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%.     

Enantioselective hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by (S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester.     

Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida

Preparation of (2S, 3R)-methyl 3-phenylglycidate via enantioselective hydrolysis of racemic phenylglycidate was carried out using whole cells of Pseudomonas putida. Under optimal conditions (2S, 3R)-methyl-3-phenylglycidate could be got with ee value 99 and 48% chemical yield.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

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%.     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

Lipase mediated resolution of γ-azidoalcohols in aqueous and organic media: Synthesis of (R)- and (S)-fluoxetine and duloxetine

A simple and efficient method for the synthesis of optically active γ-azidoalcohols is described. The lipase catalyzed kinetic resolutions of acetates of γ-azidoalcohols in aqueous as well as organic media have been studied. The enantiomerically pure γ-azidoalcohols obtained by the kinetic resolution in high enantiopurity have been utilized towards the synthesis of enantiomeric pairs of anti-depressant drugs, fluoxetine and duloxetine.     

Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-based aqueous two-phase systems

Alcohol/salt-based aqueous two-phase systems (ATPSs) were used to recover lipase derived from Burkholderia pseudomallei (B. pseudomallei). Nine biphasic systems, comprised of an alcohol-based top phase (ethanol, 2-propanol and 1-propanol) and a salt-based bottom phase (ammonium sulfate, potassium phosphate and sodium citrate), were evaluated for their effectiveness in lipase recovery. The stability of lipase in each of the solutions was tested, and phase diagrams were constructed for each system. The optimum partition efficiency for the purification of lipase was obtained in an ATPS of 16% (w/w) 2-propanol and 16% (w/w) phosphate in the presence of 4.5% (w/v) NaCl. The purified lipase had a purification factor of 13.5 and a yield of 99%.     

Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.     

Kinetic resolution of ketoprofen ester catalyzed by lipase from a mutant of CBS 5791

A biotransformation process was developed for the production of (S)-ketoprofen by enantioseletive hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1–X(1+ee)]/ln[1–X(1–ee)]; ee is enantiomeric excess, ee=(C_S–C_R)/(C_S+C_R): temperature of hydrolysis was 23°C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.     

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Serratia marcescens ECU1010

An extracellular lipase was purified to homogeneity with a purification factor of 5.5-fold from a bacterial strain Serratia marcescens ECU1010. The purified lipase is a dimer with two homologous subunits, of which the molecular mass is 65 kDa, and the pI is 4.2. The pH and temperature optima were shown to be pH 8.0 and 45 °C, respectively. Among p-nitrophenyl esters of fatty acids with varied chain length, the lipase showed the maximum activity on p-nitrophenyl myristate (C₁₄). The lipase was activated by some surfactants such as Gum Arabic, polyvinyl alcohol (PVA) and Pg350me, but not by Ca²⁺. The enzyme displayed pretty high stability in many water miscible and immiscible solvents. This is a unique property of the enzyme which makes it extremely suitable for chemo-enzymatic applications in non-aqueous phase organic synthesis including enantiomeric resolution. Several typical chiral compounds were tested for kinetic resolution with this lipase, consequently giving excellent enantioselectivities (E = 83 >100) for glycidyl butyrate (GB), 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one acetate (HMPCA), naproxen methyl ester (NME) and trans-3-(4′-methoxyphenyl) glycidic acid methyl ester (MPGM).     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

Flavobacterium johnsoniae昆布多糖酶的分离纯化及其催化性质

约氏黄杆菌Flavobacterium johnsoniae具有分泌裂解酵母细胞壁酶系的能力,经初步分析发现其发酵液中具有葡聚糖酶、几丁质酶和蛋白酶等活性。通过离子交换层析、疏水层析和凝胶过滤层析,从该菌发酵液中分离纯化到一种昆布多糖酶。该酶分子量为35 kD左右,其最适反应温度为50°C,最适反应pH为5.0。以昆布多糖和昆布寡糖为底物的反应表明,该酶以内切酶作用模式进行催化水解。     

Purification and properties of mangano-superoxide dismutase from a strain of alkaliphilic Bacillus

A mangano-superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a strain of alkaliphilic Bacillus for the first time. The purified protein, with an isoelectric point of pH 4.5, had a molecular mass of approximately 50 kDa and consisted of two identical subunits (25 kDa). The N-terminal amino acid sequence was Ala-Tyr-Lys-Leu-Pro-Glu-Leu-Pro-Tyr-Ala-Ala-Asn-Ala-Leu-Glu-Pro-His-Ile-Asp-Glu-Ala. The optimum pH and temperature for the reaction were 7.5 and 35°C, respectively. The properties of the superoxide dismutase were compared with those of the enzyme from thermophilic Bacillus stearothermophilus. Received: September 3, 1996 / Accepted: October 4, 1996     

Isolation of a Bacillus strain producing ketone reductase with high substrate tolerance

Target reaction-oriented screening from soil samples yielded a ketone reductase-producing Bacillus sp., strain ECU0013, which exhibits excellent stereoselectivity, high substrate tolerance and is capable of regenerating the required cofactor with glucose as a co-substrate. Whole-cells catalyzed the asymmetric reduction of 2-chloro-1-phenylethanone (50 mM) to (R)-2-chloro-1-phenylethanol with a 93.3% conversion rate and 99% e.e. (enantiomeric excess). A variety of ketones were enantioselectively reduced by resting cells, giving corresponding chiral alcohols with good to excellent e.e. values. These results suggest the potential of this strain for the industrial production of chiral halogenated aromatic alcohols.     

Biotransformation of the fungistatic compound (R)-(+)-1-(4′-chlorophenyl)propan-1-ol by Botrytis cinerea

The biotransformation of the fungistatic agent (R)-(+)-1-(4′-chlorophenyl)propan-1-ol (1) by the phytopathogen Botrytis cinerea has been studied. The main reaction pathways involved hydroxylations on several positions as well as condensations with secondary metabolites of the fungus. The antifungal activity of compound 1 against B. cinerea has also been determined.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Catalytic performance of a highly enantioselective (R)-ester hydrolase from a new isolate Acinetobacter sp. CGMCC 0789

A highly enantioselective (R)-ester hydrolase was partially purified from a newly isolated bacterium, Acinetobacter sp. CGMCC 0789, whose resting cells exhibited a highly enantioselective activity toward the acetate of (4R)-hydroxy-3-methyl-2-(2-propynyl)- cyclopent-2-enone (R-HMPC). The optimum pH and temperature of the partially purified enzyme were 8.0 and 60 °C, respectively. The enantioselectivity of the crude enzyme was increased by 1.2-fold from 16 to 20 when the reaction temperature was raised from 30 to 60 °C. The activity of the crude enzyme was enhanced by 4.1-fold and the enantioselectivity (E-value) was markedly enhanced by 4.3-fold from 16 to 68 upon addition of a cationic detergent, benzethonium chloride [(diisobutyl phenoxyethoxyethyl) dimethyl benzylammoniom chloride]. The hydrolysis of 52 mM (R,S)-HMPC acetate to (R)-HMPC was completed within 8 h, with optical purity of 91.4% ee_p and conversion of 49%.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.