约氏黄杆菌HSL13中新型α-淀粉酶的表达及酶学性质初探  

Expression of novalα-amylase and study of enzymatic properties from Flavobacterium johnsoniae HSL13

作　　者：丁梦瑶 谢会芳 杨江科 DING Meng-yao;XIE Hui-fang;YANG Jiang-ke(Department of Biological and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《生物技术》2020年第6期525-530,524,共7页Biotechnology

摘　　要：[目的]筛选并获得产α-淀粉酶的菌株,并初步研究其酶学性质。[方法]从红树林土壤样品中筛选到一株产α-淀粉酶的菌株约氏黄杆菌HSL13,克隆了其α-淀粉酶基因Fjamy2,构建了p PICZαA-Fjamy2重组表达载体,并在毕赤酵母中表达,然后对该淀粉酶的底物特异性、酶学性质和酶学动力学参数进行了测定。[结果]淀粉酶基因Fjamy2的最适温度为40℃,在10℃~30℃内比较稳定;最适p H为5.0,在pH 5.0~11.0内较稳定;其对多种淀粉底物以及糖原、糊精等均有良好的水解作用;对其动力学进行测定得知Vmax为0.0841μmol/min,Km为3.35 mg/mL,Kcat为45.35 s^(-1)。[结论]新型淀粉酶Fjamy2在中低温酸性条件下具有优良的活性,最适条件下比酶活为81 U/mg,成功地实现了在毕赤酵母中的异源表达。[Objective]To screen and obtain theα-amylase-producing strains and preliminarily study its enzymatic properties.[Method]An amylase-producing strain Flavobacterium johnsoniae HSL13 was screened from mangrove soil samples,itsα-amylase gene Fjamy2 was cloned and the p PICZαA-Fjamy2 recombinant expression vector was constructed and expressed in Pichia pastoris X-33,then the substrate specificity,enzymatic properties and enzymatic kinetic parameters of the amylase were determined.[Result]The optimum temperature of the amylase gene Fjamy2 was 40℃,which was relatively stable within 10℃-30℃;and the optimum p H was 5.0,which was relatively stable within p H 5.0-11.0.It has a good hydrolysis effect on a variety of starch substrates,glycogen,dextrin,etc.The kinetics were determined to find that Vmax was 0.0841μmol/min,Km was 3.35 mg/m L,Kcat was 45.35^(s-1).[Conclusion]The new amylase Fjamy2 has excellent activity under the acidic conditions of medium and low temperature,and the specific enzyme activity was 81 U/mg under the optimal conditions,which successfully realized the heterologous expression in Pichia pastoris.

关 键 词：约氏黄杆菌 淀粉酶 克隆表达 酶学性质 底物特异性 酶促动力学 

分 类 号：Q814.4[生物学—生物工程]