生物技术通报2024，Vol.40Issue(4)：278-286,9.DOI:10.13560/j.cnki.biotech.bull.1985.2023-0813
蛋白酶SpP1基因克隆、表达及酶学性质的表征
Cloning and Expression of Protease SpP1 Gene and Characterization of Enzymatic Properties
摘要
Abstract
[Objective]The protease gene of Spirulina was cloned and expressed,and the enzymatic properties of recombinant enzyme were investigated,which lays a foundation for the further research of algal protease.[Method]The protease gene SpP1 was amplified from the genome of Spirulina platensis,and the pET28a-SpP1 recombinant plasmid was constructed,which was transfected into Escherichia coli BL21(DE3)to achieve heterologous expression,and the recombinant protease was isolated and purified by using a nickel column to study its enzymatic properties.[Result]The protease SpP1 was a member of the serine protease family,with a molecular weight of 47.04 kD,and its optimal temperature and pH values were 50℃and 8.0,respectively;its thermal stability was poor,and it had a good acid-base stability in the pH=8.0-9.0 range.When casein was used as the substrate,the maximum reaction velocity Vmax=8.237 U/mL,and the Michaelis constant Km=16.369 μg/mL.The activity increased by 18-fold with the addition of 0.1 mol/L Mn 2+.Also 0.1 mol/L Fe3+,Zn2+,Ca2+,ethylenediaminetetraacetic acid(EDTA)had a significant promotion effect on the enzyme activity.[Conclusion]The protease SpP1 has the typical structure and property characteristics of serine protease family members,with good acid-base stability,and the addition of manganese ions can effectively enhance its catalytic activity.关键词
钝顶螺旋藻/蛋白酶/异源表达/酶学性质Key words
Spirulina platensis/protease/heterologous expression/enzymatic property引用本文复制引用
殷亮,王代玮,刘悦莹,刘海燕,罗光宏..蛋白酶SpP1基因克隆、表达及酶学性质的表征[J].生物技术通报,2024,40(4):278-286,9.基金项目
甘肃省重点研发计划(21YF5FA129,22YF7FG188),河西学院科研启动金(KYQD2020018),甘肃省高等学校产业支撑计划项目(2020C-25),甘肃省自然科学基金(22JR11RG222) （21YF5FA129,22YF7FG188）
