β-甘露聚糖酶异源表达及甘露寡糖制备研究

Heterologous Expression of Beta-mannanase and Preparation of Mannan Oligosaccharides

以产β-甘露聚糖酶(MAN)的枯草芽孢杆菌HD145为目的菌株,PCR扩增MAN基因片段,克隆到p HBM905A载体并转化至毕赤酵母,重组子利用甲醇进行诱导。纯化的MAN酶解槐豆胶与瓜尔豆胶,质谱检测水解产物。SDS-PAGE结果表明,MAN基因实现了表达,摇瓶酶活性为3 200 U/m L,酶最适温度与pH分别为55℃与6.0,pH 4~7时稳定性较好,在40、50、60℃的半衰期分别为165、105、42 min,Zn^(2+)、Ag^+、Co^(2+)对酶活性存在一定的抑制。槐豆胶与瓜尔胶经MAN酶解后得到二糖至十糖的寡糖。

A mannanase （MAN） DNA fragment was amplified from the total DNA of Bacillus subtilis HD145 by PCR,and cloned to plasmid pHBM905A,then the recombinant plasmid was transformed into Pichia pastoris ,and the recombinant strain was induced by methanol. The locust bean gum and guar gum were hydrolyzed by purified MAN ,and the hydrolysate was detected by Mass. The SDS-PAGE results showed that MAN gene was expressed correctly,and the enzyme activity of flask culture was 3 200 U/mL. The optimum temperature and pH were 55 ℃ and 6.0,respectively. The pH stability was 4-7, and half-life times of 40 ℃,50 ℃ and 60 ℃ were 165 min, 105 min and 42 min,respectively. Zn^2＋,Ag^＋ and Co^2＋ exhibited a preferred inhibition on enzyme activity. The enzymatic hydrolysis products of locust bean gum and guar gum were mannan oligosaccharides of dimer to decamer.