产低温弹性蛋白酶菌株的高效诱变及酶学特性

Highly Efficient Mutation of Bacillus brevis to Obtain Cold-active Elastase-producing Strain and Enzymatic Characterization

以实验室保藏的一株产低温弹性蛋白酶短芽孢杆菌Bacillus brevis XZE116为原始出发菌株，通过紫外线、亚硝基胍（NTG）复合诱变，经弹性蛋白平板水解圈初筛、摇瓶发酵复筛，成功筛选出一株高产突变菌株XZU21，其活力是出发菌株的1.67倍，最大产酶量160.3U/mL。10次传代实验表明遗传稳定性好。突变菌株最佳反应pH值为9.5，温度20℃，比原始菌株低5℃。该菌株所产弹性蛋白酶50℃以下及pH7.0～11.5之间稳定性好；Mg2＋对酶有明显激活作用，Pb2＋、Hg2＋几乎完全抑制酶活性。丝氨酸蛋白酶特异性抑制剂苯甲基磺酰氟（PSFM）强烈抑制酶活性，表明该突变菌株弹性蛋白酶属于丝氨酸蛋白酶。紫外线和NTG复合诱变对提高短芽孢杆菌弹性蛋白酶产量及降低最适反应温度效果显著。

The original strain Bacillus brevis XZE116 producing elastase preserved in our laboratory was treated with ultraviolet radiation and nitrosoguanidine （NTG） in combination. A high yield elastase-producing strain named as XZU21 was obtained according to hydrolysis zones and shaking flask fermentation. Its enzyme activity was increased to 160.3 U/mL, which exhibited 1.67 times higher than that of the original strain XZE116. Its high capacity for producing elastase remained stable after subculture for ten passages. The optimal pH of the enzyme from the mutant XZU21 was approximately 9.5 and optimal temperature was 20 ℃, showing 5 ℃ lower than that of the original strain. The enzyme of the mutant XZU21 was stable blow 60 ℃ and pH 7.0 to 11.5. The enzyme activity could be stimulated by Mg2＋, whereas Pb2＋ and Hg2＋ almost entirely inhibited the elastase activity. Its activity was strongly inhibited by serine-protease inhibitors like PSFM, suggesting that the enzyme is a serine protease. These results indicated that the combined mutation with ultraviolet radiation and nitrosoguanidine had a significant effect for increasing elastase yield and reducing reaction temperature.