Reduction Of Ethyl 4-chloroacetoacetate Research Articles

Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis.

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat /Km ) for COBE was 51.36 s-1 ·mM-1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.

An Alkali-tolerant Carbonyl Reductase from Bacillus subtilis by Gene Mining: Identification and Application

Enantiopure alcohols have received much attention due to their widespread use as pharmaceutical intermediates. In the asymmetric biosynthesis of enantiopure alcohols, the excellent performance of carbonyl reductase makes it be the best choice as the biocatalysts. In this work, an alkali-tolerant carbonyl reductase (BsCR, encoded by yueD) from Bacillus subtilis (strain 168) was obtained through gene mining, and successfully heterologously expressed in Escherichia coli with pET-32a. BsCR showed excellent alkali resistance and even can keep more than 70% of its peak activity after incubation in Tris–HCl buffer at pH 9.0 for 40 h. The Michaelis constants and maximal velocity of the BsCR to NADPH (A) and ethyl 4-chloroacetoacetate (B) are $$K_{m}^{A}$$ = 5.390 × 10−2 mmol/L, $$K_{m}^{B}$$ = 1.855 mmol/L, and $$V_{max}$$ = 147.3 μmol·min−1·mg−1, respectively. Applying the E. coli BL21(DE3)/pET-32a-yueD to catalyze asymmetric reduction of ethyl 4-chloroacetoacetate and acetophenone, the yield of S-CHBE reached 89.9% and S-1-phenyl ethanol reached 66.7%, and e.e. of both products reached more than 99%. This work provides a novel CR for asymmetric reduction. A carbonyl reductase (BsCR) and its gene were identified through gene mining, and overexpressed in Escherichia coli BL21(DE3) for whole-cell biocatalytic asymmetric reduction

Efficient production of ethyl (R)-4-chloro-3-hydroxybutanoate by a novel alcohol dehydrogenase from Lactobacillus curieae S1L19

Ethyl (R)-4-chloro-3-hydroxybutanoate ester [(R)-CHBE] is an important chiral intermediate for the synthesis of chiral drugs. In this study, a novel short-chain, NADH-dependent dehydrogenase (LCRIII) from Lactobacillus curieae S1L19 was discovered to exhibit high activity and enantioselectivity in the production of (R)-CHBE by reduction of ethyl 4-chloroacetoacetate (COBE). LCRIII was heterologously overexpressed in Escherichia coli and the protein was purified to homogeneity. Characterization of LCRIII showed broad substrate specificity towards a variety of ketones. In addition, an efficient cofactor regeneration system was constructed by co-expressing LCRIII and glucose dehydrogenase (GDH) in E. coli cells. Up to 1.5M (246.8g/L) COBE could be completely reduced to (R)-CHBE with excellent enantiomeric excess (>99% ee) in a monophasic aqueous system. Moreover, the process could be performed even without external addition of cofactors. These results demonstrate the great potential of this process in industrial applications.

Immobilized microorganisms in the reduction of ethyl 4-chloro acetoacetate

Microorganisms were used to reduce ethyl 4-chloroacetoacetate (CAAE) to ethyl ( S)-4-chloro-3-hydroxybutanoate [( S)-CHBE]. Mucor ramannianus provided 98% conversion with 84% ee. Free cells of Kluyveromyces marxianus led to 95% conversion with 81% ee. After a fractionary factorial design to study the reaction conditions, calcium alginate immobilized cells of K. marxianus furnished the product with 99% conversion with 91% ee.

Mathematical modeling of asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast

A mathematic model was developed to simulate the asymmetric reduction of ethyl 4-chloro acetoacetate (ECA) by bakers’ yeast. The model of the process considered the kinetics of enzymatic reaction, the effect of substrate inhibition and the spontaneous degradation of the substrate. The reaction kinetics of the ECA degradation was determined empirically. The inhibition by the substrate was analyzed and the apparent kinetic constants of the overall enzymatic reaction, of the S-enzymes and of the R-enzymes, were estimated individually. The system of equations was solved numerically using the Runge–Kutta method. The close correlation between the predicted and experimental results concerning product formation, reaction yield and optical purity of product under various substrate concentrations, implied the reliability of the established model.

Reaction engineering studies on β-ketoester reductions with whole cells of recombinant Saccharomyces cerevisiae

A recombinant Saccharomyces cerevisiae strain over-expressing the fatty acid synthase of S. cerevisiae and the glucose dehydrogenase of Bacillus subtilis was applied for enantioselective reduction of ethyl 4-chloro acetoacetate (CAAE) to ethyl ( S)-4-chloro-3-hydroxybutanoate ( S-CHBE) as well as the reduction of ethyl benzoylacetate (EBA) to ethyl ( S)-3-hydroxy-3-phenylpropionate ( S-HPPE). The reaction conditions for these asymmetric reductions were optimised by combining simple screening procedures and a stochastic search strategy (Genetic Algorithm). Complete conversion of 200 mM CAAE was achieved within 8 h in a two liquid phase system (water/ n-butyl acetate). The S-CHBE yield (94%) and the product/biocatalyst ratio (1.9 mmol S-CHBE/g cell dry weight) were improved by 50 and 450%, respectively, compared to published data. The enantiomeric excess was 90%. Nearly complete conversion of 55 mM EBA was achieved within 48 h without addition of an organic solvent. The S-HPPE yield (74%) and the product/biocatalyst ratio (0.7 mmol S-HPPE/g cell dry weight) were improved by 20 and 350%, respectively, compared to published data. The enantiomeric excess was >97% in the case of S-HPPE production.

Asymmetric synthesis of the chiral synthon ethyl ( S)-4-chloro-3-hydroxybutanoate using Lactobacillus kefir

Lactobacillus kefir was used as the whole cell biocatalyst for the asymmetric reduction of ethyl 4-chloro acetoacetate 1 to the chiral synthon ethyl ( S)-4-chloro-3-hydroxybutanoate 2. Ketoester 1 was obtained as micro-droplets, without the use of an organic solvent as substrate reservoir. 2 (1.2 M) was produced using 2-propanol as co-substrate with a final yield of 97% within 14 h. A high space-time yield and a high specific product capacity of 85.7 mmol/L h and of 24 mmol/g DCW were measured. The enantiomeric excess of the ( S)-alcohol 2 was 99.5%.

Stereoselective reduction of ethyl 4-chloro acetoacetate with recombinant Pichia pastoris

A recombinant strain of Pichia pastoris coexpressing the carbonyl reductase of Candida magnoliae and the glucose dehydrogenase of Bacillus subtilis was used for the stereoselective reduction of ethyl 4-chloro acetoacetate 1 to ( S)-4-chloro-3-hydroxybutanoate 2. The bioreduction was performed in a two-phase reaction system with n-butyl acetate as organic solvent. Complete conversion of 350 mM 1 with a final yield of 91% and an enantiomeric excess of 95% was achieved by continuous feeding of cell suspension and ketoester 1.

Applying the Taguchi robust design to the optimization of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast

This study examined the characteristics and operational parameters of the asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast in order to produce S-4-chloro-3-hydroxybutyric acid ethyl ester. Eight operational variables were also optimized using the Taguchi method with consideration of the freshness of yeast cells as a noise factor. An L 18 orthogonal array was used to design the experiments. The reaction yield and the product's optical purity were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Additionally, the signal-to-noise (SN) ratio was used to estimate the variability in product quality. Optimization was undertaken not only to yield the best performance, but also to minimize the variation in quality. The confirmation experiments indicated that the reaction performance and the robustness of the product quality under the optimized conditions were higher than those obtained in other experiments in this study. Our results further demonstrate that the product's optical purity could be increased to >95% by adjusting the operational level of the main factors.

Yeast Catalysed Reduction of β-Keto Esters (2): Optimisation of the Stereospecific Reduction by Zygosaccharomyces Rouxii

Zygosaccharomyces rouxii catalysed the reduction of ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) to the corresponding (S)-hydroxy ester (ethyl (S)-4-chloro-3-hydroxybutanoate) in high enantiomeric excess. The productivity of non-immobilised cells was compared to cells immobilised on a range of organic and inorganic supports. Cells immobilised in calcium alginate displayed a catalytic activity significantly higher than that of non-immobilised cells. A time dependent fall in the enantiomeric purity of the product was observed with the use of this matrix. This phenomenon was not seen in the reduction catalysed by non-immobilised cells.