一株产生淀粉酶杆菌Bacillus sp. GEL-09的筛选、鉴定及发酵条件优化

Screening, identification and fermentation optimization of a newly isolated raw starch digesting amylase-producing strain Bacillus sp. GEL-09

摘要:

【背景】生淀粉酶可以水解生淀粉颗粒，在酒精发酵、白酒、黄酒和食醋的生料酿造工业中具有广阔的应用前景。【目的】从自然环境中筛选产生淀粉酶的菌，对其发酵条件及酶性能进行考察，为淀粉生料发酵过程提供优良菌种和酶资源。【方法】取木薯田土壤，经过稀释、热处理、富集培养以及木薯淀粉平板筛选培养基初筛，摇瓶复筛得到产高效降解生淀粉酶的菌株；经过菌落形态、细胞染色观察以及16S rRNA基因序列比对进行鉴定；对筛选菌株的发酵培养基和发酵条件进行优化，并对酶蛋白进行分离纯化和酶学性质分析。【结果】分离到一株具有较高生淀粉酶水解活力的菌株GEL-09，经鉴定为芽胞杆菌Bacillus sp. GEL-09；该菌在最优发酵条件下培养96 h，胞外酶活力达到430.6 U/mL，是优化前的2.8倍；酶学性质分析发现该酶为中温、中性酶，最适温度和pH为50 °C和7.0；生淀粉降解能力对比发现，该酶的生淀粉降解能力值为62.3%，显著高于细菌α-淀粉酶、生麦芽糖淀粉酶和甘薯β-淀粉酶对生淀粉的降解能力。【结论】Bacillus sp. GEL-09在生淀粉酶生产方面具有良好的开发应用前景。

Abstract:

[Background] Raw starch digesting amylase can directly degrade raw starch granules below the gelatinization temperature. Hence, it has been wildly used in the production of bioethanol, Chinese spirit, yellow rice wine and vinegar. [Objective] This study aims at isolation and identification new raw starch digesting amylase-producing strain, to provide enzyme resource that can be used in the process of fermentation with uncooked materials. [Methods] With tapioca starch as sole carbon source, raw starch digesting amylase-high producing strain was isolated from soil of cassava field. Then, it was identified by analysis of colony morphology, photomicrographs, and 16S rRNA gene sequence. Single factor experiment was used to optimize the medium component and fermentation conditions. The enzyme was purified and characterized. [Results] Strain GEL-09 was isolated and identified as a species of the genus Bacillus, designated as Bacillus sp. GEL-09. The raw starch digesting amylase production was optimized by varying culture conditions. On the optimal culture condition, the maximal enzyme production was 430.6 U/mL, which was 2.8 folds of that before optimization. The enzyme showed highest activity at 50 °C and pH 7.0. Its raw starch digestion ability value was 62.3%, which was higher than that of the α-amylase, maltogenic α-amylase or β-amylase. [Conclusion] Bacillus sp. GEL-09 has great development potential and application prospects in the production of raw starch digesting amylase.