产β-mannanase内生菌的诱变育种和酶学特性研究

Mutation Breeding of a β-Mannanase-producing Endophytic Bacteria and its Enzymatic Characterization

摘要:

以实验室筛选的产β-甘露聚糖酶的黄豆内生菌Bacillus subtilis HD-1（54.6 U/mL）为出发菌株，分别采用紫外线，硫酸二乙酯，紫外线-光复活和紫外线-硫酸二乙酯对其进行诱变，结果表明，以紫外线-硫酸二乙酯复合诱变效果最好，获得一株高产β-甘露聚糖酶的突变菌株Bacillus subtilis UD-20，酶活达89.5 U/mL，比出发菌株提高了63.9%，遗传性能稳定。该β-甘露聚糖酶的最适反应温度和pH分别为50 ℃和7.0，该酶在50 ℃以下，pH 5.0~8.0之间稳定性较好，属于中性酶。亚铁离子和钴离子对酶有较强的激活作用，相对酶活分别提高了13.2%和31.7%，但锰离子对酶有强烈的抑制作用，相对酶活仅有对照组的55.6%；金属离子钾、钙、钴和钡都能增强酶热稳定性，其中钴离子的增强效果最为明显，残余酶活约是对照组的1.5倍。

Abstract:

The mutagenic effects of the ultraviolet ray, diethyl sulfate's single factor and ultraviolet-diethyl sulfate compound mutagenesis on Bacillus subtilis HD-1 (54.6 U/mL) were studied. Results of enzyme activity showed that ultraviolet-diethyl sulfate compound mutagenesis was better. A mutant strain, named Bacillus subtilis UD-20 with high-level β-mannanase production was obtained after ultraviolet-diethyl sulfate compound mutagenesis from Bacillus subtilis HD-1 which was an endophytic bacteria screened from soybean seeds. The β-mannanase activity of Bacillus subtilis UD-20 increased 63.9% compared with that of the original strain Bacillus subtilis HD-1 (54.6 U/ml) and was stable during the generations. The optimal temperature for β-mannanase activity was 50 ℃ and the enzyme was stable up to 50 ℃. The optimal pH for enzyme activity was 7.0 and the enzyme demonstrated broad pH stability within a pH range of 5.0~8.0. The β-mannanase activity was activated by Fe2+ (113.2%) and Co2+ (131.7%), but strongly inhibited by Mn2+ (55.6%). The β-mannanase stability was increased by K+, Ca2+, Ba2+, among which Co2+ (150%) showed the best effect on the enzyme stability.