蛋白酶SpP1基因克隆、表达及酶学性质的表征OA北大核心CSTPCD

[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.