为提高枯草芽孢杆菌工程菌产角蛋白酶的能力并降低发酵成本,对发酵培养基进行优化。首先利用单因素试验对培养基成分和培养条件进行了优化,然后设计Plackett-Burman试验筛选得到酵母浸膏、pH、温度3个显著因素,最后设计Box-Behnken中心组合试验并进行响应面分析。确定了优化后的发酵培养基为30 g/L蔗糖、40 g/L豆粕、5.72 g/L酵母浸膏、3 g/L Na2HPO4·12H2O、1.5 g/L KH2PO4、0.3 g/L MgSO4·7H2O;优化后的培养条件为温度37.69 ℃,接种量体积分数5%,pH 7.68。在此条件下,摇瓶发酵24 h角蛋白酶活性达到260 480 U/mL,较优化前提高了4.26倍。在3 L发酵罐中连续发酵26 h角蛋白酶活性达到704 400 U/mL。另外,优化后的发酵培养基原料成本降低了96%。综上,通过发酵优化有效提高了枯草芽孢杆菌产角蛋白酶的能力,极大地降低了发酵成本,为角蛋白酶的工业化生产奠定了基础。
In order to improve the ability of Bacillus subtilis engineering bacteria to produce keratinase and reduce the cost of fermentation, the fermentation medium was optimized. First, the single-factor test was used to optimize the medium composition and culture conditions. And then, the Plackett-Burman test was designed, and three significant factors, yeast extract, pH and temperature, were obtained. Finally, the Box-Behnken center combination test was designed and the response surface analysis was performed. The optimized fermentation medium was 30 g/L sucrose, 40 g/L soybean meal, 5.72 g/L yeast extract, 3 g/L Na2HPO4·12H2O, 1.5 g/L KH2PO4, 0.3 g/L MgSO4·7H2O, and the optimized culture temperature was 37.69 ℃, with the inoculation amount 5%, and pH 7.68. Under the optimized condition, the enzyme activity in flask culture reached 260 480 U/mL at 24 h, which is 4.26 times higher than before. Furthermore, the keratinase activity reached 704 400 U/mL after 26 h of batch culture in a 3 L fermenter. In addition, the cost of the fermentation medium after optimization reduced to only 4% of the origin raw materials. In summary, the optimization of fermentation effectively improves the ability of Bacillus subtilis WB600 to produce keratinase, and greatly reduces the cost of fermentation, thus lays the foundation for the industrial production of keratinase.
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