环氧树脂固定化环状芽孢杆菌糖氨基转移酶的研究  

Using epoxy resins to immobilize sugar aminotransferase from Bacillus circulans

作　　者：厉刚刚 阿合丽马·阿力木别克 冯雁[1] 李谦[2] 崔莉[1] LI Ganggang;AHELIMA·Alimubieke;FENG Yan;LI Qian;CUI Li(State Key Laboratory of Microbial Metabolism/College of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;College of Life Sciences and Technology,China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]上海交通大学生命科学技术学院/微生物代谢国家重点实验室,上海200240 [2]中国药科大学生命科学与技术学院,南京210009

出　　处：《扬州大学学报（农业与生命科学版）》2020年第2期9-15,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家重点研发计划项目(2018YFA0900702);国家自然科学基金资助项目(31770098、21977067、31620103901);政府间国际科技创新合作重点项目(2017YFE010330)。

摘　　要：使用环氧树脂MC-150EP对来自环状芽孢杆菌的糖氨基转移酶BtrR进行固定化以制备光学纯β-井冈霉烯胺,在单因素试验的基础上,采用响应面试验进一步优化固定化工艺,得到最佳固定化条件:载体与蛋白质投放比例为21, pH值8.2,温度17℃,盐离子浓度460 mmol·L^-1,固定化时间15 h,辅酶浓度0.2 mmol·L^-1,由此制得的固定化酶比活力为20.6 U·g^-1(湿载体),酶活回收率为66.5%。酶学性质研究表明,固定化酶的立体选择性与游离酶相同,最适反应温度和pH值分别为39℃和8.5,热稳定性和酸碱稳定性与游离酶相比有较大提高;同时,固定化酶具有良好的操作稳定性和储存稳定性,在连续使用9个批次后,固定化酶保留初始酶活的50.5%;4℃下连续放置20 d后,固定化酶的比活力为初始酶活的81.6%。The immobilization of sugar aminotransferase BtrR from Bacillus circulans was performed by using epoxy resin MC-150 EP for the production of optical pure β-valienamine. After the single-factor experiments, the immobilization process was further optimized by response surface methodology and the final immobilization conditions were as follows: the mass ratio of carrier to protein 21, pH 8.2, temperature 17 ℃, salt ion concentration 460 mmol·L^-1, PLP concentration 0.2 mmol·L^-1, and immobilization time 15 h. In this condition, the specific activity of immobilized enzyme was determined as 20.6 U·g^-1(wet support) with the activity recovery 66.5%. The characterization of enzymatic properties showed that immobilized enzyme kept the same high stereoselectivity with free enzyme. The optimal reaction temperature and pH of immobilized enzyme were 39 ℃ and 8.5, respectively, and immobilized enzyme also exhibited improved thermal stability and pH stability compared with free enzyme. Meanwhile, immobilized enzyme behaved good operational stability and storage stability. After 9 consecutive batches of use, immobilized enzyme remained 50.5% of the original activity, and also remained 81.6% of the original activity after storage at 4 ℃ for 20 days.

关 键 词：环氧树脂 糖氨基转移酶 固定化 β-井冈霉烯胺 响应面法 

分 类 号：Q814[生物学—生物工程]