高去酰胺蛋白酶产生菌的诱变选育及产酶条件优化

摘要

The original strain Bacillus licheniformis 2709 was mutagenized by means of nitrogen ion implantation at the optimal implantation dose of 1.5 × 1015 ions/cm2.As a result,a mutant strain with a deamidation capacity of 48.99%（increased by 85.57% compared with the original strain） but a relatively weak ability to cleave peptide bonds named as SDL-9 was obtained.The orthogonal array design method was used for the optimization of fermentation conditions for production of alkaline protease with high deamidation capacity by SDL-9.The optimal fermentation conditions were 2 g/100 mL corn meal as the carbon source,3 g/100 mL soybean protein as the nitrogen source,0.04 g/100 mL Fe3＋ with distinct promoting effect on enzyme production,0.9 g/100 mL glutamine that could notably induce enzyme production,initial pH 7.0,and temperature 35 ℃.Under these conditions,the deamidation capacity and hydrolysis degree of peptide bonds were 57.1% and 13.2%,respectively.%以地衣芽孢杆菌2709为出发菌株,采用氮离子注入技术,在最佳注入剂量为1.5×1015ions/cm2的条件下进行诱变处理,筛选得到一株去酰胺度高达48.99%的突变株SDL-9,去酰胺度提高了85.57%,而肽键水解度相对较弱。并对SDL-9的产酶条件进行优化,结果表明：最佳碳源为质量浓度为2.0g/100mL的玉米粉,最佳氮源为质量浓度3.0g/100mL的大豆蛋白,质量浓度为0.04g/100mL Fe3＋对产酶具有明显的促进作用,质量浓度为0.9g/100mL谷氨酰胺对产酶有明显的诱导作用,最佳起始pH值为7.0,最佳发酵温度35℃,在此条件下进行发酵产酶,测得去酰胺度为57.1%,肽键水解度为13.2%。