生物技术通报Issue(6)：181-186,6.
黑曲霉嗜热β-甘露聚糖酶在毕赤酵母中的克隆表达及其魔芋降解产物分析
Cloning,Expression of Thermostableβ-mannanase and the Preparation of Mannooligosaccharide
摘要
Abstract
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.关键词
β-甘露聚糖酶/密码子偏好性/毕赤酵母/酶学性质/低聚甘露糖Key words
β-mannanase/Codon preference/Pichia pastoris/Enzymatic properties/Mannooligosaccharide引用本文复制引用
倪玉佳,周旻昱,欧阳嘉,郑兆娟,勇强..黑曲霉嗜热β-甘露聚糖酶在毕赤酵母中的克隆表达及其魔芋降解产物分析[J].生物技术通报,2014,(6):181-186,6.基金项目
国家林业公益性行业科研专项（201404615），教育部新世纪优秀人才支持计划（NCET-11-0988），江苏省杰出青年基金（BK2012038），江苏省高校优势学科建设工程资助项目 （）
