一株地衣芽胞杆菌产碱性蛋白酶条件优化

Optimization of alkaline protease production by a strain of Bacillus licheniformis

摘要:

【背景】碱性蛋白酶是众多芽胞杆菌的发酵产物，是工业上极其重要的一类酶。【目的】利用酪素培养基从环境样品中筛选出一株产碱性蛋白酶的菌株，对传代次数、发酵的碳源、氮源、金属离子、磷酸盐、初始pH、接种量和温度进行优化，提高其产碱性蛋白酶的能力并降低发酵成本。【方法】采用革兰氏染色法、扫描电镜、生理生化试验、16S rRNA基因序列对分离的菌株进行鉴定；采用单因素、Plackett-Burman、最陡爬坡和响应面试验优化碱性蛋白酶的发酵条件，使用Minitab对试验数据进行分析。【结果】经鉴定分离菌株为地衣芽胞杆菌，命名为Bacillus licheniformis NWMCC0046。优化后的发酵培养基组成为(g/L)：豆粕50.00，葡萄糖10.00，酵母浸膏13.46，CaCl₂ 0.50，Na₂HPO₄·12H₂O 4.00，KH₂PO₄ 0.30；优化后的培养条件为：pH 7.5，34.81℃，接种量4.13%。在此条件下，摇瓶发酵48 h时碱性蛋白酶活性达到165.41 U/mL，较优化前提高了8.04倍。【结论】通过发酵培养基组成和条件优化有效提高了地衣芽胞杆菌产碱性蛋白酶的能力，为碱性蛋白酶的后续研究提供了参考。

关键词:地衣芽胞杆菌;碱性蛋白酶;Plackett-Burman设计;最陡爬坡试验;响应面法

Abstract:

[Background] Alkaline protease, a fermentation product of many Bacillus species, is an important enzyme in industry. [Objective] An alkaline protease-producing strain was isolated by casein medium from an environmental sample. The passage number, carbon source, nitrogen source, metal ions, phosphate, initial pH, inoculum amount and temperature of the fermentation were optimized to increase the alkaline protease production of the strain and to reduce the cost of fermentation. [Methods] The isolates were identified via Gram staining, scanning electron microscopy, physiological and biochemical tests, and 16S rRNA sequence analysis. The fermentation conditions were optimized by single factor test, Plackett-Burman experiment, the steepest ascent method, and response surface methodology. The experimental data were analyzed in Minitab. [Results] The strain was identified as B. licheniformis and named B. licheniformis NWMCC0046. The optimized formula of the fermentation medium was determined as soybean meal 50.00 g/L, glucose 10.00 g/L, yeast extract 13.46 g/L, CaCl₂ 0.50 g/L, Na₂HPO₄·12H₂O 4.00 g/L, and KH₂PO₄ 0.30 g/L. The fermentation conditions were optimized as pH 7.5, 34.81 ℃ and the inoculum amount of 4.13%. Under these conditions, the alkaline protease activity reached 165.41 U/mL in 48 h of shake flask fermentation, which was 8.04 times higher than that before optimization. [Conclusion] The optimization of the medium formula and fermentation conditions improved the ability of B. licheniformis to produce alkaline protease, providing a reference for subsequent research on alkaline protease.

Key words:Bacillus licheniformis;alkaline protease;Plackett-Burman design;the steepest ascent method;response surface methodology