重组毕赤酵母生产β-葡萄糖苷酶发酵条件优化及固定化研究
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(江南大学 食品科学与技术国家重点实验室/工业生物技术教育部重点实验室/ 教育部食品安全国际合作联合实验室, 江苏 无锡 214122)

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Optimization of Fermentation Conditions and Immobilization of β-Glucosidase Produced by Recombinant Pichia pastoris
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(State Key Laboratory of Food Science and Technology/Key Laboratory of Industrial Biotechnology, Ministry of Education/International Joint Laboratory on Food Safety, Ministry of Education, Jiangnan University, Wuxi 214122, China)

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    摘要:

    为了实现绿色木霉菌Trichoderma viride来源的β-葡萄糖苷酶在重组毕赤酵母中的高效表达,对重组菌P.pastoris KM71/pPIC9K-bgl1/pPICZ A-pdi进行3.6L罐发酵培养条件优化。结果表明,当诱导温度28℃,初始诱导菌体浓度50g/L,诱导阶段甲醇体积分数1.0%时,酶活力最高,能达到1452U/mL。同时以壳聚糖为载体、戊二醛为交联剂,采用吸附交联法对β-葡萄糖苷酶进行固定化。结果表明,当壳聚糖质量浓度0.03g/mL,戊二醛质量浓度0.008g/mL,游离酶添加量400U/g(1g壳聚糖微球加酶量为400U),固定化吸附时间20h时,固定化酶酶活回收率最高,达到65.4%。以800g/L葡萄糖为底物,优化的转化条件下连续转化6次,低聚龙胆糖产率仍达到15.2%,显示出该固定化酶具有较好的持续利用性及较高的低聚龙胆糖生产能力。

    Abstract:

    In order to achieve high expression of Trichoderma viride β-glucosidase in recombinant Pichia pastoris,the fermentation conditions of the P. pastoris KM71/pPIC9K-bgl1/pPICZ A-pdi were optimized in a 3.6L bioreactor. When the optimal conditions were the induction temperature 28℃, initial cell density 50g/L, and methanol volume fraction 1.0%, fermentation of P. pastoris KM71/ pPIC9K-bgl1/pPICZ A-pdi yielded an enzyme activity of 1452U/mL. β-glucosidase from the recombinant strain was immobilized on chitosan cross-linked with glutaraldehyde. When the optimal conditions of immobilization were 0.03g/mL chitosan, 0.008g/mL glutaraldehyde, 400U/g chitosan beads, and adsorption time 20h, the immobilization efficiency reached up to 65.4%. The yield ratio was still up to 15.2% after 6 times of continuous conversion and it showed a good operational stability and a high productivity of gentiooligosaccharides.

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汪飞,吴敬,陈晟.重组毕赤酵母生产β-葡萄糖苷酶发酵条件优化及固定化研究[J].食品科学技术学报,2019,37(3):41-47.

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  • 收稿日期:2019-04-29
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  • 在线发布日期: 2019-06-17
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