Improved production of (<Emphasis Type="Italic">S</Emphasis>)-ketoprofen ester hydrolase by a mutant of<Emphasis Type="Italic"> Trichosporon brassicae</Emphasis> CGMCC 0574

An efficient screening method following UV mutagenesis yielded a high frequency of improved mutants of Trichosporon brassicae CGMCC 0574, a wild-type esterase-producer capable of enantioselectively hydrolyzing the ethyl ester of ketoprofen [2-(3-benzoylphenyl) propionic acid]. The mutant had an activity 1.8-fold higher than the wild type and was stable in its enzyme production for ten serial transfers. As the best single carbon source, isopropanol improved the specific activity of the enzyme 5-fold; and this did not result from the effect of cell permeabilization. An 18-h culture grown on a medium containing 0.5% glucose plus 0.5% isopropanol produced 3-fold as much esterase as a culture grown on 1% glucose.     

Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011

Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-α-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-α-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima of 30°C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate. It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-α-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-α-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity.     

Extractive fermentation in cloud point system for lipase production by Serratia marcescens ECU1010

Extractive microbial fermentation for production of lipase by Serratia marcescens ECU1010 has been carried out in cloud point system. The cloud point system is composed of mixture nonionic surfactants with a ratio of Triton X-114 to Triton X-45 4:1 in aqueous solution. The lipase prefers to partition into the surfactant rich phase (coacervate phase) whereas the cells and other hydrophilic proteins retain in the dilute phase of cloud point system. Thus, a concentration factor 4.2-fold and a purification factor 1.3-fold of the lipase have been achieved in the extractive fermentation process. This is the first report about extractive fermentation of proteins in cloud point system.     

Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides

Escherichia coli BL21 as production strain for the production of cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. in high yields was developed. The expression was first optimized with a series of flask experiments testing several key parameters for their influence on the expression level and enzyme activity. The optimal process parameters found in the flask experiments were verified in a cultivation process in a 5-L bioreactor. Glycerol proved to be superior over glucose as carbon source. Low dissolved oxygen (DO) concentration (<10%) during expression was found to be critical for active P450s production, resulting in expression level of 400 nM for P450SMO. Intact cells were used to establish an efficient bioconversion system for the production of sulfoxidation product. With p-chlorothioanisole as a representative substrate, the desired product (S-sulfoxide) was afforded with 99% ee and highest production of 130 mg/L within 12 h.     

Isolation and properties of a <Emphasis Type="Italic">levo-</Emphasis>lactonase from <Emphasis Type="Italic">Fusarium proliferatum</Emphasis> ECU2002: a robust biocatalyst for production of chiral lactones

A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone (pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.     

Modified ferric hydroxamate spectrophotometry for assaying glycolic acid from the hydrolysis of glycolonitrile by Rhodococcus sp. CCZU10-1

We successfully modified a ferric hydroxamate spectrophotometry method for assaying glycolic acid. Comparable to the high-performance liquid chromatography (HPLC)-based method, ferric hydroxamate spectrophotometry can be used to accurately monitor the time course of glycolonitrile bioconversion. Glycolic acid was assayed simply and rapidly at room temperature (25 ～ 35°C). Optimum culture conditions were obtained using this method to assay the glycolonitrile-hydrolyzing activity of Rhodococcus sp. CCZU10-1. The preferred carbon and nitrogen sources and ideal inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (5 g/L), and ?-caprolactam (2 mmol/L), respectively. The optimal growth temperature and initial medium pH for Rhodococcus sp. CCZU10-1 glycolonitrile-hydrolyzing activity were 30°C and pH 7.0. Modified ferric hydroxamate spectrophotometry could potentially be employed to assay other carboxylic acids.     

生物转化对二甲苯生成对苯二甲酸的初步研究

对苯二甲酸是生产聚酯的主要原料,其生产方法主要是采用化学合成法。随着生物转化与生物催化研究的深入,其高效、环保、节能等优势越来越明显。筛选能够生物转化对二甲苯生成对苯二甲酸的菌株将会为生物催化法生产对苯二甲酸打下基础。通过建立筛选模型,利用唯一碳源法从土壤中分离筛选得到微生物16,经鉴定为嗜麦芽窄食单胞菌和睾丸酮丛毛单胞菌的混合菌株,该微生物可以利用对二甲苯为底物生物转化生成对苯二甲酸。实验中对诱导剂进行了选择,表明甲苯对该反应有明显的诱导作用,最佳诱导剂加入量为200mg／L。发酵液中对苯二甲酸及中间产物采用高效液相色谱法测定。     

Biocatalytic ketone reduction: A green and efficient access to enantiopure alcohols

Chiral secondary alcohols play an important role in pharmaceutical, agrochemical, and chemical industries. In recent years, impressive steps forward have been achieved towards biocatalytic ketone reduction as a green and useful access to enantiopure alcohols. An increasing number of novel and robust enzymes are now accessible as a result of the ongoing progress in genomics, screening and evolution technologies, while process engineering provides further success in areas of biocatalytic reduction in meeting synthetic challenges. The versatile platform of these techniques and strategies offers the possibility to apply high substrate loading and thus to overcome the limitation of low volumetric productivity of usual enzymatic processes which is the bottleneck for their practical application. In addition, the integration of bioreduction with other enzymatic or chemical steps allows the efficient synthesis of more complex chiral products.