产碱性蛋白酶菌株的筛选、分子鉴定及其酶学性质的初步研究

Screening,molecular classification of alkaline protease-producing strains and enzyme characteristics

从盐碱地土壤中筛选到5株产碱性蛋白酶的细菌,分别命名为TCCC11001、TCCC11004、TCCC11013、TCCC11024、TC-CC11029。将其革兰氏染色、16S rRNA鉴定为Bacillus subtilis、Bacillus alcalophilus、Bacillus pumilus、Bacillus subtilis、Bacillus licheni-formis。将菌株接入发酵培养基中,37℃、180r/min摇床培养48h,发酵液进行不同处理后Folin法测定酶活。结果发现:TCCC11001、TCCC11029的最适作用温度为40℃,TCCC11004、TCCC11013、TCCC11024的最适作用温度为50℃;TCCC11001、TCCC11004、TC-CC11013、TCCC11024、TCCC11029的最适作用pH值分别为10.0、11.0、10.0、9.0、11.0;TCCC11004、TCCC11013于40℃保温2h,残留酶活为最高酶活70%;TCCC11001、TCCC11024、TCCC11029的残留酶活<50%。DFP、PMSF完全抑制酶的活性,表明5种蛋白酶是典型的丝氨酸蛋白酶。

5 strains which could produce alkaline protease were isolated from soil and named TCCC11001, TCCC11004, TCCC11013, TCCC11024 and TCCC11029. They were regarded as Bacillus subtilis, Bacillus alcalophilus, Bacillus pumilus, Bacillus subtilis, Bacillus licheniformis alter determination of 16s rRNA and Gram staining. Enzyme activities were determined after incubating these strains at 37℃, 180r/min for 48h. The results showed the optimal temperature of TCCC11001 and TCCC11029 was 40℃, and for the others, the temperature was 50℃; the optimal pH of these five strains were 10.0, 11.0, 10.0, 9.0 and 11.0 respectively. Residual enzyme activities of TCCC11004 and TCCC11013 were as much as 70% compared to the maximum activity when incubated at 40℃ for 2h, and residual enzyme activities of TCCC11001, TCCC11024, TCCC11029 were less than 50%. The enzyme activity could be completely inhibited by DFP and PMSF, and it showed that these five proteases were typical serine protease.