复合诱变原生质体选育耐热碱性蛋白酶高产菌

THE STUDY OF SELECTION FOR HIGH-YIELD THERMOSTABLE ALKALINE PROTEASE PRODUCING STRAIN AFTER COMPLEX MUTAGEMISIS OF PROTOPLASTS

以地衣芽孢杆菌(Bacillus licheniformis)53号为原始菌株,在原生质体形成和再生的最佳条件下制备原生质体,对原生质体进行复合诱变,对大量再生突变株进行筛选,最终获得了高产、稳定、耐热的碱性蛋白酶产生菌53-G38-6,产酶活力由1104U/ml提高到22080U/ml。适宜的发酵条件:培养基(％)胰蛋白胨1,酵母膏0.5,玉米粉5,Na_2HP0_4·12H_20 0.4,KH_2P0_4 0.03,Na_2CO_3 0.1,自然pH。42℃旋转培养44～48h,得到的蛋白酶热稳定性强,60℃处理1h剩余酶活55％。酶反应最适条件:62℃,pH10.0,在pH9～10.5范围内稳定。

Under the optimum condition of formation and regeneration, protoplasts of primitive strain Bacillus licheniformis 53-As were prepared. After many mutagenetic treatment of protoplasts of strain 53 and selection from a lot of mutants, a mutants 53-G38-6 producing high-output, stable and thermo-stable alkaline protease was obtained. The enzyme activity was increased to 22080 U / ml from 1104 U / ml. The medium for fermentation consisted of 1% tryptone, 0.5% yeast extract, 5% sweet potato meal, 0.4% Na2HPO4 · 12H2O, 0.03% KH2PO4, 0.1% Na2CO3. The strain produced maximux alkaline protease activity after growth at 42℃ for 44-48 h on a rotary shaker. The alkaline protease produced by this strain was thermostable, it even retained 55% activity after incubation at 60℃ for 1 h. The optimum temperature and pH for the protease activity production are 61℃ , pH 10.0 and stable at pH 9 -10.5.