一株海洋中性蛋白酶高产菌S-3685的鉴定及产酶条件

Identification of a Marine Bacterium S-3685 with High Neutral Protease and Optimization of Its Fermentation Conditions

关键词:  海洋微生物  中性蛋白酶  芽孢杆菌属  鉴定  培养条件优化

Key words:  Marine microorganism  Neutral protease  Bacillus sp.  Identification  Culture optimization

摘要:

通过生理生化、分子生物学、单因子优化等方法研究了来自南海海域一株产中性蛋白酶菌株S-3685，并对其在250 ml摇瓶中的培养条件进行了优化。结果显示，该菌株初步确定为芽孢杆菌属(Bacillus)，发酵培养的最佳碳、氮源分别为葡萄糖10 g/L和豆面10 g/L，牛肉膏5 g/L，酵母膏5 g/L。无机盐MgSO4·7H2O、Na2CO3、KH2PO4最佳浓度分别为0.2 g/L、2.0 g/L、1.0 g/L；菌株在培养基起始pH=7.0、4%接种量、15 ml/250 ml (v/v)装液量和30℃的条件下，发酵72 h获得较高的酶产量。在最佳培养条件下产酶量为4250 U/ml，是优化前的5倍。

Abstract:

The S-3685 strain, a marine bacterium producing neutral protease, was screened from South China Sea. It was identified as a Bacillus sp. based on the morphology, the biochemical characteristics, and the 16S rDNA sequencing results. This strain was gram-positive, and the spore located in the middle had ellipse or columnar shapes. The bacterial colony was round, protuberance, and milk white on the agar culture-medium. The surface of the bacterial colony was smooth and moist (30℃, 24 h). Over time the colony slightly shriveled bumps in the middle. The length was 2.4−3.2 microns. The strain grew under the pH 5.0−11.0, and could grow normally at the temperature 4−40℃. We then explored the fermentation conditions using a 250 ml shake flask. The optimum sources of carbon and nitrogen were glucose (10 g/L) and pulse flour (10 g/L) respectively, plus the beef extract (5 g/L) and the yeast extract (5 g/L). The optimum concentrations of MgSO4·7H2O, Na2CO3 and KH2PO4 were 0.2 g/L, 2.0 g/L and 1.0 g/L respectively. The optimum initial pH of the culture medium for the protease production was 7.0. The optimized culture conditions were: inoculums at 4%, broth content at 15 ml/250ml(v/v), temperature at 30℃, and culture for 72 h. After the optimization, the productivity of protease was increased by 5 times (from 850 to 4250 U/ml).