（R）与（S）-羰基还原酶偶联一步法制备（S）-苯乙二醇

【目的】通过 (R) - 和(S) -羰基还原酶在大肠杆菌中偶联,实现了一步法制备（S）-苯乙二醇的生物转化过程。【方法】将来源于近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)- 羰基还原酶基因（rcr）和(S) -羰基还原酶基因（scr）串联于共表达载体pETDuetTM-1上。重组质粒pETDuet-rcr-scr转化稀有密码子优化型菌株Escherichia coli Rosetta,获得酶偶联重组菌株E. coli Rosetta / pETDuet-rcr-scr。当重组菌体培养至OD600 0.6-0.8时,添加终浓度1 mmol/L IPTG,30℃诱导蛋白表达10 h。【结果】SDS-PAGE结果表明(R)- 和(S) -羰基还原酶均明显表达,它们的相对分子质量分别为37 kDa和30 kDa。重组菌生物转化结果表明：在pH7.0的磷酸缓冲液中,添加5 mmol/L Zn2+时,获得产物(S)-苯乙二醇,产物光学纯度为91.3% e.e.,产率为75.9%。【讨论】采用分子重组技术成功整合了两种氧化还原酶的催化功能,实现了(S)- 苯乙二醇的一步法转化,为简化手性醇制备途径提供了一条崭新的思路。     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.     

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.     

多酶融合表达体系以木聚糖为辅助底物高效合成(S)-苯乙二醇

[目的] 利用木聚糖为辅助底物加强手性催化反应中的辅酶循环,构建来源于近平滑假丝酵母（Candida parapsilosis）CCTCC M203011的（S）-羰基还原酶II（SCRII）、枯草芽孢杆菌（Bacillussp.）YX-1葡萄糖脱氢酶突变体Ala258Phe/GDH和里氏木霉（Trichoderma reesei）Rut C-30木聚糖酶（XYN2）在大肠杆菌（Escherichia coli）BL21（DE3）中的融合表达体系,高效合成（S）-苯乙二醇。[方法] 调节3种酶编码基因在pET-28a载体上的位置,运用重叠延伸PCR技术,构建了E.coli/pET-SCRII-A258F-XYN2和E.coli/pET-A258F-SCRII-XYN2两种重组菌,研究了其合成（S）-苯乙二醇的最适反应条件。[结果] 重组菌株E.coli/pET-SCRII-A258F-XYN2在底物2-羟基苯乙酮与辅助底物木聚糖的比例为1：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达98.8%（W/W）;而重组菌株E.coli/pET-A258F-SCRII-XYN2在底物与辅助底物的比例为2：1、35℃、pH为7.0条件下,（S）-苯乙二醇的产率达95.6%（W/W）,两者合成产物的光学纯度均>99%。[结论] 通过构建3种酶的融合表达体系,成功将木聚糖酶和葡萄糖脱氢酶突变体介导的辅酶再生循环体系引入不对称生物合成反应,提高了手性转化效率,为将大自然中丰富的木聚糖用于手性催化奠定了较扎实的研究基础。     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.     

Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Abstract

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified with a specific activity of 8.32 U mg^?1, over 36% higher than that of Escherichia coli SCRII. The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical purity of >99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L^?1 as co-substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at >99% enantiomeric excess and >85%, respectively.     

Enzymatic resolution of 1-phenyl-1,2-ethanediol by enantioselective oxidation: overcoming product inhibition by continuous extraction

Oxidations of alcohols by alcohol dehydrogenases often suffer from low conversions and slow reaction rates due to severe product inhibition. This can be overcome by continuous product extraction, because only the concentrations, but not the kinetic parameters, can be changed. As a consequence, it is favorable to apply a differential circulation reactor with continuous product extraction, where only a small amount of product is formed per cycle. The product is then directly extracted using a microporous hydrophobic hollow fiber membrane. This results in an increase of the relative activity of the dehydrogenase at a given conversion. The reaction investigated is the kinetic resolution of racemic 1-phenyl-1,2-ethanediol by glycerol dehydrogenase (GDH). The resulting oxidation product, 2-hydroxyacetophenone, causes a strong product inhibition. Additionally, it reacts in a chemical reaction with the cofactor lowering its active concentration. Because the GDH needs beta-nicotinamide adenine dinucleotide (NAD(+)) as a cofactor, lactate dehydrogenase is used to regenerate NAD(+) from NADH by reducing pyruvate to (L)-lactate. A conversion of 50% with respect to the racemate and an enantiomeric excess >99% of the (S)-enantiomer was reached.     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.     

A novel NADH-dependent carbonyl reductase with unusual stereoselectivity for (R)-specific reduction from an (S)-1-phenyl-1,2-ethanediol-producing micro-organism: purification and characterization

AIMS: To purify and characterize the (R)-specific carbonyl reductase from Candida parapsilosis; to compare the enzyme with other stereospecific oxidoreductases; and to develop an available procedure producing optically active (R)-1-phenyl-1,2-ethanediol (PED). METHODS AND RESULTS: An (R)-specific carbonyl reductase was found and purified from C. parapsilosis through four steps, including blue-sepharose affinity chromatography. The relative molecular mass of the enzyme was estimated to be 35 kDa on gel-filtration chromatography and 37.5 kDa on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalysed the reduction of various ketones, including alkyl and aromatic ketones, and was specific to short-chain and medium-chain alkyl ketones. The enzyme activity was inhibited by divalent ion of CuSO(4) and FeSO(4), whereas zincum ion stimulated its activity. For catalysing reduction, the enzyme performed maximum activity at pH 6.0 and the optimum temperature was 45 degrees C. The carbonyl reductase catalysed asymmetric reduction of beta-hydroxyacetophenone to the corresponding (R)-PED with the optical purity of 100% enantiomeric excess (e.e.). By analysing its partial amino acid sequences, the enzyme was proposed to be a novel stereospecific carbonyl reductase. CONCLUSIONS: The purified carbonyl reductase showed unusual stereospecificity and catalysed the NADH-dependent reduction of beta-hydroxyacetophenone to (R)-PED. The enzyme was different from other stereoselective oxidoreductases in catalytic properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The discovery of (R)-specific oxidoreductase exhibiting unusual stereospecificity towards hydroxyl ketone is valuable for the synthesis of both enantiomers of useful chiral alcohols, and provides research basis for the achievement of profound knowledge on the relationship between structure and catalytic function of (R)-specific enzymes, which is meaningful for the alteration of stereospecificity by molecular methods to obtain the enzymes with desired stereospecificity.     

固定化细胞生物催化制备(S)-苯基乙二醇的研究

采用海藻酸钙固定化细胞方法能有效提高近平滑假丝酵母催化(RS)-苯基乙二醇(S-PED)去消旋化反应的稳定性,在优化反应条件下,固定化细胞催化批次由游离细胞的2次提高至6次,产物可保持高光学纯度(>95%)、高产率(>85%)。与化学合成液晶掺杂剂S-1011的理化特性对比结果表明,生物催化法制备获得的(S)-PED能够替代化学法制备的相同产品。     

Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity

An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (k_cat/K_m) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.     

猪肝酯酶催化苯乙二醇环碳酸酯水解最佳反应条件的研究

猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度（15g/L）、pH值（8.0）、温度（25~30℃）及有机溶剂种类和浓度（二氧六环,65%v/v）,为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.     

Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.     

Design,synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule destabilizers

A series of 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as microtubule destabilizers were designed, synthesised and evaluated for anticancer activity. Based on bioisosterism, we introduced the tetrazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. The key intermediates ethyl 1-aryl-1H-tetrazole-5-carboxylates 10 can be simply and efficiently prepared via a microwave-assisted continuous operation process. Among the compounds synthesised, compound 6–31 showed noteworthy potency against SGC-7901, A549 and HeLa cell lines. In mechanism studies, compound 6–31 inhibited tubulin polymerisation and disorganised microtubule in SGC-7901 cells by binding to tubulin. Moreover, compound 6–31 arrested SGC-7901cells in G2/M phase. This study provided a new perspective for development of antitumor agents that target tubulin.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Modulation of the immune response to tumors by a novel synthetic compound, (4R)-3-benzoyl-N-[(1R)-1-phenylethyl]-4-thiazolidinecarboxamide (RS-0481)

Summary RS-0481, (4R)-3-benzoyl-N-[(1R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumorspecific cytotoxic T lymphocytes,Tdth (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice.