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

作　　者：殷亮 王代玮 刘悦莹 刘海燕 罗光宏 YIN Liang;WANG Dai-wei;LIU Yue-ying;LIU Hai-yan;LUO Guang-hong(Gansu Microalgae Engineering and Technology Research Center/Gansu Microalgae Technology Innovation Center,Hexi university,Zhangye 734000;School of Agricultural and Ecological Engineering,Hexi University,Zhangye 734000)

机构地区：[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]河西学院农业与生态工程学院,张掖734000

出　　处：《生物技术通报》2024年第4期278-286,共9页Biotechnology Bulletin

基　　金：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)。

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V_(max)=8.237 U/mL,米氏常数K_(m)=16.369μg/mL。Mn^(2+)对其具有较强的激活作用,添加0.1 mol/L Mn^(2+)后其酶活提高了18倍,同时0.1 mol/L的Fe^(3+)、Zn^(2+)、Ca^(2+)、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。【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 acidbase stability in the pH=8.0-9.0 range.When casein was used as the substrate,the maximum reaction velocity V_(max)=8.237 U/mL,and the Michaelis constant K_(m)=16.369μg/mL.The activity increased by 18-fold with the addition of 0.1 mol/L Mn ^(2+).Also 0.1 mol/L Fe^(3+),Zn^(2+),Ca^(2+),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 

分 类 号：Q78[生物学—分子生物学]