高温大曲中产耐热性蛋白酶芽孢杆菌的筛选和鉴定

Screening and identification of bacillus which product heat resistance protease from high-temperature daqu

从高温大曲样品中通过分离纯化,在牛奶琼脂平板上初筛得到产耐热性蛋白酶的菌株,然后液体发酵培养测定蛋白酶酶活挑选出产酶较强的菌株A1。在经过菌株形态特征、生化鉴定、16SrDNA分子生物学鉴定为芽孢杆菌属苏云金芽孢杆菌(Bacillus thuringiensis)。选择三个主要影响因子葡萄糖、酵母浸出粉和pH,运用响应面法优化发酵培养基,最佳产酶培养基为pH为8.4,葡萄糖添加量为1.092%,酵母浸出粉添加量为0.902%,最佳酶活力为142.81u/mL。测定蛋白酶酶作用的最适温度是61℃,且在55～70℃之间有较高的酶活力,60℃相对酶活为97.63%。

The strain product heat resistance protease was screened on the milk agar plate through the separation and purification from the high-temperature daqu samples,then the strain A1 which can produce more enzyme from further purification was selected by measuring the liquid fermentation enzyme protease.The strain A1 was identified as Bacillus thuringiensis through morphological characteristics,biochemical identification,16S rDNA sequencing and molecular biology.Three main factors which have affected the choice of glucose,yeast extract and pH,was optimized for fermentation medium with response surface method.The optimum medium for enzyme production was pH value of 8.4,glucose dosage 1.092%,yeast extract dosage of 0.902%,the best enzyme activity was 142.81u/mL.Determination of protease enzyme optimum temperature was 61℃,and it was higher enzyme activity when 55 ～ 70℃,the relative activity was 97.63% at 60℃.