多粘类芽孢杆菌中L-阿拉伯糖异构酶的克隆表达及固定化研究  

Cloning,Expression and Immobilization of L-Arabinose Isomerase from Paenibacillus polymyxa

作　　者：王晓倩 丁洪勇 郁桂聪 王平 李玉梅 古鹏飞 高娟 WANG Xiaoqian;DING Hongyong;YU Guicong;WANG Ping;LI Yumei;GU Pengfei;GAO Juan(School of Biological Science and Technology,University of Jinan,Jinan 250022,Shandong,China;Shandong Zhongjing Biotechnology Co.,Ltd.,Tai’an 271411,Shandong,China)

机构地区：[1]济南大学生物科学与技术学院,山东济南250022 [2]山东中京生物科技有限公司,山东泰安271411

出　　处：《济南大学学报（自然科学版）》2024年第5期624-633,共10页Journal of University of Jinan(Science and Technology)

基　　金：国家自然科学基金项目(32270093);山东省科技型中小企业创新能力提升工程项目(2021TSGC1247)。

摘　　要：为了对D-半乳糖进行异构化制备D-塔格糖,从多粘类芽孢杆菌KF-1中克隆L-阿拉伯糖异构酶基因,并在大肠杆菌BL21(DE3)细胞中表达;重组蛋白PpoLAI通过镍柱纯化,并利用壳聚糖微球进行固定化研究。结果表明:使用D-半乳糖作为底物时,PpoLAI的最佳温度和pH分别为50℃、7.0;Mn^(2+)显著激活酶活性,当Mn^(2+)的浓度为0.8 mmol/L时,PpoLAI的活性最高;以D-半乳糖为底物的PpoLAI的米氏常数和分别为161.4 mmol/L及98.84μmol/(mg·min);PpoLAI的最优固定化条件为壳聚糖的质量分数为3%,戊二醛的体积分数为3%,游离酶添加量为0.9 mg/g,吸附温度为25℃,吸附时间为4 h,PpoLAI的固定化率为80.12%,固定化的PpoLAI的热稳定性、pH稳定性与游离酶相比有显著提升。To isomerize D-galactose for preparing D-tagatose,a novel L-arabinose isomerase gene was cloned from Paenibacillus polymyxa KF-1 and expressed in Escherichia coli BL21(DE3)cells.The recombinant protein PpoLAI was purified by Ni Sepharose 6FF column,and immobilized by chitosan-glutaraldehyde crosslinking method.The results show that the optimal temperature and pH of purified PpoLAI are 50℃and 7.0,respectively,using D-galactose as substrate.The enzyme is significantly activated by Mn^(2+),and the activity of PpoLAI reaches the highest when Mn^(2+)concentration is 0.8 mmol/L.The Michaelis constant and maximal velocity of PpoLAI with D-galactose as substrate are 161.4 mmol/L and 98.84μmol/(mg·min),respectively.The optimal immobilization conditions of PpoLAI are achieved with following conditions:mass fraction of chitosan is 3%,volume fraction of glutaraldehyde is 3%,amount of free enzyme addition is 0.9 mg/g,adsorption temperature is 25℃,and adsorption time is 4 h.Under these conditions,the immobilization rate reaches 80.12%,and the thermal stability and pH stability of immobilized PpoLAI are significantly improved compared with the free enzyme.

关 键 词：D-塔格糖 L-阿拉伯糖异构酶 表达 酶活性 固定化 

分 类 号：Q814[生物学—生物工程]