Optical Purity Of Product Research Articles

Development of a Novel D-Lactic Acid Production Platform Based on Lactobacillus saerimneri TBRC 5746.

D-Lactic acid is a chiral, three-carbon organic acid, that bolsters the thermostability of polylactic acid. In this study, we developed a microbial production platform for the high-titer production of D-lactic acid. We screened 600 isolates of lactic acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains, Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism. We investigated the effects of high temperature and the use of cheap, renewable carbon sources on lactic acid production and observed a titer of 99.4g/L and a yield of 0.90g/g glucose (90% of the theoretical yield). However, we also observed L-lactic acid production, which reduced the product's optical purity. We then used CRISPR/dCas9-assisted transcriptional repression to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional repression in this microbial host and represents progress toward efficient microbial production of D-lactic acid.

Halohydrin dehalogenase-catalysed synthesis of enantiopure fluorinated building blocks: bottlenecks found and explained by applying a reaction engineering approach

Biocatalytic modification of fluorinated styrene oxide derivatives: improvement of reaction outcome in terms of reaction yield, selectivity and product optical purity by employing reaction engineering approach.

The effect of deep eutectic solvents on the asymmetric hydrolysis of styrene oxide by mung bean epoxide hydrolases

BackgroundDeep eutectic solvents have attracted considerable attention in numerous fields. There is little information on mung bean epoxide hydrolase-catalyzed epoxides in deep eutectic solvent-containing system.ResultsAdding deep eutectic solvents with hydrogen bond donor of acids to phosphate buffer resulted in an obvious decrease in the optical purity of product; nevertheless, the relative slight change was observed by the addition of the deep eutectic solvents with hydrogen bond donor of alcohols and urea. Of the tested deep eutectic solvents, 10% additional amount of choline chloride/trithylene glycol can cause a significant improvement in the enantiopurity of product, from 83.2 ± 1.3% to 87.9 ± 0.3%. Moreover, with the increase in the addition amount of choline chloride/triethylene glycol from 10 to 30%, the enantiomeric excess of product enhanced from 87.9 to 94%, but a decline of product yield was observed. Finally, the evaluation of enzyme stability showed that the additional amount from 10% to 30% was not beneficial to the activity recovery.ConclusionIn short, adding choline chloride/triethylene glycol contributes to the improvement in the optical purity of (R)-1-phenyl-1, 2-ethanediol in the catalysis of styrene oxide by mung bean epoxide hydrolases; meanwhile, the enzyme immobilization could be essential.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency

(S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular “stapler” for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and kcat/Km values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (Tm) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved Tms and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.

Synthesis of (<i>S</i>)-<i>Tert</i>-Butyl 3-Hydroxybutyrate by Asymmetric Reduction of <i>Tert</i>-Butyl Acetoacetate with <i>Saccharomyces cerevisiae </i>B5<sup></sup>

S)-tert-butyl 3-hydroxybutyrate was synthesized by asymmetric reduction of tert-butyl acetoacetate with Saccharomyces cerevisiae B5 as catalyst. The enantiometric excess of (S)-tert-butyl 3-hydroxybutyrate increased with addition of more amount of substrate. High optical purity of product can be obtained when 6 g/L chloroform was used as inhibitor. The optimum reduction time, temperature, and initial pH of reaction mixture were 60 h, 30 °C, and 6.2. Addition of more biomass and lower amount of substrate helped to get high conversion. Conversion and enantiometric excess of product reached 100% when initial substrate concentration and biomass were 2.0 g/L and 140 g/L with 6 g/L chloroform as inhibitor.

Jerusalem artichoke powder: A useful material in producing high-optical-purity l-lactate using an efficient sugar-utilizing thermophilic Bacillus coagulans strain

Jerusalem artichoke is a low-requirement crop, which does not interfere with food chain, and is a promising carbon source for industrial fermentation. Microbial conversion of such a renewable raw material to useful products, such as lactic acid, is an important objective in industrial biotechnology. In this study, high-optical-purity l-lactate was efficiently produced from the hydrolysates of Jerusalem artichoke powder by a thermophilic bacterium, Bacillus coagulans XZL4. High l-lactate production (134gl−1) was obtained using 267gl−1 Jerusalem artichoke powder (total reducing sugars of 140gl−1) and 10gl−1 of corn steep powder in fed-batch fermentation, with an average productivity of 2.5gl−1h−1 and a yield of 0.96gg−1 reducing sugars. The final product optical purity is 99%, which meets the requirement of lactic acid polymerization. Our study represents a cost-effective and promising method for polymer-grade l-lactate production using a cheap raw bio-resource.

Fermentative l-(+)-lactic acid production from non-sterilized rice washing drainage containing rice bran by a newly isolated lactic acid bacteria without any additions of nutrients

A newly isolated lactic acid producing bacterium Lactobacillus rhamnosus M-23, from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. The maximum lactate yield attained was 59 g/l with a productivity of 1.23 g/l/h and a product optical purity of 95% corresponding to a conversion of 0.85 g of lactic acid per gram of sugar equivalent.

Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation.

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the 'ene' reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated alpha,beta-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49-99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cbeta (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18-19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Calpha is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation

AbstractWe show that pentaerythritol tetranitrate reductase (PETNR), a member of the ‘ene’ reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β‐unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme‐substrate complexes based on the crystal complex of the PETNR with 2‐cyclohexenone 4a. The optical purity of products is variable (49–99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18–19a), an enantiodivergent course of the reduction of E/Z‐forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non‐enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.

Enantioseparation via EIC‐OSN: Process design and improvement of enantiomers resolvability and separation performance

AbstractThis article presents a mathematical model to assess and optimize the separation performance of an enantioselective inclusion complexation‐organic solvent nanofiltration process. Enantiomer solubilities, feed concentrations, solvent compositions, permeate solvent volumes, and numbers of nanofiltrations were identified as key factors for process efficiency. The model was first tested by comparing calculated and experimental results for a nonoptimized process, and then, calculations were carried out to select the best operating conditions. An important finding was that the optimal configuration varied with the objective function selected, e.g., resolvability versus yield, with a boundary on product optical purity. The model also suggested that the process efficiency could benefit from diafiltration of the distomer and from the use of higher feed concentrations. However, the latter strategy would result in higher losses of eutomer. To address this drawback, a multistage process was evaluated using the verified process model. © 2009 American Institute of Chemical Engineers AIChE J, 2010

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system

An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.e. and yield of 90% after 60 h. Simultaneously, it was observed that change in cell morphology impedes the further enhancement of substrate concentration in this system but can be reversibly changed after stereoinversion or cultivation in systems without PEG.

Non-Sterilized Fermentative Production of Polymer-Grade L-Lactic Acid by a Newly Isolated Thermophilic Strain Bacillus sp. 2–6

BackgroundThe demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure l-lactic acid is essential for polymerization of PLA. The high fermentation cost of l-lactic acid is another limitation for PLA polymers to compete with conventional plastics.Methodology/Principal FindingsA Bacillus sp. strain 2–6 for production of l-lactic acid was isolated at 55°C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure l-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2–6, 118.0 g/liter of l-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum l-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%.Conclusions/SignificanceWith the newly isolated Bacillus sp. strain 2–6, high concentration of optically pure l-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade l-lactic acid production from renewable resources.

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of α‐keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.

Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol

Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereoselectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride (m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production.

Mathematical module for countercurrently fractional chiral extraction and its VB simulation

Based on chemical thermodynamics and mass balance, countercurrently fractional chiral extraction by hollow fiber membrane was investigated. The mathematical modules for the relation of chiral extraction yield and product optical purity with number of transfer units by chiral extraction with hollow fiber membrane were established, and the modules were simulated by visual basic(VB) proceduce. The results show that, the difference in free energy between two diastereomeric complexes formed by R- and S-enantiomer with chiral selector,—Δ(ΔG), is the force of separation of enantiomers. It is necessary to separate enantiomers, where one of the extraction factors is above 1, and the other is below 1. Under certain phase ratio, chiral separation depends on separation factor and number of transfer units. The experimental result is in agreement with the theoretical value.

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.

Sequential design of pH profiles for asymmetric bioreduction of ethyl 4-chloro-3-oxobutyrate using a new experimental design method

A sequential fashion experimental design algorithm for the optimal pH profile search in the asymmetric reduction of ethyl 4-chloro-3-oxobutyrate catalyzed by Saccharomyces cerevisiae was addressed. Ethyl S-4-chloro-3-hydroxybutyrate was produced to reach two important quality indices: reaction yield and product's optical purity. Instead of the traditional strategies with the fixed pH or the uncontrolled pH values during the reaction, a novel strategy for designing the optimal time-varying operating pH profile was developed based on the methodology of the data-driven method. The proposed design technique composed of the hybrid function approximation and the Taguchi's orthogonal array was used to determine the new design profiles in the next run. The hybrid type of the function approximation allows the global and local approximations to construct the profiles in the whole design space and some local regions, respectively. The optimal profile can be obtained if the location of the function coefficients is properly adjusted in the functional space. The Taguchi's approach is used to design experiments and analyze the outcomes of each experimental design to improve the optimal objective (quality) function. Without the prior knowledge of the system, the proposed method using information from the previous experiments can update and modify the profiles that would be applied to the subsequent experiments. In comparison with traditional methods, the product's ee could be significantly enhanced (from 83.8% to 91.3%) without decreasing the reaction yield.

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.

Design of the pH profile for asymmetric bioreduction of ethyl 4-chloro-3-oxobutyrate on the basis of a data-driven method.

The goal of this paper was to design the optimal time-varying operating pH profile in the asymmetric reduction of ethyl 4-chloro-3-oxobutyrate by baker's yeast. Ethyl (S)-4-chloro-3-hydroxybutyrate was produced to reach two important quality indices: reaction yield and product optical purity. The method integrated an orthogonal function approximation and an orthogonal array. The technique used a set of orthonormal functions as the basis for representing the possible profile. The optimal profile could be obtained if the orthogonal coefficients were properly adjusted. The orthogonal array was used to design and analyze the effect of each orthogonal coefficient in order to reach the optimal objective (quality) function. The performance based on the proposed strategy was significantly improved by over 10% compared with the traditional fixed pH or uncontrolled pH values during the reaction. The proposed method can be applied to the required dynamic profile in the bioreactor process to effectively improve the product quality, given good design directions and the advantage of the traditional statistical approach.