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
殷亮 1王代玮 2刘悦莹 2刘海燕 1罗光宏1
作者信息
- 1. 河西学院甘肃省微藻工程技术研究中心 甘肃省微藻技术创新中心,张掖 734000
- 2. 河西学院甘肃省微藻工程技术研究中心 甘肃省微藻技术创新中心,张掖 734000;河西学院农业与生态工程学院,张掖 734000
- 折叠
摘要
[目的]对螺旋藻来源的一个蛋白酶基因进行克隆、表达,并探究重组酶酶学性质,为藻类蛋白酶的深入研究奠定基础.[方法]从钝顶螺旋藻(Spirulina platensis)基因组扩增获得了蛋白酶基因SpP1,进而构建 pET28a-SpP1 重组质粒,将其转入Escherichia coli BL21(DE3)实现了异源表达,利用镍柱分离纯化重组蛋白酶并研究其酶学性质.[结果]螺旋藻蛋白酶SpP1 属于丝氨酸蛋白酶家族成员,分子量为 47.04 kD,最适温度和pH值分别为 50℃和 8.0.其热稳定性较差,在pH=8.0-9.0 的范围内具有良好的酸碱稳定性.以酪蛋白为底物时,最大反应速度 Vmax=8.237 U/mL,米氏常数Km=16.369 μg/mL.Mn2+对其具有较强的激活作用,添加 0.1 mol/L Mn2+后其酶活提高了 18 倍,同时 0.1 mol/L 的Fe3+、Zn2+、Ca2+、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)等对酶活也具有明显的促进作用.[结论]螺旋藻来源的蛋白酶SpP1 具有丝氨酸蛋白酶家族成员的典型结构和性质特征,具有较好的酸碱稳定性,添加金属锰离子可有效提升其催化活性.
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
基金项目
甘肃省重点研发计划(21YF5FA129)
甘肃省重点研发计划(22YF7FG188)
河西学院科研启动金项目(KYQD2020018)
甘肃省高等学校产业支撑计划(2020C-25)
甘肃省自然科学基金(22JR11RG222)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据