Ma Yuanyuan, Liu Xuewei, Yin Yanchen, Zou Chao, Wang Wanchao, Zou Shaolan, Hong Jiefang, Zhang Minhua
R&D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China; Key Laboratory for Green Chemical Technology of Ministry of Education, R&D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, China.
R&D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, China; Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
J Biotechnol. 2015 Jul 20;206:79-88. doi: 10.1016/j.jbiotec.2015.04.016. Epub 2015 Apr 30.
The β-glucosidases from Saccharomycopsis fibuligera (SfBGL1) and Trichoderma reesei (TrBGL1) were cloned and expressed in Pichia pastoris. Methanol concentration and pH significantly affected the production. The combined effects of the two factors were optimized by using the response surface method, resulting in a 137% and 84% increase in rTrBGL1 and rSfBGL1 yield compared to single-factor experiment. Structure and biochemical properties of the two enzyme were investigated and compared. They belong to glycosyl hydrolase family 3 and exhibit significant hydrolysis activity and low-level transglycosylation activity. The two enzymes show similar substrate affinity and ion-tolerance, and both of them can be activated by Cr(6+), Mn(2+) and Fe(2+). The rSfBGL1 has greater catalytic speed, higher specific activity and acid-tolerance than rTrBGL1, but rTrBGL1 is more thermostable and has higher optimal temperature than rSfBGL1. This study provides a useful and quick optimal method for recombinant enzyme production and makes a valuable comparison of biochemical properties, which opens important avenues of exploration for relationship between structure and function and further practical applications.
克隆了扣囊复膜孢酵母（Saccharomycopsis fibuligera）的β-葡萄糖苷酶（SfBGL1）和里氏木霉（Trichoderma reesei）的β-葡萄糖苷酶（TrBGL1），并在毕赤酵母中进行表达。甲醇浓度和pH对产量有显著影响。采用响应面法对这两个因素的联合效应进行了优化，与单因素实验相比，重组TrBGL1（rTrBGL1）和重组SfBGL1（rSfBGL1）的产量分别提高了137%和84%。对这两种酶的结构和生化特性进行了研究和比较。它们属于糖基水解酶家族3，具有显著的水解活性和低水平的转糖基化活性。这两种酶表现出相似的底物亲和力和离子耐受性，并且都能被Cr(6+)、Mn(2+)和Fe(2+)激活。rSfBGL1比rTrBGL1具有更高的催化速度、比活性和耐酸性，但rTrBGL1更耐热，具有比rSfBGL1更高的最适温度。本研究为重组酶的生产提供了一种有用且快速的优化方法，并对生化特性进行了有价值的比较，为结构与功能的关系及进一步的实际应用开辟了重要的探索途径。
