胱硫醚-β-合成酶的高效表达及酶学性质研究  

Efficient expression and enzymatic properties of cystathionine-β-synthase

作　　者：陈武 刘春华 何鹏辉[2] 蔡冬波 陈守文[2] 王冬 CHEN Wu;LIU Chun-hua;HE Peng-hui;CAI Dong-bo;CHEN Shou-wen;WANG Dong(Guangdong Zhaoqing Xinghu Biotechnology Co.,Ltd.,Zhaoqing 526040,Guangdong,China;School of Life Sciences,Hubei University,Wuhan 430062,China;Zhuhai Yinzhi Biotechnology Co.,Ltd.,Zhuhai 519085,Guangdong,China;Chongqing Academy of Agricultural Sciences,Chongqing 400050,China)

机构地区：[1]广东肇庆星湖生物科技股份有限公司,广东肇庆526040 [2]湖北大学生命科学学院,武汉430062 [3]珠海茵智生物技术有限公司,广东珠海519085 [4]重庆市农业科学院,重庆400050

出　　处：《湖北农业科学》2024年第11期203-207,共5页Hubei Agricultural Sciences

摘　　要：以酿酒酵母的cbs基因序列为模板,依据大肠杆菌Escherichia coli BL21(DE3)的密码子偏好性进行密码子优化,以pET28a(+)为骨架构建表达质粒pET28a-cbs,并利用Escherichia coli BL21(DE3)进行胱硫醚-β-合成酶(CBS)的表达,同时优化诱导表达条件并分析CBS的酶学性质。结果表明,在诱导温度为16℃、IPTG浓度为0.1 mmol/L、诱导时间为18 h时,CBS浓度最高,为266.67 mg/L。CBS发挥催化作用过程中也受到多种环境因素的影响,在40~55℃时,CBS相对酶活性迅速下降,CBS不适宜长期保存于温度大于40℃的环境中;CBS适宜长期保存于pH为6.5~8.5的缓冲溶液中,不宜保存在过酸或过碱的缓冲溶液中;CBS在温度40℃、pH 8.0条件下,酶活性较高。This study used the cbs gene sequence of brewing yeast as a template and optimized codons based on the codon preference of Escherichia coli BL21(DE3).The expression plasmid pET28a-cbs was constructed using pET28a(+)as the skeleton,and the cystathionine β-synthase(CBS)was expressed using Escherichia coli BL21(DE3).The inducing expression conditions were optimized and the enzymatic properties of CBS were analyzed.The results showed that the highest concentration of CBS was 266.67 mg/L at an induction temperature of 16 ℃,IPTG concentration of 0.1 mmol/L,and induction time of 18 hours.During the catalytic process of CBS,it was also influenced by various environmental factors.At 40~50 ℃,the relative enzyme activity of CBS rapidly decreased,and CBS was not suitable for long-term storage in environments with temperatures above 40 ℃;CBS was suitable for long-term storage in buffer solutions with pH ranging from 6.5 to 8.5,and should not be stored in buffer solutions that were too acidic or too alkaline;CBS exhibited high enzyme activity at a temperature of 40 ℃ and pH 8.0.

关 键 词：胱硫醚-β-合成酶(CBS) 高效表达 酶学性质 诱导表达 

分 类 号：Q819[生物学—生物工程]