纳豆激酶高产菌株的选育及其酶学活性研究

Breeding of Nattokinase-Producing Strain and its Enzymatic Activity

目的:从豆制品废液沉淀池淤泥中筛选高产纳豆激酶菌株,为降血栓药物、保健食品开发利用提供优良菌株资源。方法:采用平板涂布法分离纯化菌株,利用纤维蛋白-琼脂糖测定平皿筛选高产纳豆激酶菌株,采用形态学、分子生物学方法鉴定分离得到的功能菌株,并对该菌株产生的纳豆激酶进行酶学特性研究。结果:筛选到一株高产纳豆激酶的菌株DB-07,在37℃、150 r/min摇床培养48 h,酶活达到3 097.63 IU/mL;经鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis);酶学特性研究表明,该纳豆激酶最适作用温度30℃,最适作用pH值6.5,最适酶水浴时间为60 min,另外Ca^(2+)和Cu^(2+)两种金属离子对酶学活性具有促进作用,分别由相对酶活100%提升至101.77%和107.02%。结论:筛选获得的枯草芽孢杆菌DB-07高产纳豆激酶,酶学特性优良,在降血栓药物、保健食品行业中具备很好的开发应用前景。

Objective:To screen high-yield nattokinase strains from the sludge of bean product waste liquid sedimentation tank,so as to provide excellent strain resources for the development and utilization of antithrombotic drugs and health food.Methods:The strain is isolated and purified by plate coating method,and the strain with high nattokinase production is screened by fibrin-agarose assay plate.The functional strain is identified by morphological and molecular biological methods,and the enzymatic characteristics of nattokinase produced by this strain are studied.Results:A strain DB-07 with high nattokinase production is screened,and the enzyme activity reach 3097.63 IU/mL after 48 hours of shaking culture at 37℃and 150 r/min.The strain is identified as Bacillus subtilis.The study of enzymatic characteristics shows that the optimum temperature of the nattokinase is 30℃,the optimum pH value is 6.5,and the optimum enzyme bath time is 60 min.In addition,two metal ions,Ca^(2+)and Cu^(2+),promote the enzymatic activity from 100%to 101.77%and 107.02%respectively.Conclusion:Bacillus subtilis DB-07 has high nattokinase production,excellent enzymatic characteristics,and has a good development and application prospect in antithrombotic drugs and health food industries.