耐碱性β-甘露聚糖酶产生菌的分离鉴定及发酵条件优化

Isolation, identification and optimization of fermentation conditions of an alkali-resistant β-Mannanase producing strain

研究旨在高产β-甘露聚糖酶饲用益生芽孢杆菌的筛选及培养条件优化。用透明圈法筛选得到一株产β-甘露聚糖酶活力较高的枯草芽孢杆菌(Bacillus subtilis)WMYB-2,通过单因素实验、Box-Behnken实验及响应面分析对该菌的产酶培养基及培养条件进行了优化,并对其酶学性质进行了初步研究。结果表明,该菌株产酶的最佳培养基及发酵条件为:魔芋微粉10.0 g/l,大豆蛋白胨10.0 g/l、CaCl_2 0.6 g/l、K_2HPO_4 0.8 g/l、MgSO_4·7H_2O 1.0 g/l,pH三角瓶,37℃、220 r/min发酵36 h,酶活力达355.75 U/ml,是初始酶活力的3.5倍。该酶的最适反应温度和pH值分别为55℃和5.5。在45～55℃内酶活力稳定,55℃保温30 min和3 h后其相对酶活力保留92%和57%;在pH值5.0～10.0内酶活力稳定,在pH值9.0和10.0的环境下处理2 h其相对酶活力保留95%和81%。Li^+和K^+对该酶具有一定的激活作用。该酶具有良好的耐碱特性及应用潜力。

The experiment was conducted to screen the highly β-Mannanase producing probiotic Bacillus sp. and optimization its culture conditions. A highly β-Mannanase producing Bacillus subtilis named WMYB-2, was screened by transparent circle method. Single-factor experiment and BoxBehnken experiment of response surface analysis were performed to obtain the optimal medium and fermentation conditions for β-Mannanase production, and the enzymatic properties were studied. The result showed that culture with an inoculation size of 2% for 36 h at 37 ℃ and 220 r/min in a 250 ml shaking flask filled with 40 ml liquid medium consisting of 10.0 g/l konjac powder, 10.0 g/l soybean peptone, CaCl20.6 g/l, K2HPO4 0.8 g/l, MgSO4·7 H2O 1.0 g/l, at initial pH 7.51, the β-Mannanase activity reached 355.75 U/ml, which was 3.5 times higher than before the optimization. The optimum temperature and pH of the enzyme were 55 ℃ and 5.5. The enzyme was stable between temperature 45~55 ℃ and pH 5.0~10.0. The enzyme activity remained 92% at 55 ℃ for a treatment of 30 minutes and 57% after a treatment of 3 hours. The enzyme activity remained 95% and 81% after a treatment of 2 hours at pH 9.0 and 10.0. The enzyme was activated by Li+ and K+. The enzyme showed excellent alkali resistance characteristics, and had the potential for application.