一株共青土壤产碱性蛋白酶的地衣芽孢杆菌的分离纯化与鉴定

Purification and Identification of A Bacillus licheniformis Strain Producing Alkaline Protease from Gongqing Soil

从共青地区禽类养殖场的土壤中筛选出了4株地衣芽孢杆菌,其中1株地衣芽孢杆菌产碱性蛋白酶的酶活高达805.53 U/mL。通过酪蛋白培养基初筛,取能够产生水解圈的菌株进行平板划线获得单一菌株,再对菌株进行革兰氏染色和芽孢染色等形态学鉴定及生化特征鉴定筛选,最后对获得菌株进行分子生物学鉴定,16S rDNA测序结果显示4株菌株与地衣芽孢杆菌的同源性在97%以上。结合其形态学鉴定结果和生化特征鉴定结果,确定筛选出来的4株菌株均为地衣芽孢杆菌;最后通过Folin-酚试剂法测定各菌株产碱性蛋白酶粗酶酶活,最终获得的菌株中:菌株2-1-1酶活为805.53 U/mL、菌株2-1-2酶活为688.87 U/mL、菌株2-1-3酶活为593.91 U/mL、菌株3-1-1酶活为729.40 U/mL。

In this paper,four strains of Bacillus licheniformis were screened from the soil of poultry farms in Gongqingcheng,one of which produced alkaline protease enzyme activity as high as 805.53 U/mL.A single strain was obtained by primary screening of casein medium,plate scribing of strains that could produce hydrolytic circles,and then the strains were screened for morphological identification such as gram staining,bacteriophage staining and biochemical characteristics.Finally,the molecular biology of the obtained strains was identified,and the results of 16S rDNA sequencing showed that the homology between the four strains and Bacillus licheniformis was above 97%.Combined with the morphological identification results and biochemical characterization results,the four strains were identified as Bacillus licheniformis.Finally,the crude alkaline protease enzyme activity of each strain was determined by the Folin-phenol reagent method.The enzyme activity of strain 2-1-1,2-1-2,2-1-3and 3-1-1 was 805.53,688.87,593.91,and 729.40 U/mL,respectively.