Native Lipase Research Articles

Parallel Computations in the Development of Thermostable Lipase Mutants

The advanced high performance computing methods are used to study the stability and conformational dynamics of the bacterial enzyme lipase LipA, its mutants, and the close homologous enzyme CLE whose substrate is polylactic acid-based plastics. From the analysis of the GPU molecular dynamics of native lipases and their mutants the amino acid residues whose point substitutions can markedly improve the thermostability of the enzymes under study without deteriorating their activity are determined.

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55 °C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72 h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.

Biocatalytic Route for the Synthesis of Oligoesters of Hydroxy-Fatty acids and ϵ-Caprolactone.

Developments of past years placed the bio-based polyesters as competitive substitutes for fossil-based polymers. Moreover, enzymatic polymerization using lipase catalysts has become an important green alternative to chemical polymerization for the synthesis of polyesters with biomedical applications, as several drawbacks related to the presence of traces of metal catalysts, toxicity and higher temperatures could be avoided. Copolymerization of ϵ-caprolactone (CL) with four hydroxy-fatty acids (HFA) from renewable sources, 10-hydroxystearic acid, 12-hydroxystearic acid, ricinoleic acid, and 16-hydroxyhexadecanoic acid, was carried out using commercially available immobilized lipases from Candida antarctica B, Thermomyces lanuginosus, and Pseudomonas stutzeri, as well as a native lipase. MALDI-TOF-MS and 2D-NMR analysis confirmed the formation of linear/branched and cyclic oligomers with average molecular weight around 1200 and polymerization degree up to 15. The appropriate selection of the biocatalyst and reaction temperature allowed the tailoring of the non-cyclic/cyclic copolymer ratio and increase of the total copolymer content in the reaction product above 80%. The catalytic efficiency of the best performing biocatalyst (Lipozyme TL) is evaluated during four reaction cycles, showing excellent operational stability. The thermal stability of the reaction products is assessed based on TG and DSC analysis. This new synthetic route for biobased oligomers with novel functionalities and properties could have promising biomedical applications.

The Effect of N-Terminal Domain Removal towards the Biochemical and Structural Features of a Thermotolerant Lipase from an Antarctic Pseudomonas sp. Strain AMS3.

Lipase plays an important role in industrial and biotechnological applications. Lipases have been subject to modification at the N and C terminals, allowing better understanding of lipase stability and the discovery of novel properties. A thermotolerant lipase has been isolated from Antarctic Pseudomonas sp. The purified Antarctic AMS3 lipase (native) was found to be stable across a broad range of temperatures and pH levels. The lipase has a partial Glutathione-S-transferase type C (GST-C) domain at the N-terminal not found in other lipases. To understand the influence of N-terminal GST-C domain on the biochemical and structural features of the native lipase, the deletion of the GST-C domain was carried out. The truncated protein was successfully expressed in E. coli BL21(DE3). The molecular weight of truncated AMS3 lipase was approximately ~45 kDa. The number of truncated AMS3 lipase purification folds was higher than native lipase. Various mono and divalent metal ions increased the activity of the AMS3 lipase. The truncated AMS3 lipase demonstrated a similarly broad temperature range, with the pH profile exhibiting higher activity under alkaline conditions. The purified lipase showed a substrate preference for a long carbon chain substrate. In addition, the enzyme activity in organic solvents was enhanced, especially for toluene, Dimethylsulfoxide (DMSO), chloroform and xylene. Molecular simulation revealed that the truncated lipase had increased structural compactness and rigidity as compared to native lipase. Removal of the N terminal GST-C generally improved the lipase biochemical characteristics. This enzyme may be utilized for industrial purposes.

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2 h at 37 °C and 160 rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4 °C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8 h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t1/2 = 693 h) and a negligible decay rate in the ability to transform substrate (Dr = 0.08% h−1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.

Role of Q177A and K173A/Q177A substitutions in thermostability and activity of the ELBn12 lipase.

Thermostable lipases have many applicationsin detergent industries and in organic synthesis.Thereare many ways to improve thermal stability of enzymes,for example, higher hydrophobicity, greater structural packing, higher content of the charged residues, and lower thermolabile ones. In this study, thermolabile Gln (sensitive to higher temperatures)was substituted with Alain native ELBn12 and mutated K173A lipases to examine its effect on thermal stability and activity of the lipases. Single (Q177A) and double mutants (K173A/Q177A) were expressed inEscherichia colipLysS. The Q177A variant increased both activity and thermostability of the lipase, whereas K173A/Q177A had a negative effect on the lipase activity and only had better thermal stability than the native at 50°C. pH stability of the double mutant between 9.0 and 11 was also lower than the other variants. Stability analysis in the presence of chemicals showed that Q177A mutant had better activity with 50% (v/v) organic solvents. On the other hand, K173A lipase showed increased activity with 1% (w/v) nonionic surfactant, and finally K173A/Q177A had better stability with 10 mM metal ions compared to the native lipase.

Lipase‐Catalyzed Kinetic Resolution of N‐Substituted 1‐(β‐Hydroxypropyl)indoles by Enantioselective Acetylation

A new, concise, and efficient route towards both antipodes of N‐(β‐hydroxypropyl)indoles is described. The key step is an enantioselective lipase‐mediated acetylation of racemic indolic alcohols [1‐(1H‐indol‐1‐yl)propan‐2‐ol, 1‐(2‐methyl‐1H‐indol‐1‐yl)propan‐2‐ol, and 1‐(5‐bromo‐1H‐indol‐1‐yl)propan‐2‐ol] with enol esters (vinyl acetate and isopropenyl acetate) as acyl group donors, performed under kinetically controlled conditions. A substantial influence of enzyme type, acyl group donor, and organic solvent on conversion rates and enantioselectivity of the enzymatic kinetic resolutions (EKRs) of the respective racemates was examined in detail. The best results in terms of enantioselectivity were obtained for the resolutions performed with native lipase from Pseudomonas fluorescens (PFL, Amano AK) in diisopropyl ether (DIPE) or tert‐butyl methyl ether (TBME) solution. To support the assignment of the absolute configurations of EKR products, chemical correlations with enantiomerically enriched analytical standards and single‐crystal X‐ray diffraction analysis were applied.

Kinetics of Enantiomerically Enriched Synthesis of Solketal Esters Using Native and SBA-15 supported P. Fluorescens Lipase

Abstract The studies showed that alkaline lipase from Pseudomonas fluorescens enables an irreversible transesterification of vinyl esters to give enantiomeric excess (eeR) of about 80% using vinyl butyrate as acyl donor and diisopropyl ether as a solvent, at partially optimized conditions. For the native lipase the process was adequately described by a five-parameter Ping-Pong Bi Bi model for both enantiomers plus expression accounting for the formation of enzyme-acyl donor complex, but for the same lipase supported on mesoporous materials of SBA-15-Oc type, R-product inhibition also had to be taken into account. The use of hydrophobic support increased by more than two-fold the rate of the S-solketal conversion but even more that of R-solketal. Thus the immobilization of lipase had very positive effect on the process kinetics but decreased its enantioselectivity.

Modification of fatty acid selectivity of Candida antarctica lipase A by error-prone PCR

ObjectiveTo generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).ResultsA Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.ConclusionDirected molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase.

The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

Enhanced Promiscuity of Lipase-Inorganic Nanocrystal Composites in the Epoxidation of Fatty Acids in Organic Media.

In the present study, Candida antarctica lipase B (CALB) was encapsulated in inorganic nanocrystal composites with flower-like shapes, retaining 92% of its catalytic activity compared to that of native lipase. Surprisingly, CALB-inorganic crystal nanoflowers exhibited promiscuous activity at levels 25- and 4-fold higher than those of native lipase and the commercial immobilized lipase Novozym 435, respectively, as demonstrated by the chemoenzymatic epoxidation of fatty acids conducted in organic media. To the best of our knowledge, we showed for the first time that the promiscuity of enzymes can be significantly improved by enzyme immobilization, suggesting that the enzyme-inorganic nanocrystal composites are a very promising type of immobilized enzyme that can be used to address the challenge of the extremely low efficiency of enzymatic promiscuity.

Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance.

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel.

Lipase catalysed conversion of triglycerides to amides in liquid ammonia

AbstractImmobilised lipase B (Candida antarctica lipase B) is, perhaps unexpectedly, catalytically active in liquid ammonia and exhibits chain length selectivity in the ammonolysis of triglycerides. Using glycerol triacetate as the substrate, the ammonolysis of only the first ester linkage is catalysed by the enzyme, whereas for glycerol tributyrate, the cleavage of all three ester groups is catalysed and converted to the corresponding amide. These observations represent the first example of the use of enzymes in pure liquid ammonia, exemplifying both catalysis and selectivity. Glycerol trioleate is relatively stable in liquid ammonia but, with Candida antarctica lipase B present, readily undergoes ammonolysis to give oleamide and glycerol even at 25 °C. There is no enzyme‐catalysed ammonolysis with the native lipase as it is insoluble in liquid ammonia. The solvolysis of triglycerides in liquid ammonia occurs by a stepwise conversion of the triester to diester to monoester to glycerol with one equivalent of carboxylic acid amide produced at each stage. The pseudo first‐order rate constants for each of the three steps of the uncatalysed ammonolysis of triglycerides in liquid ammonia decrease with increasing alkyl chain length in the carboxyl residue as expected from a small steric effect. Copyright © 2016 John Wiley & Sons, Ltd.

Luffa cylindrica sponges as a thermally and chemically stable support for Aspergillus niger lipase.

The use of biopolymer compounds as matrices for enzyme immobilization is currently a focus of increasing interest. In the present work we propose the use of Luffa cylindrica vegetable sponges as a support for the lipase extracted from Aspergillus niger. Effectiveness of immobilization was analyzed using Fourier transform infrared spectroscopy, elemental analysis and the Bradford method. An initial enzyme solution concentration of 1.0 mg/mL and an immobilization time of 12 h were selected as the parameters that produce a system retaining the highest hydrolytic activity (84% of free enzyme). The resulting biocatalyst system also exhibited high thermal and chemical stability, reusability and storage stability, which makes it a candidate for use in a wide range of applications. Kinetic parameters for the native and immobilized lipase were also calculated. The value of the Michaelis-Menten constant for the immobilized lipase (0.47 mM) is higher than for the free enzyme (0.21 mM), which indicates that the adsorbed enzyme exhibits a lower affinity to the substrate than native lipase. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:657-665, 2016.

A two step process for production of omega 3-polyunsaturated fatty acid concentrates from sardine oil using Cryptococcus sp. MTCC 5455 lipase

The omega-3 polyunsaturated fatty acid concentrates rich in eicosapentaeonic acid (EPA) and docosahexaenoic acid (DHA) were prepared by a two-step process involving the hydrolysis of sardine oil using lipase obtained from Cryptococcus sp. MTCC 5455 followed by urea complexation. The lipase was produced in a 3.7L fermenter with an agitator speed of 300rpm and airflow rate of 1vvm at 25°C. A maximum activity of 60±2U/mL and a productivity of 0.8U/mL/h was obtained at 72h, which was 1.6 times greater than at shake flask level. The concentrated enzyme could hydrolyse sardine oil effectively and the reaction conditions were optimized by central composite rotatable design. A hydrolysis ratio of 83.7% was obtained at 72 h with 1000 U of lipase and 1% (w/v) Triton X-100 concentration. Urea complexation of the hydrolysed sardine oil enriched the contents of EPA and DHA to 44.3% and 11.6%, which were 2.6 and 1.8 times higher than the initial content of the oil. Spectral properties of the enriched PUFA concentrates were evaluated by ATR-FTIR and 1H NMR spectroscopy. PUFA enrichment using native lipase from yeast Cryptococcus sp. was attempted for the first time in this study.

The Influence of Organic Medium and Supports in the Resolution of (R,S)-Menthol Catalyzed by Lipases

In this study, various commercial and native lipases, free or immobilized, were used in the enantioselective resolution of (R,S)-menthol with vinyl acetate for the preparation of menthyl acetate. Lipase from Candida rugosa (AYL) was found to be the most appropriate. When the reaction was carried out at 35 oC, using 40 mg of AYL and a molar ratio of 1:2 (alcohol:ester), the values for the conversion degree, the enantiomeric excess of the substrate (ees) and product (eep) and the enantiomeric ratio (E) were 33.6, 50.3, > 99% and > 200, respectively. The results showed that the reaction was more efficient using toluene in comparison with other organic solvents or mixtures of toluene and ionic liquids (ILs). The best support for the AYL immobilization, was corn starch film. This system was used 4 times over 71 days of storage, and menthyl acetate was still obtained with good values of conversion degree, ees, eep, and E.

Cellulose binding domain assisted immobilization of lipase (GSlip–CBD) onto cellulosic nanogel: characterization and application in organic medium

A cbd gene was cloned into the C-terminal region of a lip gene from Geobacillus stearothermophilus. The native lipase (43.5kDa) and CBD-Lip fusion protein (60.2kDa) were purified to homogeneity by SDS-PAGE. A highly stable cellulosic nanogel was prepared by controlled hydrolysis of microcrystalline cellulose onto which the CBD-lip fusion protein was immobilized through bio-affinity based binding. The nanogel-bound lipase showed optimum activity at 55°C, and it remains stable and active at pH 10–10.5. Furthermore, the immobilized lipase showed an over two-fold increase of relative activity in the presence of DMSO, isopropanol, isoamyl alcohol and n-butanol, but a mild activity decrease at a low concentration of methanol and ethanol. The immobilized biocatalyst retained ∼50% activity after eight repetitive hydrolytic cycles. Enzyme kinetic studies of the immobilized lipase showed a 1.24 fold increase in Vmax and 5.25 fold increase in kcat towards p-NPP hydrolysis. Additionally, the nanogel bound lipase was tested to synthesize a biodiesel ester, ethyl oleate in DMSO. Kinetic analysis showed the km 100.5±4.3mmol and Vmax 0.19±0.015mmolmin−1 at varied oleic acid concentration. Also, the values of km and Vmax at varying concentration of ethanol were observed to be 95.9±13.9mmol and 0.22±0.013mmolmin−1 respectively. The maximum yield of ethyl oleate 111.2±1.24mM was obtained under optimized reaction conditions in organic medium. These results suggest that this immobilized biocatalyst can be used as an efficient tool for the biotransformation reactions on an industrial scale.

Improving Catalytic Performance of Candida rugosa Lipase by Chemical Modification with Polyethylene Glycol Functional Ionic Liquids

Candida rugosa lipase was chemically modified with various polyethylene glycol functional ionic liquids to increase its catalytic performance. All tested polyethylene glycol functional ionic liquids improved lipase catalytic activity, thermostability, organic solvent tolerance, and adaptability to temperature and pH changes in olive oil hydrolysis reaction. In particular, lipase modified by [HOOCEPEG350Im][H2PO4] increased the catalytic activity by 1.7-fold, thermostability by 5.0-fold at 50 °C for 2.5 h, organic solvent tolerance by 13.3-fold in 60% dimethyl sulfoxide and 3.4-fold in 5% methanol compared with the native lipase. Investigations by ultraviolet, fluorescence, and circular dichroism spectroscopy revealed microenvironment changes, the increase in β-sheet and the decrease in α-helix content of secondary structures in the modified lipases. These changes were correlated with enzymatic properties alteration, including improved activity and stability of modified lipases. Compared with the results o...

Native lipase dissolved in hydrophilic green solvents: A versatile 2‐phase reaction system for high yield ester synthesis

Native lipase B from Candida antarctica was evaluated for ester production in the presence of hydrophilic green solvents including DES. Ester yields of up to 98% were achieved without removing water from the system in single step transformations. Analysis of the lipid phase exhibited a low water content of 0.29 mg/mL in the presence of DES in contrast to 2.5 mg/mL without solvent. Thus, water adsorbing properties of the hydrophilic solvents are responsible for the equilibrium shift towards ester synthesis, which was even observed in the presence of more than 20% water in total. Non‐immobilized CalB was surprisingly active in hydrophilic solvents with logP‐values below −1, when 5–10% water was added to the solvent phase and initial reaction velocities were significantly higher than with other organic solvents. Optimum reaction conditions were in the range of 40–50 °C with a minimum content of 50 mg/kg pure CalB needed for full conversion. The solvent phase was easily separated by decantation and final product purification with established refinement procedures yielded esters of high quality. Stability testing of soluble lipase B revealed that enzyme stability in glycerol and ChCl:Gly is exceptionally high and stabilization of lipase in denaturing ChCl:U was possible in the presence of a lipid interface. The hydrophilic solvents were superior to less polar organic solvents in regards to ester yield and initial reaction velocity. A drawback of some hydrophilic solvents in lipase‐catalyzed reactions is byproduct formation due to their chemical structure containing hydroxyl groups.Practical applications: The proposed biocatalytic process is suited for the preparation of green esters for cosmetic and technical applications in “low‐cost” production equipment. As no water removal is needed to obtain high yields and no complex downstream processing is applied, the process may be conducted in simple stirred tank reactors. The recycling of the non‐volatile hydrophilic solvent by example decantation can easily be done in most production units. The utilization of soluble lipases in small quantities even allows for single‐use enzyme application, though the active enzyme may be recovered with the solvent and used again to minimize catalyst costs.Lipase CalB is dissolved in the hydrophilic phase consisting of a hydrogen bonding network of solvent and water molecules. Lipase catalysis takes place at the solvent/lipid interface and water is adsorbed into the solvent phase, thus shifting the equilibrium towards ester formation in the water‐depleted lipid phase.

Immobilization and Stabilization of Lipase (CaLB) through Hierarchical Interfacial Assembly

Nanostructure-enabled hierarchical assembly holds promise for efficient biocatalyst immobilization for improved stability in bioprocessing. In this work we demonstrate the use of a hierarchical assembly immobilization strategy to enhance the physicochemical properties and stability of lipase B from Candida antarctica (CaLB). CaLB was complexed with iron oxide nanoparticles followed by interfacial assembly at the surface of an oil-in-water emulsion. Subsequent ring opening polymerization of the oil provided cross-linked microparticles that displayed an increase in catalytic efficiency when compared to the native enzyme and Novozym 435. The hierarchical immobilized enzyme assembly showed no leakage from the support in 50% acetonitrile and could be magnetically recovered across five cycles. Immobilized lipase exhibited enhanced thermal and pH stability, providing 72% activity retention after 24 h at 50 °C (pH 7.0) and 62% activity retention after 24 h at pH 3.0 (30 °C); conditions resulting in complete deactivation of the native lipase.