高对映选择性环氧化物水解酶产生菌的筛选及特性研究

从土壤中分离的芽孢杆菌Bacillus megaterium ECU1001所产五氧化物水解酶能高对映选择性水解缩水甘油苯基醚（对映选择率E值可达47.8）,当转化率为55.9%时,剩余的（S）－缩水甘油苯基醚的光学纯度（对映体过量值,ee）可达99.5%;当底物浓度提高到60mmol/L时,光学纯（S）－缩水基油苯基醚的收率达到25.6%。     

Biocatalytic asymmetric hydroxylation of hydrocarbons by free and immobilized Bacillus megaterium cells

Screening of soil bacteria with allylbenzene resulted in a Bacillus megaterium strain, which hydroxylates simple hydrocarbons in high enantiomeric excess (ee up to 99%). Benzylic and nonbenzylic hydroxylation products were obtained, without the usually observed high preference for the benzylic position. The immobilization of the B. megaterium cells in alginate gel effectively improved the stability of the cells and increased the amounts of products formed, without loss of enantioselectivity. The product ratio ( vs. β hydroxylation) was shifted towards benzylic hydroxylation, which suggests that at least two hydroxylating enzymes with distinct regioselectivity are involved. Comparison to free-cell fermentations in small- and large-scale bioreactors (up to 2000 ml) showed that the use of immobilized cells is advantageous, as they are easier to handle and yield higher amounts of oxidation products.     

Biocatalytic resolution of nitro-substituted phenoxypropylene oxides with Trichosporon loubierii epoxide hydrolase and prediction of their enantiopurity variation with reaction time

Biocatalytic resolution of 3-(2′-nitrophenoxy)propylene oxide (1a), 3-(3′-nitrophenoxy)propylene oxide (1b) and 3-(4′-nitrophenoxy)propylene oxide (1c) were exploited by using lyophilized cells of yeast Trichosporon loubierii ECU1040 with epoxide hydrolase (EH) activity, which preferentially hydrolyzes (S)-enantiomers of the epoxides (1a–c), yielding (S)-diols and (R)-epoxides. The activity increased as the nitro group in the phenyl ring was shifted from 4′-position (1c) to 2′-position (1a). When the substrate concentration of 1a was increased from 10 to 80 mM, the E-value increased at first, until reaching a peak at 40 mM, and then decreased at higher concentrations (>40 mM). The optically active epoxide (R)-1a was prepared at gram-scale (97% ee, 41% yield). Furthermore, a simple method was developed to predict the enantiomeric excess of substrate (ee_s) at any time of the whole reaction course based on the ee_s value determined at a certain reaction time at a relatively lower substrate concentration. This will be helpful for terminating the reaction at a proper time to get both higher optical purity and higher yield of the remaining epoxides.     

Enantioselective epoxide hydrolase activity of a newly isolated microorganism, Sphingomonas echinoides EH-983, from seawater

A marine microorganism, Sphingomonas echinoides EH-983, which possesses epoxide hydrolase (EH) activity was isolated from seawater and characterized. The EH of S. echinoides EH-983 preferentially metabolized (R)-enantiomer when the racemic styrene oxides were supplied as substrates. The optimal pH and temperature for the enantioselective hydrolysis by whole-cells ofS. echinoides EH-983 were 7.0 and 20 °C, respectively. When kinetic resolution was conducted with a racemic mixture of styrene oxides at an initial concentration of 40 mM, enantiopure (S)-styrene oxide was obtained in 180 min with a yield of 21.3%. To our best knowledge, S. echinoides EH-983 is the first marine microorganism that is reported to have EH activity.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

定点突变改善PvEH1对邻甲基苯基缩水甘油醚的催化特性

环氧化物水解酶能够对外消旋环氧化物进行动力学拆分保留单构型的环氧化物。测定了菜豆环氧化物水解酶(Pv EH1)针对苯基缩水甘油醚及其甲基衍生物的催化特性,并基于分子对接及多序列比对分析确定7个突变位点,通过单点和组合突变对Pv EH1进行改造,以期改善Pv EH1对邻甲基苯基缩水甘油醚(1a)的催化特性。底物谱分析表明Pv EH1对1a的催化活性(157.2U/g湿细胞)和对映选择性(E=5.6)最高。单点突变结果显示E.coli/pveh1~(L105I)和E.coli/pveh1~(V106I)对1a的催化活性和对映选择性均有明显提高;L105I和V106I位组合突变菌株E.coli/pveh1~(L105I/V106I)的催化活性(493.8U/g湿细胞)是E.coli/pveh1的3.1倍,对映选择性(E=8.3)也提高至E.coli/pveh1的1.5倍。纯化后Pv EH1~(L105I/V106I)的催化活性为17.6U/mg,是Pv EH1的1.5倍,对1a的催化效率提高至Pv EH1的2.1倍。SDS-PAGE分析表明提高了蛋白质的可溶性表达量。利用E.coli/pveh1~(L105I/V106I)全细胞催化100mmol/L 1a水解动力学拆分获得手性纯(R)-1a(ee96%)的产率和时空产率分别为31.2%和5.12g/(L·h),因此,在手性纯(R)-1a的制备中,E.coli/pveh1~(L105I/V106I)是一种颇具潜力的生物催化剂。     

Biocatalytic resolution of glycidyl aryl ethers by Trichosporon loubierii: cell/substrate ratio influences the optical purity of (R)-epoxides

Glycidyl aryl ethers were resolved by using lyophilized cells of Trichosporon loubierii ECU1040 having epoxide hydrolase activity. The activity and enantioselectivity depended on the structure of the aryl group. Different cell/substrate ratios also influenced the optical purity of remaining substrate. An additional stability test of the whole-cell enzyme suggests that rapid deactivation of the epoxide hydrolase was the potential reason. (R)-Epoxides were prepared in gram amounts with optical purity of 87% - 99% ee.     

酶法拆分制备(R)-苯基缩水甘油醚及缓冲体系对产物光学纯度的影响

新筛选出一株能选择性水解外消旋苯基缩水甘油醚(GPE)的菌株Bacillus megateriumZJUZQ-001,研究了其动力学拆分苯基缩水甘油醚条件,包括时间、温度、缓冲液类型和离子强度的影响。相比其他体系,硼酸缓冲液(100 mmol/L,pH 8.2)e.e._s值可以从91.2%提高到99.5%,E值从25.0提高到46.8,底物浓度从60 mmol/L增加到90 mmol/L。     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida

Preparation of (2S, 3R)-methyl 3-phenylglycidate via enantioselective hydrolysis of racemic phenylglycidate was carried out using whole cells of Pseudomonas putida. Under optimal conditions (2S, 3R)-methyl-3-phenylglycidate could be got with ee value 99 and 48% chemical yield.     

Chemoenzymatic synthesis of (1S,2R)-1-amino-2-indanol, a key intermediate of HIV protease inhibitor, indinavir

The synthesis of (1S,2R)-1-amino-2-indanol, a key component of HIV protease inhibitor is accomplished in four steps starting from indanone efficiently and with high levels of diastereo- and enantioselectivity. The starting material is converted into 2-acetoxy-1-indanone involving Manganese (III) acetate oxidation . The 2-acetoxyketone is hydrolyzed to 2-hydroxy-1-indanone enantioselectively using Rhizopus oryzae. Selective reduction of 2-hydroxyoxime derivative, derived from the 2-hydroxyketone, gives the amino alcohol up to 98% diastereo- and enantioselectivity.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Lipase mediated resolution of γ-azidoalcohols in aqueous and organic media: Synthesis of (R)- and (S)-fluoxetine and duloxetine

A simple and efficient method for the synthesis of optically active γ-azidoalcohols is described. The lipase catalyzed kinetic resolutions of acetates of γ-azidoalcohols in aqueous as well as organic media have been studied. The enantiomerically pure γ-azidoalcohols obtained by the kinetic resolution in high enantiopurity have been utilized towards the synthesis of enantiomeric pairs of anti-depressant drugs, fluoxetine and duloxetine.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Biological control of Tipula paludosa (Diptera: Nematocera) using entomopathogenic nematodes (Steinernema spp.) and Bacillus thuringiensis subsp. israelensis

Tipula paludosa (Diptera: Nematocera) is the major insect pest in grassland in Northwest Europe and has been accidentally introduced to North America. Oviposition occurs during late August and first instars hatch from September until mid-October. Laboratory and field trials were conducted to assess the control potential of entomopathogenic nematodes (EPN) (Steinernema carpocapsae and S. feltiae) and Bacillus thuringiensis subsp. israelensis (Bti) against T. paludosa and to investigate whether synergistic effects can be exploited by simultaneous application of nematodes and Bti. Results indicate that the early instars of the insect are most susceptible to nematodes and Bti. In the field the neonates prevail when temperatures tend to drop below 10 °C. S. carpocapsae, reaching >80% control, is more effective against young stages of T. paludosa than S. feltiae (<50%), but the potential of S. carpocapsae might be limited by temperatures below 12 °C. Mortality of T. paludosa caused by Bti was not affected by temperature even at 4 °C but the lethal time increased with decreasing temperatures. Synergistic effects of Bti and EPN against T. paludosa were observed in 3 out of 10 combinations in laboratory assays but not in a field trial. The potential of S. carpocapsae was demonstrated in field trials against early instars in October reaching an efficacy of >80% with 0.5 million nematodes m⁻² at soil temperatures ranging between 3 and 18 °C. Results with Bti were strongly influenced by the larval stage and concentration. Against early instars in autumn between 74 and 83% control was achieved with 13 kg ha⁻¹ Bti of 5,700 International Toxic Units (ITUs) and 20 kg ha⁻¹ of 3,000 ITUs. Applications in spring against third and fourth instars achieved between 0 and 32% reduction. The results indicate that application of Bti and nematodes will only be successful and economically feasible during the early instars and that the success of S. carpocapsae is dependent on temperatures >12 °C. Synergistic effects between S. carpocapsae and Bti require more detailed investigations in the field to determine maximal effect.     

Production of (<Emphasis Type="Italic">R</Emphasis>)-ethyl-3,4-epoxybutyrate by newly isolated <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> containing epoxide hydrolase

Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate. Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species. Cells were cultivated under aerobic condition at 30°C using glucose as carbon source and resting cells were used as biocatalysts for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (>99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate.