Abstract:

A Gram-negative bacterium was isolated from the soil and sludge samples, which had high activity for enantioselective hydrolysis of (R, S)-metalaxyl. The strain was identified as Albibacters by 16S rDNA sequence analysis. It was named Albibacters sp. zjut528. At 37℃, 15 g·L^-1 of the wet cells (81% water content) catalyzed the hydrolysis of (R, S)-metalaxyl at the concentration of 50 g·L^-1 for 28h, the yield reached 47.1% and the main product was R-metalaxyl acid with ee_p > 99.9%. In order to isolate its intracellular esterase, the cells were disrupted by ultrasonic. After centrifugation, the supernatant was treated by ammonium sulfate precipitation, followed by ion-exchange chromatography and gel filtration chromatography. A SDS-PAGE electrophoresis-pure esterase protein was obtained with a molecular mass of about 40×10³. Its N-terminal amino acid sequence was determined as NH₂-Ala-Ala -Lys-Ala-Pro-Leu-Arg-Leu-Lys-Glu. Its optimum catalytic temperature and pH were 40℃ and 9.0, respectively. The enzyme was stable at 20-40℃ and pH 5.0-10.0. 0.2 mmol·L^-1 of Fe²⁺, Mn²⁺, Zn²⁺ can improve its activity, but Fe³⁺ inhibited it. Its activity was highly improved when some organic solvents, such as acetonitrile, isopropanol, acetone, isopropyl ether, cyclohexane and n-hexane, were added at the concentration of 20% (vol). The Michaelis-Menten kinetics parameters for the enzyme were V_m=0.18 mmol·L^-1·min^-1, K_m=2.29 mmol·L^-1 and K_cat=0.85 min^-1. A new process for producing R-matalaxyl from R,S-matalaxyl was established by using Albibacters sp. zjut528 cells as the catalyst. First, the cells catalyzed enantioeletive hydrolysis of (R,S)-matalaxyl to produce R-metalaxyl. After reaction, the R-metalaxyl acid and the unreacted S-matalaxyl were separated by extraction. R-Metalaxy acid was esterified with methanol to produce R-metalaxyl. Followed by some separation steps, the final product of R-metalaxy was obtained. Its purity was 96.2% and ee value was 99.3%. The unreacted S-metalaxyl can be reused by racemization. After heated for 6 h at 60℃, its ee value reduced from 99.4% to 4.2%.

Key words: biocatalysis, bioprocess, hydrolysis, R,S-metalaxyl, R-metalaxyl, strain screening and identification, esterase, Albibacters

摘要：

从土壤和污泥样品中分离得到一株能不对称水解甲霜灵的革兰阴性菌。通过分子生物学鉴定，命名该菌株为Albibacters sp.zjut528。当底物浓度为50 g·L^-1，湿菌体（含水量81%）：底物=0.3：1（质量比），反应28 h，产物得率为47.1%，主产物为R-甲霜灵酸，对映体过量值ee_p > 99.9%。将菌株细胞破碎得到的粗酶液分离纯化得到一个SDS-PAGE电泳纯的酯酶，分子量约为40×10³，酶的N端10个氨基酸序列为NH₂-Ala-Ala-Lys-Ala-Pro-Leu-Arg-Leu-Lys-Glu。该酶最适温度为40℃，20~40℃稳定性较好。最适pH为9.0，在pH 5.0~10.0范围内稳定。Fe²⁺、Mn²⁺、Zn²⁺对酶活有促进作用，Fe³⁺对酶活有一定的抑制作用。在含有20%的乙腈、异丙醇、丙酮、二异丙醚、环己烷、正己烷的水溶液中，酶活均比在纯水相中有明显提高。该酶反应动力学符合米氏方程，V_m为0.18 mmol·L^-1·min^-1，K_m为2.29 mmol·L^-1，K_cat为0.85 min^-1。建立了一条利用Albibacters sp.zjut528细胞作催化剂拆分甲霜灵制备R-甲霜灵的工艺路线，终产品R-甲霜灵的纯度是96.2%，ee值为99.3%。

关键词: 生物催化, 生物过程, 水解, R,S-甲霜灵, R-甲霜灵, 菌种筛选和鉴定, 酯酶, Albibacters