Kinetic resolution of aminoacid ester with immobilized cells ofSulfolobus solfataricus

Summary Cells ofSulfolobus solfataricus trapped in sodium alginate have been used to hydrolyze a range of aminoacid esters with useful stereoselectivity for L-enantiomer at different temperatures.     

Preparation of both enantiomers of ethyl 4,4,4-trifluoro-3-hydroxy butanoate by enantioselective microbial reduction

The effect of some parameters, i.e. temperature, time, pH and concentration, on the baker's yeast reduction of ethyl 4,4,4-trifluoroacetoacetate is presented. The enantiomeric excess of the R enantiomer appeared to increase up to 76% when the temperature of the reduction decreased. The other factors do not appear to improve the enantioselectivity of the reaction. Reduction with Candida utilis allowed preparation of the S enantiomer in higher optical purity than previously reported.     

Asymmetric reduction of ketones with resting cells of Sulfolobus solfataricus

The method of resting cells has been of interest in the development of biocatalysts applied to organic reactions.This article deals with the use of resting cells of a thermophilic archaebacterium Sulfolobus solfataricus, in the asymmetric reduction of acyclic, cyclic, and aromatic ketones. The system allows the continuous regeneration of endogenous coenzyme with the coupled substrate approach. The results indicate that the direction of hydride attack was equatorial on the re face of the carbonyl group of substrates producing (S)-alcohols with a good optical yield. A convenient system for the reuse of resting cells has been set out to synthesize (S)-alcohols on a preparative scale.     

Double kinetic resolution of (±) ethyl 4 - chloro - 3- 0-acetyl butanoate

Summary Single pot double kinetic resolution of (±) ethyl 4-chloro-3-O-acetyl butanoate 1 by transesterification with n-butanol in diisopropyl ether using the enzyme Candida Cylindracea Lipase (CCL) is investigated. This technique is found to give products with better optical purity.     

Production of 2-keto-3-deoxygluconate by immobilized cells of Sulfolobus solfataricus

Summary 2-Keto-3-deoxygluconate, an intermediate of glucose breakdown inSulfolobus solfataricus, was produced by enzymic dehydration of gluconate using whole cells of the micro-organism immobilized in crude egg white. The degradation of 2-keto-3-deoxygluconate to pyruvate and glyceraldehyde was avoided by inhibiting the aldolase activity in the cells by sodium borohydride treatment.     

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.     

Enhancement of asymmetric bioreduction of ethyl 4-chloro acetoacetate by the design of composition of culture medium and reaction conditions

This study investigated the feasibility of improving the stereospecificity of yeast by the adjustment of the culture medium composition and the reaction conditions. The investigation was performed systematically, using an approach that integrates the Taguchi's array method and the steepest ascent method. The reaction yield and the product's ee were the two indexes of reaction performance. A desirability function was applied to combine these two indexes as a single objective function. The removal of peptone and malt extract from the YM medium increased the yeast's stereoselectivity, without reducing the production of biomass. The medium composition and the reaction conditions were then simultaneously optimized. The resulting optimal conditions were 30 g/l glucose for cultivation, 12 g/l yeast extract, a cultivation time of 12 h, 15 g/l glucose for reaction, 150 g/l yeast for reaction, a reaction buffer concentration of 0.2 M and a buffer pH of 8.5. Compared to the one before this study, the product's ee was improved from 82.1 to 92.3%, and the reaction yield was enhanced from 77.3 to 82.3%. Furthermore, the biomass production was increased considerably from 5.94 to 10.14 g DCW/l.     

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 L₁₈ 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.     

Production of 2-hydroxy 4-methoxy benzaldehyde using root cultures of Hemidesmus indicus

Nodal explants of Hemidesmus indicus plants cultured in half strength Murashige and Skoog medium fortified with 2 mg indole-3-butyric acid l–1 in the dark produced 10–12 roots (1–2 cm) with minimal callusing in 10 days. Roots, 20 mg dry wt, cultured in Gamborg et al. medium supplemented with 2 mg IBA l–1, sucrose (4% w/v), pH (5.6) and agitation (70 rpm) in the dark yielded 550 mg root dry wt/flask and 0.18% 2-hydroxy 4-methoxy benzaldehyde in the roots after 30 days. The maintenance of normal morphology of the roots, consistent biomass and product yields over a 21-month period indicated the stability of the cultures. © Rapid Science Ltd. 1998     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Production of L-phenylalanine from phenylpyruvate using resting cells of Escherichia coli

Summary We describe the production of L-phenylalanine from phenylpyruvate using resting cells of a genetically modified strain of Escherichia coli. Fermentations were carried out by continuously raising the feeding rate of D-glucose. We reached a biomass of 10 g/l dry weight and an aminotransferase activity of 14000 U/l The maximum phenylalanine concentration achieved was 173 mmol/l with a phenylpyruvate molar conversion yield of 95%.Dedicated to Prof. Dr. Heinz Harnisch on the occasion of his 60th birthday     

Production of biosurfactants by the resting cells ofPseudomonas aeruginosa CFTR-6

A resting cell system was used for the production of glycolipids byPseudomonas aeruginosa CFTR-6. In this, the growth phase was separated from the production phase to overcome the inhibition of glycolipid production by inorganic phosphate. It was shown that when the cells were transferred after the growth phase into a medium devoid of phosphate, glycolipid production was increased nearly twofold. The maximum glycolipid concentration was attained much more rapidly than the conventional batch fermentation system, thus increasing the productivity.     

Production of mannosylerythritol lipids as biosurfactants by resting cells ofCandida antarctica

Summary The resting cells ofCandida antarctica strain T-34 was found to produce a large amount of mannosylerythritol lipids as biosurfactants when incubated in the medium containing only the carbon source. The resting cells prepared from different water-soluble carbon sources were able to produce the lipids abundantly from water-insoluble carbon sources. Under the optimal conditions in a shake culture, the concentration of the total lipids amounted to about 47 g/l after 6 days, and the yield of the lipids became higher than that obtained by using the growing cells of the strain.     

Production of vanillic acid from vanillin by resting cells of Serratia marcescens

Resting-cell suspensions of Serratia marcescens were able to convert, quantitatively, 0.3% vanillin to vanillic acid. The vanillic acid-producing activity reached a maximum after 28 h of incubation with 0.01% vanillin as an inducer.     

Lysosomal inhibitors stimulate resting NIH 3T3 cells to proliferate

Lysosomal inhibitors (amino acid methyl esters) and platelet-derived growth factor stimulate resting NIH 3T3 cells to enter the S period. Incubation of cells in medium containing lysosomal inhibitors causes an increase in protein accumulation and does not disrupt lysosomes. The results indicate that proliferative homeostasis depends partially on the metabolic status of the cell and that catabolic processes activated in resting cells negatively influence prereplicative reactions.     

Lysosomal inhibitors stimulate resting NIH 3T3 cells to proliferate

Abstract. Lysosomal inhibitors (amino acid methyl esters) and platelet-derived growth factor stimulate resting NIH 3T3 cells to enter the S period. Incubation of cells in medium containing lysosomal inhibitors causes an increase in protein accumulation and does not disrupt lysosomes. The results indicate that proliferative homeostasis depends partially on the metabolic status of the cell and that catabolic processes activated in resting cells negatively influence prereplicative reactions.     

Polyadenylate-protein complexes in resting and growing 3T3 cells.

The properties of the ribonuclease resistant cytoplasmic ribonucleoprotein particles were studied in contact-inhibited and serum induced proliferating 3T3 cells. The RNP particles were fractionated by oligo (dT)-cellulose chromatography and banded in CsSO4 gradients. The main RNP fraction, eluted with 25% formamide, contained the major ribonuclease resistant RNA sequences in both resting and growing cells. The protein component of this fraction had a molecular weight of about 72,000 in contact-inhibited cells and 81,000 in serum induced cells.     

Production of vanillic acid from vanillin by resting cells of Serratia marcescens.

Resting-cell suspensions of Serratia marcescens were able to convert, quantitatively, 0.3% vanillin to vanillic acid. The vanillic acid-producing activity reached a maximum after 28 h of incubation with 0.01% vanillin as an inducer.     

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by fungi

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).