重离子诱变技术选育碱性蛋白酶高产菌株

The Selection for High-yield Alkaline Protease Producing Strain by Heavy-ion Irradiation

摘要:

从采集的土壤样品中分离筛选出一株碱性蛋白酶产生菌G-41, 经16S rRNA分子鉴定为芽孢杆菌属菌株。该菌株在发酵培养基中能产生较高产量的胞外碱性蛋白酶(1.7 × 104 U/mL)。以G-41为出发菌株, 对其进行重离子辐照诱变处理, 获得突变株G-41-68, 将该突变株再次经重离子诱变, 从大量突变株中筛选出碱性蛋白酶高产菌株15Gy-54, 其酶活力达到6.22 × 104 U/mL。与出发菌株相比较, 突变株G-41-68和15Gy-54的酶活力分别提高了1.58倍和2.65倍。对突变株15Gy-54的发酵条件进行了优化研究, 结果表明, 该菌株的碱性蛋白酶活力得到进一步提高, 达到7.18 × 104 U/mL, 其最适发酵条件为: 培养基(g/100 mL)为胰蛋白胨1、酵母膏 0.5、乳糖5、Na2HPO4·12H2O 0.4、KH2PO4 0.03、Na2CO3 0.1、MgSO4 0.0481 (4 × 10-3 mol/L)、pH 8.0, 培养温度41°C, 振荡培养时间42-48 h。实验结果表明, 重离子辐照诱变技术是一种非常有效的微生物诱变育种新技术。

Abstract:

Strain G-41 was isolated from the soil. The strain was identified as Bacillus by 16S rRNA method. Growing in fermentative medium, the strain can produce high-yield alkaline protease (1.7×104 U/mL). We delt with the original strain (G-41) by the heavy-ion irradiation and obtained the mutant G-41-68. The mutant G-41-68 was again treated by the heavy-ion irradiation. We screened a high-yield alkaline protease producing strain 15Gy-54 from many of mutants and its enzyme activity reached to 6.22 × 104 U/mL. Compared with the original strain, the enzyme activity of mutant strains G-41-68 and 15Gy-54 increased by 1.58 and 2.65 times, respectivity. The fermentation conditions of the mutant strain 15Gy-54 were optimized and its enzyme activity further increased, reaching to 7.18 × 104 U/mL. The mutant’s optimum conditions for enzyme production consisted of 1% tryptone, 0.5% yeast extract, 5% lactose, 0.4% Na2HPO4·12H2O, 0.03% KH2PO4, 0.1% Na2CO3, 4 × 10-3 mol/L MgSO4, the initial pH 8.0, shaking culture for 42-48 h, at 41°C. These results showed that the heavy-ion irradiation is an effective method for microbe mutagenesis.