Template-based modeling of a psychrophilic lipase: Conformational changes,novel structural features and its application in predicting the enantioselectivity of lipase catalyzed transesterification of secondary alcohols

In order to fully explore the structure–function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79–Asp232–H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol.     

Lipase immobilized in ordered mesoporous silica: A powerful biocatalyst for ultrafast kinetic resolution of racemic secondary alcohols

Although Burkholderia cepacia lipase (BCL) has been proved to be a potential catalyst for chiral resolution, it is rarely applied in industry because of the low catalysis activity and poor stability of the free form. In this article, BCL was immobilized on the phenyl-modified ordered mesoporous silica (Ph-OMMs) to obtain a novel immobilized lipase. Benefits from the bottle-neck mesoporous structure, high loading of BCL could be completed within only 15 min. When BCL@Ph-OMMs was used as a catalyst for the resolution of 1-phenylethanol, up to 50% conversion with more than 99% ee_s was obtained within only 25 min, which is about 65-folds faster than that of the free lipase. Stabilized BCL@Ph-OMMs was successfully used for the ultrafast resolution of six secondary alcohols by selectivity transesterification, which reached high conversion (50%) and high enantioselectivity (≥99%) within 20–180 min. The activity of BCL@Ph-OMMs was kept relatively constant in 50 consecutive cycles, which is the best result among the reported immobilized lipases. The study suggests that BCL@Ph-OMMs is an attractive catalyst in industrial applications.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate

Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil^® support via Schiff base formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization performance was evaluated in terms of bound protein per gram of support (%) and recovered activity (%). A 4-factor and 3-level Box–Behnken design was applied for the acylation of (±)-2-(propylamino)-1-phenylethanol, a model substrate, with vinyl acetate and the asymmetric acylations of other (±)-2-amino-1-phenylethanols with different alkyl substituents onto nitrogen atom such as (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol were performed under the optimized conditions. The optimal conditions were bulk water content of 1.8%, reaction temperature of 51.5 °C, initial molar ratio of vinyl acetate to amino alcohol of 1.92, and immobilized lipase loading of 47 mg mL^?1. (R)-enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Enantiomeric ratio values were >200 for all the reactions. Our results demonstrate that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

A novel mono- and diacylglycerol lipase highly expressed in Pichia pastoris and its application for food emulsifier preparation

A mono- and diacylglycerol lipase (MDL) was cloned from Penicillium cyclopium and expressed in Pichia pastoris strain GS115. The recombinant enzyme was named Lipase GH1. High cell density fermentation was performed by culture in a 7.5-L fermenter using BSMG medium, in which the phosphate in basal salt medium was replaced by sodium glycerophosphate (Na₂GP). The maximal lipase activity detected was 18,000 U per mL, and total protein content in the fermentation supernatant was 3.94 g per L. The activity of the liquid enzyme remained stable under alkaline conditions at 4 °C for 6 months and was 50% after one year. Lipase GH1 was used for the synthesis of mono- and diacylglycerols (MAGs and DAGs), which are commonly used emulsifiers for industrial applications. A conversion rate of 84% after 24 h of reaction was obtained using glycerol/oleic acid molar ratio 11:1, water content 1.5 wt%, enzyme dosage 80 U per g, and reaction temperature 35 °C. Lipase GH1 was more efficient for the synthesis of MAGs and DAGs than was Lipase G50 (a similar, commercially available lipase derived from Penicillium camemberti) when oleic acid was used as an acyl donor. Lipase GH1 has potential for food emulsifier preparation.     

Efficient regioselective acylation of andrographolide catalyzed by immobilized Burkholderia cepacia lipase

For the first time, PSL-C, an immobilized lipase from Burkholderia cepacia, was successfully applied to the regioselective acylation of andrographolide by vinyl acetate in acetone. FT-IR spectra demonstrated the occurrence of acylation reaction. The ¹³C NMR, ESI-MS and elemental analysis confirmed that the 14-acetylandrographolide was formed exclusively. Water activity and reaction temperature had a significant effect on the initial rate and the substrate conversion, but little effect on the regioselectivity of the reaction. The optimal water activity and reaction temperature were 0.11 and 50 °C, respectively. Under these conditions, the initial rate and substrate conversion were 50.2 mM h⁻¹ and 99.0%, respectively, after a reaction time of around 4 h. Besides, immobilized lipase also displayed higher operational stability and 83.5% of its original activity was maintained after being reused for eight batches.     

An organic solvent and thermally stable lipase from Burkholderia ambifaria YCJ01: Purification,characteristics and application for chiral resolution of mandelic acid

A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S-isomer of mandelic acid with a theoretical conversion yield of 50%, ee_p of 99.9% and ee_s of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from Thermomyces lanuginosus

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.     

Enantioselective enzymatic hydrolysis of racemic glycidyl butyrate by lipase from Bacillus subtilis with improved catalytic properties

The lipase from Bacillus subtillus (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of glycidyl butyrate. A high enantiomeric ratio (E = 108) was obtained by using 1,4-dioxane as co-solvent (18%, v/v) and decreasing the reaction temperature to 5 °C. The ratio is about 16-fold more than that (E = 6.52) obtained in pure buffer solutions (25 °C, pH 7.8). Under the optimum conditions, the remained (R)-glycidyl butyrate with high enantiopure (ee > 98%) was obtained when the conversion was above 52%.     

Lipase immobilization on modified zirconia nanoparticles: Studies on the effects of modifiers

Zirconia nanoparticles modified with carboxylic acid with a long alkyl chain can significantly enhance the activity of immobilized lipases used for asymmetric synthesis in organic media. In this study, various carboxylic acids with different alkyl chain lengths were grafted to zirconia. Lipase from Pseudomonas cepacia (PCL) was immobilized on the modified zirconia nanoparticles and used for the resolution of (R,S)-1-phenylethanol through acylation in isooctane. The results showed that among the straight-chained carboxylic acids that formed a closely packed layer on the support, stearic acid with a long alkyl chain gave the best activity/enantioselectivity. The initial activity obtained was about 10 times higher than that from lipase loaded onto unmodified zirconia. This improvement could be attributed to the interaction between the long hydrophobic chain of the modifier and the lipase, leading to an interfacial activation effect. The stability of the immobilized PCL was investigated, and the lipase was found to be stable for at least ten consecutive runs with a substrate concentration of 5 g/L. At a high substrate concentration of ca. 100 g/L, the activity of the lipase gradually decreased, which suggests that deactivation was induced by substrate toxicity.     

Enhanced biotransformation of l-phenylalanine to 2-phenylethanol using an in situ product adsorption technique

An in situ product adsorption technique was used to enhance the biotransformation of l-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae BD. As a suitable adsorbent, the non-polar macroporous resin D101, selected from several resins tested, showed high adsorption capacity for 2-phenylethanol but not l-phenylalanine. Product inhibition was effectively alleviated by the addition of macroporous resin D101 to the biotransformation medium. When 2 g of hydrated resin D101 was added to 30 mL of the biotransformation medium, the total 2-phenylethanol concentration achieved was 6.17 g/L, of which 3.15 g/L remained in the aqueous phase and 3.02 g/L was adsorbed onto the resin. The molar yield of 2-phenylethanol reached 0.70 after 24 h cultivation. Addition of the macroporous resin greatly increased the volumetric productivity of 2-phenylethanol, and made the downstream processing more feasible and easier to perform in an industrial application.     

Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane

A biocatalyst with high activity retention of lipase was fabricated by the covalent immobilization of Candida rugosa lipase on a cellulose nanofiber membrane. This nanofiber membrane was composed of nonwoven fibers with 200 nm nominal fiber diameter. It was prepared by electrospinning of cellulose acetate (CA) and then modified with alkaline hydrolysis to convert the nanofiber surface into regenerated cellulose (RC). The nanofiber membrane was further oxidized by NaIO₄. Aldehyde groups were simultaneously generated on the nanofiber surface for coupling with lipase. Response surface methodology (RSM) was applied to model and optimize the modification conditions, namely NaIO₄ content (2–10 mg/mL), reaction time (2–10 h), reaction temperature (25–35 °C) and reaction pH (5.5–6.5). Well-correlating models were established for the residual activity of the immobilized enzyme (R² = 0.9228 and 0.8950). We found an enzymatic activity of 29.6 U/g of the biocatalyst was obtained with optimum operational conditions. The immobilized lipase exhibited significantly higher thermal stability and durability than equivalent free enzyme.     

Combination of two lipases more efficiently catalyzes methanolysis of soybean oil for biodiesel production in aqueous medium

A combination of two lipases was employed to catalyze methanolysis of soybean oil in aqueous medium for biodiesel production. The two lipase genes were cloned from fungal strains Rhizomucor miehei and Penicillium cyclopium, and each expressed successfully in Pichia pastoris. Activities of the 1,3-specific lipase from R. miehei (termed RML) and the non-specific mono- and diacylglycerol lipase from P. cyclopium (termed MDL) were 550 U and 1545 U per ml respectively, and enzymatic properties of these supernatant of fermentation broth (liquid lipase) were stable at 4 °C for >3 months. Under optimized conditions, the ratio of biodiesel conversion after 12 h at 30 °C, using RML alone, was 68.5%. When RML was assisted by addition of MDL, biodiesel conversion ratio was increased to >95% under the same reaction conditions. The results suggested that combination of lipases with different specificity, for enzymatic conversion of more complex lipid substrates, is a potentially useful strategy for biodiesel production.     

The effect of substitution of Phe181 and Phe182 with Ala on activity,substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase

Steric hindrance leads to limitation in the access of substrate into the enzyme active site. In order to decrease steric hindrance, two conserved residues, Phe¹⁸¹ and Phe¹⁸², in the lid domain of Bacillus thermocatenulatus lipase were substituted with alanine by using site-directed mutagenesis. As a result, three mutant lipases were produced. Circular dichroism (CD) spectroscopy showed that the secondary structure of all lipases is similar to one another. F181A mutation increased the distance between phe¹⁸¹ and catalytic ser¹¹⁴, which is buried in the active site by 3.24 Å. It can be suggested that such an increase in distance may lead to a decrease in steric hindrance. F181A mutation increased overall lipase activity by up to 2.6-fold (4670 U mg⁻¹) toward C8 substrate. It also resulted in optimal lipase activity at 65 °C rather than 55 °C. F182A mutation increased the distance between phe¹⁸² and catalytic ser¹¹⁴ by 1.54 Å but failed to induce any significant effect on lipase activity. However, F181A–F182A mutation significantly decreased the activity due to decreased van der Waals interactions between the phenyl group of phenylalanines and the acyl chain of triacylglycerol. These results indicate that presence of one of the two residues, Phe¹⁸¹ or Phe¹⁸², is important for stabilizing triacylglycerols in active site.     

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.     

Pancreatic α-amylase and lipase inhibitory activity of polyphenolic compounds present in the extract of black chokeberry (Aronia melanocarpa L.)

The aim of this study was to investigate the effect of black chokeberry (Aronia melanocarpa L.) extract on the activity of porcine pancreatic α-amylase and lipase. An in vitro study demonstrated that three kinds of chokeberry extracts: methanolic, water and acetic caused inhibition of α-amylase and lipase. The methanolic and acetic extracts exhibited the highest inhibitory activities against α-amylase with the IC₅₀ values of 10.31 ± 0.04 mg/ml and pancreatic lipase 83.45 ± 0.50 mg/ml, respectively. In order to identify the compounds which may be the potential inhibitors of α-amylase and lipase, chokeberry extract was analyzed by preparative reverse phase chromatography and high performance liquid chromatography–mass spectrometry (HPLC–MS). These studies have shown that both anthocyanins and phenolic acids are compounds which inhibit the ability of the reaction catalyzed by α-amylase and lipase. The most effective inhibitor of pancreatic α-amylase was chlorogenic acid (IC₅₀ = 0.57 ± 0.16 mg/ml). In the group of anthocyanins the most potent inhibitor of α-amylase was cyanidin-3-glucoside (IC₅₀ = 1.74 ± 0.04 mg/ml), which also showed an ability to inhibit the reaction catalyzed by pancreatic lipase (IC₅₀ = 1.17 ± 0.05 mg/ml). These findings seem to indicate the use of chokeberry as a functional food component, contributing to its anti-obesity activities.