摘要
【目的】筛选鉴定一株产酯酶用于选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,利用该菌株固定化细胞催化拆分外消旋底物。【方法】通过富集培养、罗丹明B平板初筛及复筛培养获得一株选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,通过对其形态、生理生化特征及16S r DNA序列分析,确立该菌株系统发育地位。优化了利用硅藻土-戊二醛吸附交联法对该菌体细胞固定化的条件,研究固定化细胞催化性质及操作稳定性。【结果】该菌为革兰氏阴性菌,鉴定其为甲基球状菌属(Methylopila)。固定化体系最优条件:聚乙烯亚胺0.15%(V/V),戊二醛0.2%(V/V),硅藻土6 g/L,菌体质量浓度100 g/L。与游离细胞相比,固定化细胞最适p H由8.0变为8.5,最适温度由35°C变为40°C,p H稳定性和温度稳定性都有所提高。Cu^(2+)、Mn^(2+)、Ca^(2+)能促进酶活,Zn^(2+)、Fe^(2+)抑制酶活。固定化细胞的有机溶剂耐受性较游离细胞有所提高。动力学分析细胞固定化后Km值变大,底物亲和力降低。利用固定化细胞水解(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯,底物浓度200 g/L,反应20 h,保留构型为S型,得率47.8%,对映体过量值ees为99.4%,重复使用12次后仍保留初始酶活的80%以上。【结论】开发了利用Methylopila sp.cxzy-L013固定化细胞择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的工艺,该工艺具有良好的工业应用前景。
[Objective] We screened and identified a strain capable of enantioelectively hydrolyzing rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester,followed by using immobilized cells to catalyze the racemic ester.[Methods] We used rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester as esterase induction in the medium inoculated with soils to enrich the target microorganism.The strain with high activity for enantioselective hydrolysis of rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester was identified by 16 S r RNA sequence analysis,morphological observation,physiological and biochemical properties.We optimized the immobilization conditions of the strain,studied the catalytic properties and operated stability of immobilized cells.[Results] We isolated a gram-negative bacterial strain which was identified as Methylopila.The optimal immobilization conditions were shown as follows：0.15%（V/V） of polymine,0.2%（V/V） of glutaraldehyde,6 g/L diatomite and 100 g/L cells.Compared with free cells,the optimum p H of immobilized cells changed from 8.0 to 8.5,the optimum temperature changed from 35 °C to 40 °C,the stability of p H and temperature was improved.Cu2＋,Mn2＋ and Ca2＋ can improve enzyme activity,but Zn2＋ and Fe2＋ inhibited it.The organic solvent tolerance of immobilized cells was higher than that of free cells.Affinity of immobilized cells with substrates decreased with the increased Km value.We used the immobilized cells to catalyze rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester at the concentration of 200 g/L for 20 h,the remaining substrate was（S）-ester with ees99.4% and the yield reached 47.8%.The relative activity of the immobilized cells remained 80% after 12 batches.[Conclusion] We established a process to produce（S）-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester from rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester by Methylopila sp.cxzy-L013 immobilized cells as the catalyst,which has a great potential for industrialization.
作者
张利坤
肖延铭
钱敏帆
严燕兵
李敬亚
Likun Zhang;Yanming Xiao;Minfan Qian;Yanbing Yan;Jingya Li(Zhejiang Engineering Research Center of Industrial Biocatalysis and Transformation,Changxing Pharmaceutical Co.,Ltd.,Changxing 313100,Zhejiang Province,China;College of Chemistry and Molecular Engineering,Zhengzhou University,Zhengzhou 450001,Henan Province,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2018年第9期1672-1682,共11页
Acta Microbiologica Sinica
基金
国家高技术研究发展计划(2015AA021004)
