关键词: 蛋白酶, 细菌, 鉴定, 发酵条件优化, 中试放大

Abstract:

In order to screen and isolate a strain with high-yield neutral protease ability and study the fermentation conditions of the strain， we collected samples during the composting period of cow manure and pig manure， and 19 protease producing strains were screened and isolated on solid medium with casein as the sole carbon source. One of the strains PC2 with the best enzyme-producing effect was selected. Its hydrolysis circle D/d value is 4.25， and the enzyme activity is 10.74 U·mL^-1. Combined with the results of morphology， physiology and biochemistry， and 16S rDNA molecular biology identification， we identified it as Bacillus subtilis， and it is a Gram-positive bacterium. In the follow-up， we optimized the conditions of shake flask and pilot scale-up， which were activated with LB medium at 37 ℃ for 24 h， and fermented with casein fermentation medium at 30 ℃ for 48 h at a speed of 180 r·min^-1. In the specific parameters of the pilot scale-up， the speed of the 50 L seed tank was 180 to 240 r·min^-1， and the aeration rate was 20 to 30 L·min^-1. Add 1% wheat protein powder to the medium of the 500 L fermenter， the rotation speed was 100 to 180 r·min^-1， and the aeration rate was 10 to 25 L·min^-1. After fermentation， the viable bacteria content of PC2 was 4.458 billion·mL^-1， and the enzyme activity was 29.48 U·mL^-1， which was 2.745 times of the initial enzyme activity. The results of this study can lay a theoretical foundation for exploring microbial agents containing neutral protease strains and guide the industrial production of microbial agents.

Key words: protease, bacteria, identification, optimization of fermentation conditions, pilot scale-up