.

An unique approach of applying magnetic nanoparticles attached commercial lipase acrylic resin for biodiesel production

A new strategy to attach magnetic Fe3O4 nanoparticles (NPs) onto the commercially available immobilized lipase on acrylic resin is demonstrated. Magnetic Fe3O4 NPs-loaded lipase was successfully utilized as an effective and recyclable heterogeneous catalyst for producing biodiesel from soybean oil. In addition, the effects of different organic solvents on the yield of fatty acid methyl ester (FAME) and the stability of the Fe3O4-immobilized lipase were investigated. A maximum FAME yield of 44% can be achieved after the optimization of reaction conditions.

Cocoa pod husk, a new source of hydrolase enzymes for preparation of cross-linked enzyme aggregate.

Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase, glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries. The extraction of high active lipase from CPH has been done under optimum conditions. The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvents.

Epoxy-silanization - tool for improvement of silica nanoparticles as support for lipase immobilization with respect to esterification activity

BACKGROUND In this study, in order to obtain the best support for the immobilization of lipase from Candida rugosa (CRL), the procedure for chemical modification of fumed silica nanoparticles (FNS) with (3-Glycidyloxypropyl)trimethoxysilane (GOPTMS) was optimized by varying the amount of GOPTMS used and the duration of support modification. To evaluate the prospects of chemical modification of FNS surface on the immobilization process, the activity and thermal stability of lipase immobilized on GFNS was compared with lipase immobilized on FNS and cyanuric chloride functionalized amino silica (CCAFNS). RESULTS The epoxy functionalized silica nanoparticles (GFNS) with 400–500 µmol of introduced epoxy groups per g of support were the most efficient, since immobilized preparations in 1 mol L−1 buffer had the highest activity of approximately 1495 IU g−1 and a loading capacity of 294 mg g−1. Lipase immobilized on GFNS exhibited 1.5-fold higher hydrolytic activity and 2-fold higher esterification activity in the synthesis of aroma esters than lipase immobilized on CCAFNS, while its thermal stability was similar to CCAFNS preparations. CONCLUSION These results indicated that the type of modification of the FNS surface has an influence on the specificity of the immobilized lipase, since lipase immobilized on GFNS showed improved properties for ester synthesis which is a promising area for immobilized lipase application. © 2015 Society of Chemical Industry

Study on the Stability and Enzymatic Property Improvement of Porcine Pancreas Lipase Modified by Ionic Liquids Using Molecular Simulation

Study on the Stability and Enzymatic Property Improvement of Porcine Pancreas Lipase Modified by Ionic Liquids Using Molecular Simulation

Effects of Triacylglycerol Molecular Species on the Oxidation Behavior of Oils Containing α-Linolenic Acid.

Two kinds of oils, pure perilla oil and a blend of perilla oil with palm oil, and their enzymatically interesterified oils having the same fatty acid compositions but with different compositions of triacylglycerol (TAG) species, were studied. In particular, the effects of TAG molecular species on the oxidation resistance of oils containing α-linolenic acid (Ln) were investigated. The content of TAG binding to three Ln molecules (3Ln-TAG) was found to be different between perilla oil (38.7%) and the interesterified oils (14.5-28.9%) , which were generated using Lipozyme RM-IM(®) (regiospecificity: sn-1, 3 positional). Oils with lower 3Ln-TAG contents were more stable to oxidation as determined by the conductometric determination method (CDM; 90°C, 20 L/h) than oils with higher 3Ln-TAG contents. This result was also supported by heating oxidation tests (180°C, 7 h) using the interesterified blended oils; the residual ratio of Ln-TAGs in the oils was found to be in the order of 3Ln-TAG<2Ln-TAG<1Ln-TAG. Oxidation stability of Lipase OF(®) (regiospecificity: random)-interesterified blended oils also improved on lowering the 3Ln-TAG content. In addition, the oxidation stabilities of Lipozyme RM-IM(®)-interesterified oils were slightly higher than those of the Lipase OF(®)-interesterified oils. We found that the content of 3Ln-TAG was almost the same in both oils, and the content of unsaturated fatty acid at the sn-1, 3 positions of Lipase OF(®)-interesterified oils was higher than that of Lipozyme RM-IM(®)-interesterified oils. These results indicate that oxidation stabilities of oils containing TAG with unsaturated fatty acid such as Ln at sn-2 position were higher than those having unsaturated fatty acids at the sn-1, 3 positions. From these results, the oxidation stability of oils rich in Ln, such as perilla oil and linseed oil, can be improved not only by decreasing 3Ln-TAG but also by enzymatically reducing the unsaturated fatty acid content at the sn-1, 3 positions.

Immobilization of Lipases on Heterofunctional Octyl-Glyoxyl Agarose Supports: Improved Stability and Prevention of the Enzyme Desorption.

Lipases are among the most widely used enzymes in industry. Here, a novel method is described to rationally design the support matrix to retain the enzyme on the support matrix without leaching and also activate the enzyme for full activity retention. Lipases are interesting biocatalysts because they show the so-called interfacial activation, a mechanism of action that has been used to immobilize lipases on hydrophobic supports such as octyl-agarose. Thus, adsorption of lipases on hydrophobic surfaces is very useful for one step purification, immobilization, hyperactivation, and stabilization of most lipases. However, lipase molecules may be released from the support under certain conditions (high temperature, organic solvents), as there are no covalent links between the enzyme and the support matrix. A heterofunctional support has been proposed in this study to overcome this problem, such as the heterofunctional glyoxyl-octyl agarose beads. It couples the numerous advantages of the octyl-agarose support to covalent immobilization and creates the possibility of using the biocatalyst under any experimental conditions without risk of enzyme desorption and leaching. This modified support may be easily prepared from the commercially available octyl-agarose. Preparation of this useful support and enzyme immobilization on it via covalent linking is described here. The conditions are described to increase the possibility of achieving at least one covalent attachment between each enzyme molecule and the support matrix.

Improved immobilization and stabilization of lipase from Rhizomucor miehei on octyl-glyoxyl agarose beads by using CaCl2

A strategy to stabilize octyl (OC)-Rhizomucor miehei lipase (RML) and OC-Candida rugosa lipase (CRL) at pH 10 is necessary to take full advantage of the immobilization of these enzymes on OC-glyoxyl (OCGLX) supports. CaCl2 and MnCl2 have been reported to stabilize OC-RM and OC-CRL at pH 5.0 and 7.0. After screening different buffers, 5mM CaCl2 was found to be fully soluble in Gly at pH 10. OC-RML was 15 folds stabilized at this pH value by CaCl2, while OC-CRL was not. This salt was used in the preparation of octyl-glyoxyl (OCGLX)-RML, permitting to maintain almost unaltered the OC-RML activity that was 3 fold higher than that of the free. This preparation was 30% and 3 folds more stable in thermal or acetonitrile inactivations respectively than the standard OCGLX one.The stabilizing effect of CaCl2 and MnCl2 on the OCGLX preparations was studied. These salts stabilized both OCGLX-RML preparations, although the one prepared using Ca2+ during the covalent attachment was more stabilized than the standard one by the presence of Ca2+, even 7-8 folds in the presence of aceonitrile. Thus, this additive permits to recover an OCGLX-RML preparation more stable and active than the standard protocol.

Development of a catalytically stable and efficient lipase through an increase in hydrophobicity of the oxyanion residue

In-silico and empirical site-directed substitutions of oxyanion Q114 of the wildtype T1 lipase with that of hydrophobic Leu and Met residues were carried out to afford the Q114L and Q114M lipases, respectively. Using the esterification production of menthyl butyrate as a reaction model, evaluation on the catalytic efficiency of the three lipases was performed. It was found that Leu evidently improved the catalytic activity of the Q114L lipase, achieving the highest conversions of menthyl butyrate under varying experimental conditions that may be attributable to its lower total energy when compared with both the T1 and Q114M lipases. The diminishing catalytic activity of T1 lipase observable following substitution with Met (Q114M lipase) may be due to formation of three additional cavities within the vicinity of the mutation, lesser density of the protein core and susceptibility to high temperature, particularly under prolonged reaction time. Therefore, it can be concluded that the substitution of Leu into the oxyanion-114 had rendered favorable structural changes that enhanced the catalytic activity of the T1 lipase, envisaging that such approach may also be of applied value for improving the catalytic activity of other bacterial lipases within the I.5 family.

Stabilization of Chromobacterium viscosum Lipase (CVL) Against Ultrasound Inactivation by the Pretreatment with Polyethylene Glycol (PEG).

Although ultrasound has been used to accelerate many enzymatic reactions, the low stability of enzymes in such a system still remains a critical issue, limiting its industrial application. Here, we have reported that polyethylene glycol (PEG) pretreatment stabilized Chromobacterium viscosum lipase (CVL) in ultrasound-assisted water-isooctane emulsion. PEGs of different molecular weights and concentrations were used to pretreat CVL, and the pretreated lipase activities for olive oil hydrolysis were investigated at different ultrasonic powers. The best result was attained with PEG400 at 100mg/ml for a lipase concentration of 0.02mg/ml and an ultrasonic power of 106W. The half-life time of PEG400-treated lipase at 106W was 54min, a 27-fold higher than that attained using untreated lipase. Circular dichroism (CD) spectra suggested that PEG increased the rigidity of CVL structure, which favored the lipase stability against ultrasound inactivation. These results have important implications for the exploitation of ultrasound in biocatalytic process.

State of fungal lipases of Rhizopus microsporus, Penicillium sp. and Oospora lactis in border layers water—solid phase and factors affecting catalytic properties of Enzymes

We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus microsporus, Penicillium sp. and Oospora lactis and their ability to absorb on different sorbents depended on the nature of groups on the solid phase surface in the model systems water: lipid and water: solid phase. Thus, the stability of Penicillium sp. lipases increased 85% in the presence ofsorsilen or DEAE-cellulose, and 55% of their initial activity respectively was preserved. In the presence of silica gel and CM-cellulose, a decreased rate of lipid hydrolysis by Pseudomonas sp. enzymes was observed in water medium, and the hydrolysis rate increased by 2.4 and 1.5 times respectively in the presence of aminoaerosil and polykefamid. In an aqueous-alcohol medium, aminoaerosil and polykefamid decreased the rate of substrate hydrolysis by more than 30 times. The addition of aerosil to aqueous and aqueous-alcohol media resulted in an increase in the hydrolysis rate by 1.2-1.3 times. Sorsilen stabilized Penicillium sp. lipase activity at 40, 45, 50 and 55 degrees C. Either stabilization or inactivation of lipases was observed depending on the pH of the medium and the nature of chemical groups localized on the surface of solid phase. The synthetizing activity of lipases also changed depending on the conditions.

Organic solvent stable lipase from Cryptococcus diffluens D44 isolated from petroleum sludge

Microbial lipases, especially yeast lipases, are extensively used for biodiesel production. In order to reduce biodiesel production cost, development of stable enzymes under extreme conditions is one of the important prominences. To this end, the properties of a lipase of Cryptococcus diffluens D44, isolated from petroleum sludge, has been investigated. Crude extracellular D44 lipase showed maximal activity at pH 9.0 and 45°C. Half-life of the enzyme at 30, 40, 50 and 60°C was 79.67, 45.90, 39.16 and 2min, respectively. Activation energy of p-nitrophenyl palmitate (pNPP) hydrolysis was 30.13kJmol⿿1. D44 lipase was active within a broad range of pH from 6.0 to 10.0 with the optimum pH at 9.0. SDS-PAGE and MALDI-TOF fingerprint analyses revealed the molecular weight of the enzyme to be 45.7kDa. Its inhibition with ethylenediaminetetraacetic acid (EDTA) indicated that D44 lipase requires metal ions as cofactors. Km and Vm values were 1.92ÿ10⿿4mM (0.000073mg/ml) pNPP and 96.15U/min, respectively. Lipase activity was enhanced in the presence of Ca2+, Mg2+, K+ and Mn+, while Na+ and Ni+ has not altered enzyme activity. Methanol, tert⿿butyl alcohol, acetonitrile and ethanol resulted in an increase in percentage residual activity after 1 and 12h of incubation. Highest residual activity, 276.43%, was obtained in 10% methanol following 12h of incubation. The extensive stability of D44 lipase in aqueous methanol makes C. diffluens D44 lipase suitable for biodiesel production, since moderate reaction conditions and less organic solvent concentrations are required for oil based fuel production. This is the first report of the biochemical characterization of a lipase from C. diffluens.

Sequence‐based proline incorporation improves the thermostability of Candida albicans lipase Lip5

Increasing kinetic stability of industrial enzymes is a key objective of protein engineering. In this study, a cold‐active Candida albicans lipase Lip5 was selected as a model to improve its stability. With no 3D structure available, multiple‐sequence alignment of homologous lipases with different kinetic stabilities in the same superfamily was used to identify the mostly likely positions relevant to protein stability; these positions (V39, I52, and I290) were selected for proline incorporation in Lip5 as a strategy to increase the protein stability. Of the mutants generated, two of them (V39P and I290P) displayed a twofold increase in kinetic stability and improved thermodynamic stability as well. None of the mutants exhibited secondary or tertiary structure changes as shown by circular dichroism and fluorescence spectroscopy analyses. These results demonstrate that in the absence of 3D structure, multiple‐sequence alignment could be a useful tool to direct proline incorporation for lipase stability engineering.Practical applications: In the absence of a 3D structure, multiple‐sequence alignment‐based proline incorporation is a useful tool to engineer lipase towards improved stability.The kinetic stability of Lip5 and mutants showed that t1/2 values of mutants V39P and I290P were approximately twice as high as that of the wild type. The ΔΔGs of these two mutants increased by 2.35 and 2.03 KJ/mol, respectively, compared with wild type, suggesting higher energy barriers against thermal inactivation for V39P and I290P. Proline incorporation at position 39 or 290 increases the kinetic stability of Lip5.