一株产碱性蛋白酶菌株的筛选鉴定及酶学特性研究

Isolation and identification of an alkaline protease producing strain and study on enzymatic properties

摘要:

[目的] 从丝茅草中筛选得到产蛋白酶菌株并研究驯化过程中微生物群落结构，以及探究该菌株的生长特性和蛋白酶的酶学特性。[方法] 通过高通量测序探究来源于丝茅草的菌株在不同培养条件下细菌种类及丰度，通过选择性培养基来筛选能够分解酪素并产生蛋白酶的菌株，通过单因素试验方法确定环境因子对菌株生长和蛋白酶活性的影响。[结果] 微生物群落结构在基础培养基和牛肉膏蛋白胨培养基中不同。通过含酪素的选择性培养基里筛选到1株产蛋白酶菌株H-16，经生理生化试验和16S rDNA鉴定知该菌株属于Escherichia marmotae，菌株H-16能产生分子量为70 kDa左右的单亚基蛋白酶。胰蛋白胨、蔗糖、30℃或35℃、pH 7分别为菌株生长的最适氮源、碳源、温度和pH。菌株H-16分泌的蛋白酶最适pH为6-8，在50℃及6%盐度以下酶活性几乎不受影响。此外，Cu(II)和Ag(I)等金属离子能够抑制蛋白酶的活性。[结论] 该菌株H-16为嗜中温菌株，能够产生碱性蛋白酶。

关键词:蛋白酶;菌株筛选;分子鉴定;酶学性质

Abstract:

[Objective] We isolated and identified a protease producing bacterium from Rhizoma Imperatae. [Methods] The species and abundances of bacteria from Rhizoma Imperatae were determined by high-throughput sequencing. The protease producing strain was screened by using selective medium containing casein. Besides, the effects of environmental factors on bacterial growth and protease activity were determined by single factor experiment. [Results] A protease producing strain H-16 was isolated from selective medium and identified as Escherichia marmotae by physiological-biochemical experiments and 16S rDNA sequence analysis. Strain H-16 could produce a protease with molecular weight of about 70 kDa. Tryptone, sucrose, 30℃ or 35℃, and pH 7 were the optimum nitrogen source, carbon source, temperature, and pH, respectively, for the growth of strain H-16. The protease produced by strain H-16 exhibited optimum activity for casein between pH 6 and 8, and it did not lose much enzyme activity under the conditions of below 50℃ and less than 6% salinity. In addition, Cu(II), Ag(I), and other metal ions could inhibit the activity of the protease. [Conclusion] Strain H-16 could be a good candidate for protease production.

Key words:protease;strain screening;molecular identification;enzymatic properties