根据毕赤酵母密码子偏好性优化设计合成一段来自黑曲霉BK01的嗜热β-甘露聚糖酶基因，通过构建表达载体pPICZαA-man线性化后电转化入不同的毕赤酵母宿主，获得最佳重组菌KM71-MAN，其发酵罐发酵酶活最高达2318.85 IU/mL。表达产物纯化后的分子量约为40 kD，最适反应温度为80℃，最适pH为5.0。该酶在70℃（pH5.0）保温44 h仍能保留43%的酶活力且在pH3.0-7.0范围内保温70 h（50℃）酶活力仍能保留85%以上。利用发酵罐所产重组酶酶解魔芋胶制备甘露低聚糖，产物以甘露二糖和甘露六糖为主，甘露低聚糖得率为55.6%。该重组β-甘露聚糖酶具有良好的热稳定性和pH稳定性，在魔芋制备甘露低聚糖中具有较好的应用潜能。%According to the Pichia pastoris’s codon preference, a DNA sequence encoding Aspergillus niger BK01 thermophilicβ-mannanase gene was designed and synthesized. Firstly, it was inserted into pPICZαA and resulted in recombinant expression vector pPICZαA-man. Then, pPICZαA-man was linearized and transformed into different hosts by electrotransformation. An optimal recombinant stain KM71-MAN was obtained by screening activity. Using recombinant strain KM71-MAN, recombinant mannanase was overexpressed and its activity in the culture medium reached 2 318.85 IU/mL in a 3 L fermentor. Recombinant enzyme had an apparent molecular size of about 40 kD by SDS-PAGE, and optimal activity at pH 5.0 and 80℃. It was highly thermostable, retaining 43%of enzyme activity after 44 h of exposure at 70℃and pH 5.0. Moreover, it remained over 85%activity from pH 3.0 to pH 7.0 after treating at 50℃for 70 h. Using this crude enzyme, the main hydrolysis products yielded from konjak gum were mannobiose and mannohexaose and the yield of mannooligosaccharides was 55.6%. The recombinant enzyme exhibited good thermal and pH stability, which indicated that the recombinant yeast has potential value in preparation of konjac gum mannooligosaccharides.
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