The influence of cosolvent concentration on enzymatic kinetic resolution of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives

A method to improve the enantioselectivity of lipase-catalyzed kinetic resolution (KR) of trans-2-phenyl-cyclopropane-1-carboxylic acid derivatives in water–acetone solution is presented. Two different approaches were compared: enzyme-catalyzed esterification and enzymatic hydrolysis of the target ester. A substantial influence of enzyme type, ethoxy group donor, and solvent on conversion and enantioselectivity of the enzymatic esterification was noted. While enzymatic esterification proceeds with poor enantioselectivity, the hydrolysis of target ester proceeds efficiently. Studies on the influence of cosolvent used for the enzymatic hydrolysis reaction showed that kinetic resolution can be performed in acetone and water buffer mixture predominantly containing organic solvent. Any change in organic solvent content resulted in a substantial decrease in enantioselectivity from almost E = 150 to less than 5.     

Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: significantly enhanced enantioselectivity and stability

The lipase selective hydrolysis of Naproxen methyl ester was explored in both water-saturated isooctane and water-saturated ionic liquid 1-butyl-3-methylimidazolium hexafluoro-phoshate ([bmim]PF₆) to see any significant differences in terms of enantioselectivity and stability between two different classes of reaction media. It is shown that polar and hydrophobic of [bmim]PF₆ made it an unearthly reaction medium for hydrolysis of Naproxen methyl ester. It not only decreases the equilibrium constant (K) and enhances the enantiomeric ratio (E), consequently improves the equilibrium conversion (C_Eq) of the hydrolysis reaction and enantiomeric excess of product (ee_p), but also maintains the lipase activity. Because the lipase would not dissolve in the 1-butyl-3-methylimidazolium hexafluoro-phoshate, it can be filtrated up from 1-butyl-3-methylimidazolium hexafluoro-phoshate and recycled for several runs. The stability of lipase was improved due to the higher solubility of methanol in 1-butyl-3-methylimidazolium hexafluoro-phoshate than 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.     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

Bio‐preparation of (R)‐DMPM using whole cells of Pseudochrobactrum asaccharolyticum WZZ003 and its application on kilogram‐scale synthesis of fungicide (R)‐metalaxyl

Methyl (R)‐N‐(2,6‐dimethylphenyl)alaninate ((R)‐DMPM) is a key chiral intermediate for the production of (R)‐metalaxyl, which is one of the best‐selling fungicides. A new strain, Pseudochrobactrum asaccharolyticum WZZ003, was identified as a biocatalyst for the enantioselective hydrolysis of (R,S)‐DMPM. The key parameters including pH, temperature, rotation speed and substrate concentrations were optimized in the enantioselective hydrolysis of (R,S)‐DMPM. After the 48 h hydrolysis of 256 mM (R,S)‐DMPM under the optimized reaction conditions, the enantiomeric excess of product (e.e._p) was up to 99% and the conversion was nearly 50%. Subsequently, the unhydrolyzed (S)‐DMPM was converted to (R,S)‐DMPM through the n‐butanal‐catalyzed racemization. Furthermore, stereoselective hydrolysis of (R,S)‐DMPM catalyzed by whole cells of P. asaccharolyticum WZZ003 was scaled up to kilogram‐scale, offering (R)‐MAP‐acid with 98.6% e.e._p and 48.0% yield. Moreover, (R)‐metalaxyl was prepared at kilogram scale after subsequent esterification and coupling reactions. Therefore, a practical production process of (R)‐DMPM and (R)‐metalaxyl with the prospect of industrialization was developed in this study. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:921–928, 2018     

Lipase-catalysed two-step desymmetrization of 2-methylmalonic dipyrazolide for preparation of optically pure enantiomer in organic solvents

A lipase-catalysed two-step butanolytic desymmetrization process for the preparation of pyrazolidyl butyl (S)-2-methylmalonate from 2-methylmalonic dipyrazolide was developed. The best reaction condition of using lipase PS-D in anhydrous n-hexane at 55?°C was first selected, leading to high yield and enantiomeric excess for the remained (S)-enantiomer. The kinetic analysis by considering the competitive inhibition from butanol was then carried out for obtaining the stereoselectivity of E₁?=?11.2 for the first desymmetrization and enantiomeric ratio of E₃E₂^?1=11.8 for the subsequent kinetic resolution. The thermodynamic analysis furthermore revealed that the enzyme stereodiscrimination was enthalpy-driven for the desymmetrization step, but changed as entropy-driven for the kinetic resolution step.     

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.     

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%.     

Kinetic resolution of drug intermediates catalyzed by lipase B from Candida antarctica immobilized on immobead‐350

Novozyme 435, which is a commercial immobilized lipase B from Candida antarctica (CALB), has been proven to be inadequate for the kinetic resolution of rac‐indanyl acetate. As it has been previously described that different immobilization protocols may greatly alter lipase features, in this work, CALB was covalently immobilized on epoxy Immobead‐350 (IB‐350) and on glyoxyl‐agarose to ascertain if better kinetic resolution would result. Afterwards, all CALB biocatalysts were utilized in the hydrolytic resolution of rac‐indanyl acetate and rac‐(chloromethyl)‐2‐(o‐methoxyphenoxy) ethyl acetate. After optimization of the immobilization protocol on IB‐350, its loading capacity was 150 mg protein/g dried support. Furthermore, the CALB‐IB‐350 thermal and solvent stabilities were higher than that of the soluble enzyme (e.g., by a 14‐fold factor at pH 5–70°C and by a 11‐fold factor in dioxane 30%–65°C) and that of the glyoxyl‐agarose‐CALB (e.g., by a 12‐fold factor at pH 10–50°C and by a 21‐fold factor in dioxane 30%–65°C). The CALB‐IB‐350 preparation (with 98% immobilization yield and activity versus p‐nitrophenyl butyrate of 6.26 ± 0.2 U/g) was used in the hydrolysis of rac‐indanyl acetate using a biocatalyst/substrate ratio of 2:1 and a pH value of 7.0 at 30°C for 24 h. The conversion obtained was 48% and the enantiomeric excess of the product (e.e._p) was 97%. These values were much higher than the ones obtained with Novozyme 435, 13% and 26% of conversion and e.e.p, respectively. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:878–889, 2018     

Enantioselective behavior of lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4°C and could be reused at least six times.     

Asymmetric hydrolysis of R-(−),S(+)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216

Whole cells and cell-free extracts derived from Rhodococcus rhodochrous NCIMB 11216 were shown to hydrolyse both aliphatic and aromatic nitriles, when the organism had been grown on either propionitrile or benzonitrile as the source of carbon and nitrogen. Whole cell suspensions and cell-free extracts derived from bacteria grown on either substrate were able to biotransform R-(-),S-(+)-2-methylbutyronitrile. The S-(+) enantiomer was biotransformed more rapidly than the the R-(-) enantiomer. For whole cell biotransformations at 30°C, the maximum enantiomeric excess (ee) of the remaining R-(-)-2-methylbutyronitrile was 93% when 70% of the R-(-) enantiomer had been converted to the product, 2-methylbutyric acid. For the corresponding biotransformation at 4°C, there was an ee of 93% for the residual R-(-) enantiomer of the substrate when only 60% of it had been converted to product. For biotransformations by cell-free extracts at 30°C the 2-methylbutyric acid product had an ee of 17% for the S-(+) enantiomer at the time of optimal ee for the remaining R-(-) enantiomer of the substrate. In contrast, when the reaction was carried out by whole cells, the ee for the product acid was 0.36%. This was probably due to further, non-selective metabolism of the acid, which was especially significant at the beginning of the reaction. At both temperatures, the ee for the S-(+) enantiomer of 2-methylbutyric acid was at a maximum in the early stage of the biotransformation; for example, at 4°C the maximum detectable ee was 100% when the yield was 11%.Abbreviations EDTA Ethylenediaminetetraacetic acid - ee enantiomeric excess - FID flame ionisation detector - GC gas chromatography - ¹HNMR H nuclear magnetic resonance - K _m Michaelis constant - NCIMB National Collection of Industrial and Marine Bacteria - td doubling time - V _max Maximum velocity     

Kinetic resolution of α-lipoic acid via enzymatic differentiation of a remote stereocenter

Kinetic resolution of α-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-α-lipoic acid, leaving the target product (R)-α-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50°C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of α-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.     

Kinetic biosynthesis of l-ascorbyl acetate by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM)

Thermomyces lanuginosus lipase (Lipozyme TLIM)-catalyzed esterification of l-ascorbic acid was studied. It was suggested that Lipozyme TLIM was a suitable biocatalyst for enzymatic esterification of l-ascorbic acid. Three solvents were investigated for the reaction, and acetone was found to be a suitable reaction medium. Furthermore, it was found that water activity could notably affect the conversion. Moreover, pH memory of Lipozyme TLIM lipase for catalyzing l-ascorbic acid esterification in acetone was observed and the effect of pH on the reaction was estimated. In addition, the influences of other parameters such as substrate mole ratio, enzyme loading, and reaction temperature and reusability of lipase on esterification of l-ascorbic acid were also analyzed systematically and quantitatively. Kinetic characterization of Lipozyme TLIM showed that K _m,a and V _max were 80.085 mM and 0.747 mM min⁻¹, respectively. As a result, Lipozyme TLIM-catalyzed esterification of l-ascorbic acid gave a maximum conversion of 99%.     

The characteristic smell emitted from two scale insects,Ceroplastes japonicus and Ceroplastes rubens

ABSTRACT

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC–MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were β-selinene (10.3%) and β-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects.     

Temperature as an enantioselective parameter in enzymatic resolutions of racemic mixtures

Summary In the field of enhancement of the enantiomeric excess, the effect of temperature on the enantiomeric ratio, E, was investigated in lipase catalysed hydrolysis and transesterification. It was found that the equation (E₁)^T1=(E₂)^T2 correlated well with the experimental data obtained.     

Enzyme analyses demonstrate that β-methylbutyric acid is converted to β-hydroxy-β-methylbutyric acid via the leucine catabolic pathway by Galactomyces reessii

Galactomyces reessii accomplishes the enzymatic transformation of β-methylbutyric acid (isovaleric acid) to β-hydroxy-β-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas β-methylbutyric acid hydroxylase, α-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, β-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to β-hydroxy-β-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to β-hydroxy-β-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into β-hydroxy-β-methylbutyric acid, thus demonstrating that β-methylbutyric acid is part of the leucine catabolic pathway. The rate of β-methylbutyric acid conversion to β-hydroxy-β-methylbutyric acid with cell-free extract was 0.013 μmol β-hydroxy-β-methylbutyric acid (mg protein)^–1 h^–1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 μmol β-hydroxy-β-methylbutyric acid (g cells)^–1 h^–1] was also observed with β-methylbutyric acid. The results indicate that β-methylbutyric acid is transformed to β-hydroxy-β-methylbutyric acid through the leucine catabolic pathway. Received: 18 July 1997 / Accepted: 12 November 1997     

Kinetic resolution of esters of amino acids in t-butanol containing 5% water catalyzed by a stable industrial alkaline protease

We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L -amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester. The D -antipode precipitated at pH 6.2–6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. © 1994 Wiley-Liss, Inc.     

Reactivity of pure Candida rugosa lipase isoenzymes (Lip1, Lip2, and Lip3) in aqueous and organic media. influence of the isoenzymatic profile on the lipase performance in organic media

Three pure isoenzymes from Candida rugosa lipase (CRL: Lip1, Lip2, and Lip3) were compared in terms of their stability and reactivity in both aqueous and organic media. The combined effect of temperature and pH on their stability was studied applying a factorial design. The analysis of the response surfaces indicated that Lip1 and Lip3 have a similar stability, lower than that of Lip2. In aqueous media, Lip3 was the most active enzyme on the hydrolysis of p-nitrophenyl esters, whereas Lip1 showed the highest activity on the hydrolysis of most assayed triacylglycerides. The highest differences among isoenzymes were found in the hydrolysis of triacylglycerides. Thus, a short, medium, and long acyl chain triacylglyceride was the preferred substrate for Lip3, Lip1, and Lip2, respectively. In organic medium, Lip3 and Lip1 provided excellent results in terms of enantioselectivity in the resolution of ibuprofen (EF value over 0.90) and conversion, whereas initial esterification rate was higher for Lip3. However, the use of Lip2 resulted in lower values of conversion, enantiomeric excess, and enantioselectivity. In the case of trans-2-phenyl-1-cyclohexanol (TPCH) resolution, initial esterification rates were high except for Lip3, which also produced poor results in conversion and enantiomeric excess. The performance of the pure isoenzymes in the enantioselectivity esterification of these substrates was compared with different CRL crude preparations with known isoenzymatic content and the different results could not be explained by their isoenzymatic profile. Therefore, it can be concluded that other factors can also affect the catalysis of CRL and only the reproducibility between powders can ensure the reproducibility in synthesis reactions.     

Kinetics of enzymatic synthesis of L-ascorbyl acetate by Lipozyme TLIM and Novozym 435

L-ascorbyl acetate was synthesized through lipase-catalyzed esterification using Lipozyme TLIM and Novozym 435. Four solvents, including methanol, ethanol, acetonitrile, and acetone were investigated for the reaction, and acetone and acetonitrile were found to be suitable reaction media. The influences of several parameters such as water activity (a _w), substrate molar ratio, enzyme loading, and reaction temperature on esterification of L-ascorbic acid were systematically and quantitatively analyzed. Through optimizing the reaction, lipase-catalyzed esterification of L-ascorbic acid gave a maximum conversion of 99%. The results from using Lipozyme TLIM and Novozym 435 as biocatalysts both showed that a _w was an important factor for the conversion of L-ascorbic acid. The effect of pH value on lipase-catalyzed L-ascorbic acid esterification in acetone was also investigated. Furthermore, results from a kinetic characterization of Lipozyme TLIM were compared with those for Novozym 435, and suggested that the maximum reaction rate for Lipozyme TLIM was greater than that for Novozym 435, while the enzyme affinity for substrate was greater for Novozym 436.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.