米曲霉孢子原生质体复合诱变及高活力蛋白酶菌株选育

Multiple protoplast mutagenesis of Aspergillus oryzae 3.042 spore and screening of high protease-producing mutant

以沪酿3.042米曲霉的孢子原生质体为诱变对象,经溶菌酶2%+蜗牛酶2%+纤维素酶2%,33℃水浴酶解时间5h的孢子原生质体最佳制备条件作用后,对其进行紫外线-氯化锂,NTG复合诱变,并利用酪蛋白平板初筛和固体发酵测定中性蛋白酶活力复筛,筛选了8株高产中性蛋白酶突变株群,为后续的细胞融合、基因组改组等实验提供了优良的候选文库。其中最高产菌株UN97,经37℃培养40h产酶活力为6834U/g(干基),为原菌株的1.62倍,传代培养8次,遗传性能稳定。

The spore protoplast of Aspergillus oryzoz 3.042 was used as the experimental material to screen high neutral protease-producing mutants. The spore of Aspergillus oryzae 3.042 was first subjected to the optimized protoplast preparation system： 2% lysozyme ＋ 2% snailase ＋ 2% novozyme188, incubated at 33℃ for 5 hours. Then UV-LiCI induced mutagenesis and NTG chemomorphosis on the spore protoplast were carried out. Finally, eight high protease-producing mutants were screened out by casein- degradating plate experiments and calculated protease activity. They could be used as the candidate mutant library for genome shuffling and cellular fusion. The protease activity of the best protease-producing mutant UN97 reached 6834U/g after cultivation at 37℃ for 40h, which is 1.62 times higher than that of the original strain, And the protease activity of the mutants was verified to be genetically stable by cultivating for eight consecutive generations.