Thermal Stability Of Lipase Research Articles

The influence of residue interaction on thermal stability of lipase based on dynamic graph embedding

Lipases with high thermal stability have greater application in industrial production. Focusing on wild-type lipase and their thermotolerant mutants, this study employs dynamic graph embedding to explore the change of spatiotemporal characteristics in residue-residue interactions, analyzing the relationship between residue mutations and the thermal stability of lipase. K-means clustering is used to analyze the synergistic relationships between residues, revealing that thermotolerant mutants achieve higher thermal stability by enhancing the synergistic interactions between secondary structures, resulting in a more balanced and tightly-knit internal structure. Critical residues are identified using anomaly node detection methods, showing that residues with spatiotemporal instability are mainly distributed on highly flexible loop regions. Specifically, mutations at Tyr89 and Asp132 restrict the motion range of the loops, making the lipase structure more stable. The study demonstrates that mutating highly flexible regions to increase structural rigidity can effectively enhance the thermal stability of lipases.

Development of a Tailored Sol-Gel Immobilized Biocatalyst for Sustainable Synthesis of the Food Aroma Ester n-Amyl Caproate in Continuous Solventless System.

This study reports the synthesis of a hybrid sol-gel material, based on organically modified silanes (ORMOSILs) with epoxy functional groups, and its application in the stabilization of lipase type B from Candida antarctica (CalB) through sol-gel entrapment. The key immobilization parameters in the sol-gel entrapment of lipase using epoxysilanes were optimized by the design of numerous experiments, demonstrating that glycidoxypropyl-trimethoxysilane can allow the formation of a matrix with excellent properties in view of the biocatalytic esterifications catalyzed by this lipase, at an enzyme loading of 25 g/mol of silane. The characterization of the immobilized biocatalyst and the correlation of its catalytic efficiency with the morphological and physicochemical properties of the sol-gel matrix was accomplished through scanning electron microscopy (SEM), fluorescence microscopy (FM), as well as thermogravimetric and differential thermal analysis (TGA/DTA). The operational and thermal stability of lipase were increased as a result of immobilization, with the entrapped lipase retaining 99% activity after 10 successive reaction cycles in the batch solventless synthesis of n-amyl caproate. A possible correlation of optimal productivity and yield was attempted for this immobilized lipase via the continuous flow synthesis of n-amyl caproate in a solventless system. The robustness and excellent biocatalytic efficiency of the optimized biocatalyst provide a promising solution for the synthesis of food-grade flavor esters, even at larger scales.

An effect of choline lactate based low transition temperature mixtures on the lipase catalytic properties

A new series of low transition temperature mixures (LTTM) based on choline lactate quaternary ammonium salt and various hydrogen bond donors was prepared and characterized towards their physicochemical properties and usability as an enzymatic reaction mixture for lipase-catalyzed transesterification reactions. Studies of low transition temperature mixtures have shown a long-term stabilizing effect for lipase as well as a positive influence on lipase thermal stability. In the case of Ch[Lac]:Gly: EthGly increasing the stability of lipase by 8 °C (up to 55.2 °C) compared to the control sample. Conducted transesterification reactions were characterized by high yields – up to 98% – and high purity of the obtained products.

INVESTIGATION OF THE FAT FRACTION ENZYMATIC HYDROLYSIS OF THE WASTE FROM PRODUCTION OF HYDROGENATED FAT BY THE LIPASE RHIZOPUS JAPONICUS

. In the article the conditions of enzymatic hydrolysis of fat fraction of waste from production of hydrogenated fat by the lipase Rhizopus japonicus are considered, namely, the influence of pH of the medium (pH-optimum, pH-stability) and temperature (thermal optimum, thermal stability). The scope of applications of lipases in various branches of the national economy, including for utilization of numerous fatty waste and by-products of oil and fat industry, is disclosed. The main reasons of biotechnological potential of microbial lipases are considered. Objects of research were the lipase Rhizopus japonicus and waste from the demetallization stage of the hydrogenated fat production. Detected, that the optimum pH value for Rhizopus japonicus lipase is 7.0, reducing the pH of the medium from the optimum to pH 6.0 is accompanied by a decrease in activity by 30%, and an increase from 7.0 to 9.0 – decrease by 20%. The maximum activity of the enzyme is observed in the region of physiological values of the temperature. It has been established that the lipase optimal temperature is 40°C. The results of the Rhizopus japonicus lipase stability study showed that incubation of the enzyme at pH 2.5 resulted in a complete loss of lipolytic activity after 30 minutes, and at alkaline pH, the enzyme was more stable. Incubation of lipase Rhizopus japonicus for 30 min at pH 9.0 leads to loss of lipolytic activity by 25% of the maximum, and total loss of activity occurs after 2.5 h. The study of pH-stability of Rhizopus japonicus lipase at an optimal pH of 7 showed that after 60 min of incubation, the enzyme lost 15% of the lipolitical activity, and after 60 min – 50%. Complete loss of Rhizopus japonicus lipase activity at pH 7.0 takes place after 150 minutes of incubation. The results of the study of thermal stability of lipase showed that at a temperature of 40°C and 60°C, the lipase activity remained rather stable for 50 minutes and completely lost after 150 minutes of incubation. At 80°C and 100°C, lipase activity was lost after 40 minutes and 50 minutes of incubation, respectively. The results of the study indicate the prospect of enzymatic hydrolysis of fat fraction of waste by Rhizopus japonicus lipase. The results obtained should be used to improve the processing technology of waste oil and fat industry food and processing industries.

Effects of β-cyclodextrin on the enzymatic hydrolysis of hemp seed oil by lipase Candida sp.99–125

To enhance the stability of lipases, the additive method is attractive because of its high efficiency and simplicity, of which β-cyclodextrin (β-CD), as an additive has attracted attention. In the present study, the effects of β-CD on enzymatic hydrolysis of hemp seed oil by lipases were investigated. The stability, spectroscopy, and reusability of Candida sp.99–125 were compared by the addition of β-CD, respectively. The results showed that Candida sp.99–125 could produce the highest yield of α-linolenic acid (ALA, 18.27%, w/w) in the presence of β-CD after 24h, and the thermal stability of lipase was also improved. The UV spectroscopy showed that the absorbance of lipase decreased with increasing concentrations of β-CD and the fluorescence result was similar. Moreover, the reusability of lipase with β-CD was better than free lipase. This study revealed that β-CD could increase hydrolysis activity and stability of the lipase, thereby improving the hydrolytic process of hemp seed oil by Candida sp.99–125.

Effect of metal ions on the enzymatic hydrolysis of hemp seed oil by lipase Candida sp. 99–125

In order to study the effect of metal ions on the enzymatic hydrolysis of hemp seed oil by Candida sp. 99–125, the spectroscopy, stability and hydrolytic activity of the biocatalyst were investigated in presence of Ca2+, Mg2+, Fe2+, Fe3+, Cu2+, Sn2+, Pb2+, Zn2+ and Ba2+ metal ions, respectively. The UV spectroscopy showed that all the metal ions enhanced the absorbance but the decrease of fluorescence intensity was observed. All the metal ions could improve the lipase thermal stability except Cu2+ and Ba2+. Hydrolysis of hemp seed oil proved that Ca2+, Fe3+, Pb2+ and Ba2+ could significantly improve the hydrolytic rate, and metal ions could influence lipase selectivity. The study revealed that metal ions could improve lipase stability, hydrolysis activity in the hydrolytic process of hemp seed oil by Candida sp. 99–125.

Effects of Triton X-100 and PEG on the Catalytic Properties and Thermal Stability of Lipase from Candida Rugosa Free and Immobilized on Glyoxyl-Agarose.

Background: Candida rugosa Lipase (CRL) shows a very low alkaline stability that comprises its immobilization on glyoxyl-agarose, which requires pH above 10. In this way, an adaptation from the original method was used; an enzyme solution at pH 7 was slowly added at a suspension of glyoxyl-agarose prepared in bicarbonate buffer, pH 10. This change of protocol was enough for allowing the preparation of derivatives actives of CRL on glyoxyl-agarose and verifying the effect of this modified procedure on the properties of the immobilized enzyme. The effect of the additives Triton-X-100 and polyethylene glycol (PEG) on the enzymatic activity recovery and immobilized enzyme stability was evaluated.Methods:The glyoxyl-agarose support was prepared by etherification of 6% agarose beads with glycidol and further oxidation with sodium periodate. CRL was immobilized covalently on glyoxyl-agarose support in the absence and presence of 1% (w/v) Triton-X-100 or 5 g L-1 polyethylene glycol (PEG). The lipolysis activity of the free and immobilized enzyme was determined at 37ºC and pH 7.0, using p-nitrophenyl palmitate (p-NPP) as substrate. Profiles of temperature-activity (37-65ºC, pH 7.0) and pH-activity (6.0-9.5, 37ºC) were evaluated as well as thermal (45ºC and pH 8.0) and operational (15 min batches of p-NPP hydrolysis at 50ºC and pH 8.0) stabilities of free and immobilized CRL.Results:Using a single modification of the original protocol, the CRL poorly stable under alkaline conditions could be immobilized on glyoxyl-agarose in its active conformation (recovered activity varying from 10.3 to 30.4%). Besides, the presence of a detergent (Triton-X-100) and an enzyme stabilizer (PEG) contributed to the preparation of more active and more stable biocatalysts, respectively. CRL immobilized on glyoxyl-agarose in the presence of PEG was around 5 times more stable than the free CRL and around 3 times more stable than the CRL immobilized on glyoxyl-agarose in absence of PEG. The higher stability of the CRL-glyoxyl derivative prepared in the presence of PEG allowed its reuse in four successive 15 min-batches of p-nitrophenyl palmitate hydrolysis at 50ºC and pH 8.0.Conclusion:The technique of immobilizing enzymes covalently on glyoxyl-agarose showed promising results for Candida rugosa lipase (CRL). The derivatives prepared in the presence of the additives retained two to three times more activity than those prepared in the absence of additives. The enzyme immobilized in presence of PEG was about three times more stable than the enzyme immobilized in absence of this additive. Maximum catalytic activity of the immobilized CRL (in absence of additives) was observed in a temperature 10ºC above that for the free enzyme and the pH of the maximum activity was maintained in the range 6.5-7.5 for free and immobilized CRL.

Effect of high salt concentrations on the stability of immobilized lipases: Dramatic deleterious effects of phosphate anions

We have analyzed the effects of the buffer nature on the stability of immobilized lipases. Commercial phospholipase Lecitase Ultra (LU), lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl-glyoxyl agarose beads. The enzymes were readily inactivated using 4 M sodium phosphate but 6 M NaCl did not inactivate them. Using 2 M of sodium phosphate, the inactivation of the 3 immobilized enzymes still was very significant even at 25 °C but at lower rate than with higher phosphate concentration. Thermal stress inactivations of the immobilized enzymes revealed that even 100 mM sodium phosphate produced a significant decrease in enzyme stability; this effect was less pronounced for Lecitase but dramatic for CALB. While 6 M NaCl presented slightly positive (LU) or negative (TLL) effects on their thermal stabilities of, CALB was thermally stabilized under the same conditions. Results were very different using free enymes. Fluorescence spectroscopy revealed dramatic structural rearrangements of the immobilized enzymes in the presence of high phosphate concentration. From these results, the use of sodium phosphate does not seem to be recommended for studies on thermal stability of lipases, although this should be verified for each enzyme and immobilized preparation.

A Calcium-Ion-Stabilized Lipase from Pseudomonas stutzeri ZS04 and its Application in Resolution of Chiral Aryl Alcohols.

An extracellular organic solvent-tolerant lipase-producing bacterium was isolated from oil-contaminated soil samples and was identified taxonomically as Pseudomonas stutzeri, from which the lipase was purified and exhibited maximal activity at temperature of 50°C and pH of 9.0. Meanwhile, the lipase was stable below or at 30°C and over an alkaline pH range (7.5-11.0). Ca2+ could significantly improve the lipase thermal stability which prompts a promising application in biocatalysis through convenient medium engineering. The lipase demonstrated striking features such as distinct stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v), and DMSO could activate the lipase dramatically. In the enzyme-catalyzed resolution, lipase ZS04 manifested excellent enantioselective esterification toward the (R)-1-(4-methoxyphenyl)-ethanol (MOPE), a crucial chiral intermediate in pharmaceuticals as well as in other analogs with strict substrate specificity and theoretical highest conversion yield. This strong advantage over other related schemes made lipase ZS04 a promising biocatalyst in organic synthesis and pharmaceutical applications.

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.

Effect of two series ionic liquids based on non-nutritive sweeteners on catalytic activity and stability of the industrially important lipases from Candida rugosa and Rhizopus delemar

This is the first study on the effect of ionic liquids (ILs) based on non-nutritive sweeteners on the catalytic efficiency and the structural and the thermal stability of lipases from Candida rugosa (CRL) and Rhizopus delemar (RhDL). Initially, we synthesized two series of ILs, namely 1-akyl-3-methylimidazolium saccharinates {[CnC1im][Sac]} and 1-akyl-3-methylimidazolium acesulfamates {[CnC1im][Ace]}, where the alkyl substituent was butyl, hexyl, octyl or decyl. The activities of the two enzymes were tested in two hydrolytic reactions using walnut oil and 4-nitrophenyl acetate as substrates. The activity of CRL toward walnut oil was strongly influenced by the chain-length of the N-alkyl substituents at the imidazolium cation of the compounds. The ILs with short alkyl substituents (butyl or hexyl) activated moderately (up to 30%) or had no effect on CRL activity, while those with medium chain-length substituents strongly inhibited CRL. In the case of CRL, we observed the same behavior pattern for the two tested series of ILs, while for RhDL the same tendency was displayed only for the series of saccharinates. The coating of RhDL with 0.1M [C8C1im][Sac] or [C8C1im][Ace] resulted in up to 1.5-fold increase in the enzyme initial activity. In contrast, CRL was strongly inhibited by these two ILs. We found that the effect of [C8C1im][Ace] on enzyme activity adopted concentration- and time-dependent manner for the two lipases.We observed a moderate increase in hydrolytic activity (by 1.2-fold factor) and good storage stability of RhDL (more than 30 days at 25°C) when small quantities of all ionic liquids (<0.04M), were added to the enzyme solutions. At the same time, by adding of 0.02M [C4C1im][Sac] or [C4C1im][Ace], CRL was also stabilized and its activity was preserved for more than 25 days at room temperature. For comparison, the activities of the stored at 25°C non-treated with ILs soluble CRL and RhDL were reduced by half for 7 days and 5 days, respectively. In contrast, we observed rapid inactivation at higher temperatures for all ionic liquid-coated enzymes. For example, half-lives of 25min and 57.3min at 50°C were estimated for the pre-treated with [C4C1im][Ace]-CRL and for the non-treated-CRL, respectively. In addition, applying Fourier transform infrared spectroscopy, we monitored the changes in the secondary structure of the two enzymes in presence of the tested ILs which explained the altered stability and the activities of the two enzymes. Interestingly, coating with small amounts of ILs prevents aggregation of the two lipases even at high protein concentration.

Isolation, Purification and Characterization of Secondary Structure and Kinetic Study of Lipase from Indian Major Carp, Catla catla (Catla)

Lipases are ubiquitous enzymes which catalyze the hydrolysis of fats into fatty acids and glycerol at the waterlipid interface and reversing the reaction in non-aqueous media. Lipases occupy a place of prominence among biocatalysts owing to their novel and multifold applications in oleochemistry, organic synthesis, detergent formulation and nutrition.Lipase was extracted and isolated from the alimentary canal and digestive gut of Catla catla (catla). The tissue was homogenized in a ratio of 1:3 with starting buffer (0.01 M TrisHCl, pH 7.2). The crude extract thus obtained was precipitated using ammonium sulphate (20-80%). Excess salt was removed by Dialysis and the resultant dialysate (Desalted Enzyme) was subjected to DEAE-Cellulose column for Ion Exchange Chromatography at a flow rate of 0.5 ml/min. Elution was carried out by a step gradient of NaCl (100-800 mM) in the starting buffer. Active fractions were pooled as Purified Fraction (PF) and were used for physical characterization of pH, temperature and effect of calcium on the enzyme activity, structural characterization, molecular characterization and for kinetic studies. The Purified Fraction (PF) showed final specific activity of 1438.72 U/mg. The optimum pH was 7.8 and the optimum temperature was found to be 20EšC. Melting Temperature (Tm) value was 42EšC and the activation energy of purified lipase was 34.82 KJ/mol/K. Thermal stability of lipase was found to be at 20EšC. Lipase activity retained up to 3 h of incubation with 10 mM and 20 mM of CaCl2 in starting buffer. This shows that Calcium has enhancing property from the denaturation of enzyme. Michaelis-Menten constant (Km) of lipase from Indian major carp, catla for the hydrolysis of pNPP was 6.695 mM. Turnover number (kcat) of lipase (catla) was 0.0022 s-1. Catalytic efficiency (kcat/Km) of lipase was 0.0003412 s-1 mM-1. SDS-PAGE of purified lipase (PF) revealed a homogenous single band with molecular mass of 70 kDa. Secondary structural arrangement of α helices and β strands of purified lipase results 48.51% and 9.74%, respectively using Circular Dichroism.

Polyols’ effect on thermal stability of lipase and its application in cotton fabrics’ enzymatic treatment

In this paper, the effect of polyols on thermal stability of lipase was investigated. Lipase, with or without polyols was heated at 75°C for different periods of time. Half-life (t1/2, min) of lipase at 75°C was prolonged from 8.74 to 12.62, 11.66, 9.45, and 8.95 with the addition of d-sorbierite, d-fructose, mannite, and maltose, respectively. Conformational changes of lipase after the heat treatment were studied by both Fluorescence spectra test and UV spectra test. The UV values (as the unfolding ratios of lipase) calculated from UV spectra were related to the proportion of inactivated lipase. Residual activityUV (originated from UV values) reflected residual lipase activity from the experimental data accurately. Compared with cotton fabrics treated by the crude lipase at 75°C, the appearance of polyols enhanced the capillary effect and weight loss of cotton fabrics to some extent.

Characterization of tributyrin hydrolysis by immobilized lipase on woolen cloth using conventional batch and novel spinning cloth disc reactors

Optimal loading and operating conditions for a new, superior immobilization of amano lipase from Pseudomonas fluorescens on woolen cloth were determined. The optimal enzyme loading was 46.8mggdrycloth−1 with activity of 200U. A batch reactor was used to characterize process conditions important to industrial application of the wool immobilized lipase. The optimal pH for immobilized lipase in tributyrin hydrolysis was 7, slightly lower than that of free lipase (pH 8). The optimal temperature for both free and immobilized lipase was 45°C. The immobilized lipase was more stable to reuse than some other lipase immobilizations, maintaining 85% of its activity after 6 long term runs and 75.8% of the original activity after storage of 40 weeks at 4°C. The thermal stability of lipase was improved by 2.4 times after immobilization. The thermal deactivation rate of immobilized lipase followed the Arrhenius law with Ed=199kJmol−1. The Michaelis–Menten constant (Km) of the lipase increased from 1.63mM to 4.48mM after immobilization. The immobilized lipase was also successfully applied for tributyrin hydrolysis in a novel enzyme process intensification technology – the spinning cloth disc reactor (SCDR): conversion increased by around 13% under similar conditions compared to a conventional batch stirred tank reactor. The SCDR is therefore key to exploiting the advantages of the wool immobilized lipase developed in this work.

Activation and Characterization of Trypsin-Treated Lipase

Effect of trypsin hydrolysis on lipase in activation, characteristics and change in thermal stability were studied. Lipase was found to be increased in activity from 584U mL-1 to 759U mL-1 via trypsin treatment at the concentration of 1.5mg mL-1, 30oC and pH7.0 for 30min. The trypsin-treated lipase showed a lower Km value (79mg mL-1 olive oil substrate) than the native lipase (100mg mL-1), indicating an improved affinity for olive oil substrate. The optimum pH value of the trypsin-treated lipase maintained basically unchanged while the optimum temperature (45oC) showed lower than the native lipase (50oC). The half-inactivation time for the trypsin-treated lipase at 45oC, 50oC and 60oC was calculated as 131min, 35.5min and 4min respectively, while for the native lipase at 50oC and 60oC was calculated as 128min and 13min respectively, indicating that the thermal stability of lipase is lowered after trypsin treatment.

Novel nanostructured tin dioxide as promising carrier for Candida rugosa lipase

This is the first report on lipase immobilization on nanostructured tin dioxide. The material was applied as a support for Candida rugosa lipase. All biocatalytic characteristics obtained for this novel biocatalyst (nano-SnO2-CRL) were compared with those for lipase immobilized on polypropylene (PP-CRL). Nano-SnO2-CRL has shown a specific activity eight times higher than that found for PP-CRL. The tin dioxide preparation preserved up to 45% of the initial lipase activity after 1h incubation at pH 10.0, while PP-CRL was completely inactivated. The immobilization on the inorganic carrier enhanced lipase thermal stability. Upon heating for 1h at 55°C, nano-SnO2-CRL retained 77% active, while PP-CRL was half inactivated. The synthetic activity and the effect of several parameters on isoamyl acetate production for both biocatalysts were evaluated. Among four tested acyl donors, acetic anhydride was the most efficient for nano-SnO2-CRL. The reaction proceeded with a high reaction rate and resulted in 88.2% yield of the target ester for 2h. Among various solvents, n-decane proved to be the best for both catalysts. The increase in the reaction temperature from 50°C to 60°C enhanced the reaction rate 1.5 and 1.7 fold, for nano-SnO2-CRL and PP-CRL, respectively. A considerable decline in activity for both enzyme preparations was observed after the second esterification run due to prolonged heating and probably also to inhibition by substrates and product.

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning, expression and characterization

Abstract An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP0380006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca2+ and inhibited by Fe2+, Zn2+, K+, and Ag+. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.

Activation and Characterization of Trypsin-Treated Lipase

Effect of trypsin hydrolysis on lipase in activation, characteristics and change in thermal stability were studied. Lipase was found to be increased in activity from 584U mL -1 to 759U mL -1 via trypsin treatment at the concentration of 1.5mg mL -1 , 30°C and p H 7.0 for 30min. The trypsin-treated lipase showed a lower K m value (79mg mL -1 olive oil substrate) than the native lipase (100mg mL -1 ), indicating an improved affinity for olive oil substrate. The optimum p H value of the trypsin-treated lipase maintained basically unchanged while the optimum temperature (45°C) showed lower than the native lipase (50°C). The half-inactivation time for the trypsin-treated lipase at 45°C, 50°C and 60°C was calculated as 131min, 35.5min and 4min respectively, while for the native lipase at 50°C and 60°C was calculated as 128min and 13min respectively, indicating that the thermal stability of lipase is lowered after trypsin treatment.

Nano‐Encapsulation of Lipase by Self‐Assembled Nanogels: Induction of High Enzyme Activity and Thermal Stabilization

The effects of self-assembled polysaccharide nanogels on colloidal and thermal stability of lipase from Pseudomonas cepacia were investigated. The enzyme activity, especially k(cat), drastically increased in the presence of nanogels of cholesterol-bearing pullulan (CHP). The thermostability of lipase complex increased because the denaturation temperature of lipase increased by more than 20 degrees C by complexation with CHP nanogels. Lipase denaturation and aggregation upon heating was effectively prevented by complexation with CHP nanogels. Moreover, complexation with CHP nanogels protected lipase from lyophilization-induced aggregation. Nano-encapsulation with CHP nanogel is a useful method for colloidal and thermal stabilization of unstable enzyme.

Characterization of Burkholderia lipase immobilized on celite carriers

In this work, a lipase originating from an indigenous Burkholderia sp. C20 strain was immobilized onto celite carriers via covalent bonding to enhance its feasibility in practical applications. The enzyme immobilization efficiency was optimal when the lipase concentration, reaction pH, and reaction time were set at 70 g/L, pH 7.0, and 3 h, respectively. The maximum immobilized lipase activity was about 273.5 U/g celite while the lipase loading was around 62.9 mg per gram of celite carriers used. The optimal temperature and pH of the immobilized lipase (denoted as celite-lipase) in terms of lipolytic activity was 55 °C and 10.0, respectively, similar to that for the free lipase. The celite-lipase remained 83% of its original activity after keeping at 55 °C for 120 min, while the free lipase lost nearly 40% of its activity under the same conditions, indicating the improvement of lipase thermal stability after enzyme immobilization. The kinetics of the immobilized lipase appeared to follow Michaelis–Menten model with the kinetic parameter ( v max and K m ) of 11.29 U/mg protein and 12.06 mM, respectively.