1种高产凝乳酶菌株复合诱变选育及所产酶酶学性质研究

Composite Mutation Breeding of High Yield Rennet Strain and Enzymatic Characterization of Its Enzyme

以蓝色刺孢霉为出发菌株,利用复合(紫外和硫酸二乙酯)诱变处理出发菌株,筛选凝乳酶高产菌株,并对其做遗传稳定性试验,研究蓝色刺孢霉产酶的酶学性质。研究表明,先UV后DES复合诱变初筛,最可能为正突变株的有A1,A7,A15和A18;先DES后UV复合诱变初筛,最可能为正突变株的有B9,B12,B20,B22和B27。通过对上述菌株进行复筛,A18,B9和B20的凝乳酶活比出发菌株分别提高了20.80%,104.32%和37.51%,其中以先DES后UV诱变的菌株B9相对酶活最大,达到(115.95±7.20)SUm L-1,酶活增加104.32%。遗传稳定性试验表明B9具有较好的遗传稳定性。通过酶抑制剂试验,确认蓝色刺孢霉所产酶为天冬氨酸蛋白酶。采用SDS-PAGE电泳,制定标准曲线,由其方程得出该凝乳酶相对分子质量为32 400 ku。通过与小牛皱胃酶酶切位点比较,得出水解产物和小牛皱胃酶的基本相同。

A rennet producing strain was screened from Red kojic rice by our laboratory; it was identified as Quambalaria cyanescens by the Institute of Microbiology of Chinese Academy of Sciences. In this research, a composite way （UV or diethyl sulfate） was carried out to deal with the original strain, in order to get a rennet producing strain. In the meantime, the genetic stability was analyzed and the enzymatic characterization of rennet was studied. The results showed that, through UV/DES-induced mutation or DES/UV-induced mutation, the corresponding possible positive mutant strains were A1, A7, A15 and A18 or B9, B12, B20, B22 and B27. Then, secondary screenings were carried out onthose strains, the relative rennet enzyme activity of A18, B9 and B20 had increased by 20.80%, 104,32 and 37.51% respectively, especially, the relative rennet enzyme activity of B9 was at the highest level（l15.95 SUmL-1 ＋7.20 SUmL-1, which had increased by 104.32%）. Genetic stability test showed that B9 had a good genetic stability. Through the en- zyme inhibitors experiment, it was confirmed that the Quambalaria cyanescens rennet was aspartic protease. Then make the standard curve after adopting SDS-PAGE eleetrophoresis, which concluded the relative molecular mass was 32 400 ku. By comparing with the enzyme cutting site of calf rennet, it could make a conclusion that its hydrolysate was basically same with calf rennet.