D-泛解酸内酯水解酶的固定化及酶学性质
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摘要
为有效提高D-泛解酸内酯水解酶的利用效率,笔者选择合适的载体对酶进行固定化,在优化固定化条件的同时研究固定化酶的最佳反应条件和酶学性质。结果表明,选择的最佳固定化载体为树脂D380,最佳固定化条件为:酶的吸附添加量为30 U(以1 g湿树脂计),吸附pH 7.0,吸附温度30℃,吸附时间5 h,戊二醛体积分数0.1%,交联时间1 h。在最优条件下得到的固定化酶的比酶活为(11.5±0.12) U/g。固定化酶的最适反应温度为37℃,最适反应pH为7.5。游离酶和固定化酶的动力学常数K_m分别为170.25和207.60 mmol/L。Ca~(2+)对酶促反应有激活作用,Mn~(2+)对该酶有较强的抑制作用。
To improve the efficiency of D-lactonohydrolase,the most suitable carrier was selected to immobilize the enzyme.The immobilization conditions were optimized.At the same time,the optimal reaction conditions and enzymatic properties of the immobilized enzyme were studied.The best immobilized carrier was resin D380 and the optimal conditions for immobilization were as follows:enzyme adsorption amount was 30 U per gram wet resin,adsorption pH was 7.0,adsorption temperature was 30 ℃,adsorption time was 5 h. At a concentration of 0.1% for glutaraldehyde and 1 h for glutaraldehyde crosslinking,the specific enzyme activity of the immobilized enzyme was(11.5±0.12) U/g under optimal conditions. The optimal reaction temperature of the immobilized D-lactonohydrolase was 37 ℃,and the optimal pH was 7.5.The kinetic constants of the free enzyme and the immobilized enzyme were 170.25 and 207.60 mmol/L,respectively.The immobilized enzyme was activated by Ca~(2+),and inhibited by Mn~(2+).
To improve the efficiency of D-lactonohydrolase,the most suitable carrier was selected to immobilize the enzyme.The immobilization conditions were optimized.At the same time,the optimal reaction conditions and enzymatic properties of the immobilized enzyme were studied.The best immobilized carrier was resin D380 and the optimal conditions for immobilization were as follows:enzyme adsorption amount was 30 U per gram wet resin,adsorption pH was 7.0,adsorption temperature was 30 ℃,adsorption time was 5 h. At a concentration of 0.1% for glutaraldehyde and 1 h for glutaraldehyde crosslinking,the specific enzyme activity of the immobilized enzyme was(11.5±0.12) U/g under optimal conditions. The optimal reaction temperature of the immobilized D-lactonohydrolase was 37 ℃,and the optimal pH was 7.5.The kinetic constants of the free enzyme and the immobilized enzyme were 170.25 and 207.60 mmol/L,respectively.The immobilized enzyme was activated by Ca~(2+),and inhibited by Mn~(2+).
引文
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[2] TANG Y X,SUN Z H,HUA L,et al.Kinetic resolution of dl-pantolactone by immobilized Fusarium moniliforme SW-902[J].Process Biochem,2002,38(4):545-549.
[3] 陈建龙,祁建城,曹仪植,等.固定化酶研究进展[J].化学与生物工程,2016,23(2):7-9.
[4] 许超群,万云宝,张小龙,等.吸附交联法固定化黑曲霉β-葡萄糖苷酶[J].中国酿造,2016,35(4):83-87.
[5] SAKAMOTO K,YAMADA H,SHIMUZU S,et al.Progress for the preparation of D-pantolactone:US5275949[P].1994-01-04.
[6] SAKAMOTO K,HONDA K,WADA K,et al.Practical resolution system for DL-pantoyl lactone using the lactonase from Fusarium oxysporum[J].J Biotechnol,2005,118(1):99-106.
[7] 汤一新.微生物酶法拆分制备D-泛解酸内酯[D].无锡:江南大学,2001.
[8] 汤一新,孙志浩,华蕾,等.D-泛解酸内酯水解酶产生菌的筛选及产酶条件研究[J].微生物学报,2002,42(1):81-87.
[9] 汤一新,孙志浩,华蕾,等.微生物酶拆分方法生产D-泛酸的手性中间体D-泛解酸内酯[J].工业微生物,2001,31(3):1-5.
[10] YU M R,TAN T W.Optical resolution of racemic pantolactone by Fusarium sp.BU-011 with high lactonohydrolase activity[J].Process Biochem,2005,40(8):2609-2614.
[11] 黎明,张建中,别松涛,等.吸附-交联法固定D-泛解酸内酯水解酶的研究[J].工业微生物,2012,42(1):28-33.
[12] XUAN M J,ZHANG J Z,YUAN H Y,et al.Optimization of process for optical resolution of DL-pantolactone using immobilized D-lactonohydrolase[J].Adv Appl Biotechnol,2017,444:459-467.
[13] 汪钊,黄美娟,高亮,等.化学酶法合成D-泛解酸内酯的研究进展[J].发酵科技通讯,2016,45(4):193-198.
[14] CHEN Y,XIN Y,YANG H,et al.Immobilization and stabilization of cholesterol oxidase on modified sepharose particles[J].Int J Biol Macromol,2013,56:6-13.
[15] ALTINKAYNAK C,TAVLASOGLU S,YZDEMIR N,et al.A new generation approach in enzyme immobilization:organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability[J].Enzyme Microb Technol,2016,93:105-112.
[16] 张楚富.生物化学原理[M].北京:高等教育出版社,2011:96-103.
[17] 华蕾,汤一新,孙志浩,等 .固定化细胞拆分DL-泛解酸内酯的初步研究[J].工业微生物,2001,31(4):5-8.