Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.     

Decarboxylative aldol reaction catalysed by lipases and a protease in organic co-solvent mixtures and nearly anhydrous organic solvent media

Abstract

The effects of the choice of lipase, reaction medium, immobilization, presence of additives and temperature on conversion and stereoselectivity during a lipase catalysed decarboxylative aldol reaction were examined. It was shown that Candida antarctica lipase B (CALB) catalysed a decarboxylative aldol reaction between 4-nitrobenzaldehyde and ethyl acetoacetate in a 60% acetonitrile–40% aqueous buffer co-solvent mixture. Interestingly, free and immobilized forms of CALB showed opposite enantioselectivity in this media. The addition of 30 mol% imidazole increased the reaction rate from 8.5 to 55.7 μM min^??1 mg^??1. A 98% conversion could be achieved in 14 h (instead of 168 h) by adding imidazole. Other lipases also catalysed this reaction in different reaction media to a varying extent. With Mucor javanicus lipase in 30% DMSO, 20% enantiomeric excess (ee) of the (R)-product was observed. CALB also catalysed this reaction in nearly anhydrous acetonitrile. In the presence of cross-linked protein coated microcrystals of CALB, 90% conversion was obtained in this media in 24 h. A commercially available protease, alcalase, was also found to catalyse this reaction. While low water media gave poor conversion, the reaction in aqueous–60% acetonitrile co-solvent mixture gave 99% conversion in 72 h, provided imidazole was used as an additive.     

The mechanism of solvent effect on the positional selectivity of Candida antarctica lipase B during 1,3-diolein synthesis by esterification

We investigated the influence of solvent on the positional selectivity of Novozym 435 which was the immobilized Candida antarctica lipase B (CALB) during the esterification of oleic acid with glycerol for 1,3-diolein preparation previously. Herein, molecular modeling was used to elucidate the underlying mechanism of the solvent effect on the positional selectivity of the enzyme. The results showed that the binding energy of sn-1 hydroxyl of glycerol molecular with CALB became higher, and the binding energy of sn-2 hydroxyl of glycerol molecular with CALB became lower along with the increase of the solvent log P. It was demonstrated that, increasing log P of the solvent, the enzyme selectivity to sn-1 hydroxyl of glycerol molecular grew weaker, and the selectivity to sn-2 hydroxyl of glycerol molecular grew stronger.     

Coprecipitation of Pseudomonas fluorescens Lipase with Hydrophobic Compounds As an Approach to Its Immobilization for Catalysis in Nonaqueous Media

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Production of (<Emphasis Type="Italic">R</Emphasis>)-ethyl-3,4-epoxybutyrate by newly isolated <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> containing epoxide hydrolase

Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate. Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species. Cells were cultivated under aerobic condition at 30°C using glucose as carbon source and resting cells were used as biocatalysts for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (>99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells

(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.     

Purification and characterization of a recombinantCaulobacter crescentus epoxide hydrolase

ACaulobacter crescentus epoxide hydrolase (CCEH) from a recombinantEscherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overall activity. The optimal reaction temperature and pH were determined to be 37°C and pH 8.0, respectively. The addition of 10% (v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene oxide.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Oxidation of propene and 1-butene byMethylococcus capsulatus andMethylosinus trichosporium

Summary Methane-grown cells ofMethylococcus capsulatus andMethylosinus trichosporium readily oxidized propene and various isomers of butene to their respective epoxides. When examined in a proton NMR spectrum using tris([3-trifluoromethylhydroxymethylene]-d-camphorato), europium III derivative as an optically active chemical shift reagent, the products propylene oxide and 1,2-epoxybutane were found to contain equal amounts of both isomers. Methane-grown cells of both bacteria had considerable levels of reducing equivalents to catalyze the epoxidation of gaseous olefins. Cells depleted of reductants catalyzed the oxidation in the presence of low levels of methanol or formaldehyde with a stoichiometry of about 2:1. The rates of epoxidation of propene and 1-butene in a continuous reactor were 2–3-times that of a batch-wise reaction; the epoxidation activity, however, was lost within 3 h. The inactivation was attributed to the reactivity of the accumulated epoxides in the reactor. Propene and 1-butene oxidation by both bacteria were drastically inhibited by the respective products. Thus, the major problem in the application of microorganisms for production of epoxides from gaseous olefins is the rapid separation of the reactive products.     

Enhancement of Enantioselectivity in the Bacillus subtilis Protease-catalyzed Hydrolysis of N-free Amino Acid Esters using the Ester Grouping-modification Approach

The generality of enantioselectivity enhancement through the modification of the alcohol moiety of a substrate ester was ascertained, for in the Bacillus subtilis protease-catalyzed hydrolysis of N-unprotected amino acid esters the enantioselectivity was enhanced largely by switching the conventional methyl ester to esters with a longer alkyl chain such as the isobutyl ester (from E = 3 to E = 130–170 in the case of 4-fluorophenylalanine esters) as in the enzymatic hydrolysis mediated by Aspergillus oryzae protease. There was indeed a profound dependence of E on the nature of the ester grouping.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

Environmentally friendly, efficient resolution of racemic secondary alcohols by lipase-catalyzed enantioselective transesterification in ionic liquids in the presence of organic bases

The lipase-catalyzed enantioselective transesterification of racemic secondary alcohols was studied using vinyl acetate as acyl donor in two imidazolium-based ionic liquids vs. hexane (Scheme), both in the absence and presence of catalytic amounts of organic bases such as triethylamine (Et(3)N) or pyridine. The organic bases generally enhanced both the rate and enantioselectivity of the reaction. Further, the system 1-butyl-3-methyl-1H-imidazolium hexafluorophosphate/Candida antarctica lipase B ([bmim][PF(6)]/CALB) could be readily recycled four times without significant loss in activity or enantioselectivity.     

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris , was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g^?1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi‐Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L^?1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.