舟山海域一株产碱性蛋白酶海洋放线菌的鉴定、选育及发酵条件的初步研究

从舟山海域潮间带海泥筛选到产碱性蛋白酶的海洋放线菌,利用Folin-酚法进行酶活测定,选取酶活最高的菌株进行鉴定,绘制系统进化树。再通过紫外线和DES诱变从而得到高产碱性蛋白酶的海洋放线菌菌株,并进行初步的发酵条件研究。结果表明:分离筛选得到2株菌株,在脱脂奶筛选平板上能产生较大的水解透明圈,通过Folin-酚法进行酶活测定,挑选出酶活较高的菌株A20(初始酶活为104.7U/mL),生理生化试验和16S rDNA试验结果显示该菌株为Streptomyces roseus。对菌株A20进行紫外线和硫酸二乙酯(DES)诱变,最终得到高产碱性蛋白酶的菌株,酶活为227.5U/mL,酶活提高117.3%,传代试验显示该菌株具有较好的遗传稳定性。单因素试验显示最适发酵温度为50°C、pH值为9.0、培养时间为72h。

BREEDING OF A MARINE ACTINOMYETES STRAIN PRODUCING ALKALINE PROTEASE

Marine actinomycetes able to produce alkaline protease were isolated from an intertidal zone in Zhoushan Islands region. Enzyme activity was determined by the Lowry method, and the strain of highest enzyme activity was studied. After ultraviolet and DES mutation, a marine actinomycete Strain A20 in high yield of alkaline protease was obtained. We found that Strain A20 was more capable of producing alkaline protease. Morphological, physiological, and biochemical characteristics, as well as 16S rDNA sequence homology show that Strain A20 is relative to Streptomyces roseus by sharing 99.8% in the sequence under phylogenetic tree. The result of mutation shows that enzyme activity increased by 117.3% from 104.7 to 277.5 U/mL. Genetic stability test showed the Strain A20 had good genetic stability. The optimal conditions for producing alkaline protease was temperature 50?C, pH 9.0, and culture period for 72h.