中性植酸酶在毕赤酵母中的表达及其分子改造

Expression and molecular modification of neutral phytase in Pichia pastoris

关键词:  毕赤酵母 中性植酸酶 克隆表达 酶学性质 分子改造

Key words:  Pichia pastoris, neutral phytase, expression?cloning,enzyme properties, molecular modification

摘要:

为了开发适用于水产领域的中性植酸酶，本研究对本实验室保藏的植酸酶基因进行克隆表达，探究重组酶的酶学性质并进行分子改造。从本实验室保藏的菌种pUC-Nphy/TOP10中克隆植酸酶基因Nphy，构建重组表达质粒PIC-Nphy并在毕赤酵母Pichia pastoris中诱导表达，研究重组酶酶学性质；为进一步提高野生酶Nphy的酶活力和热稳定性，采用Alphafold2对Nphy进行建模，根据单点与多位点组合突变相结合的方法对野生酶Nphy进行分子改造。实验结果表明，重组酶Nphy的最适反应温度为50℃，最适反应pH为6.5，为中性植酸酶；对野生酶进行分子改造，获得一个比活力比野生酶Nphy提高23.5%、耐热比Nphy提高10.69%的突变体Nphy1，其最适反应温度和pH与野生酶Nphy一致，经50 L液体发酵罐发酵获得的单位体积酶活比野生酶提高32.9%。本研究获得的中性植酸酶酶活力和热稳定性均有所提升，丰富了中性植酸酶数据库，为其应用研究提供参考。

Abstract:

The purpose of this study was to develop?a?neutral phytase suitable for the aquaculture field, the phytase gene stored in our laboratory was cloned and expressed, the enzymatic property of the recombinant enzyme were investigated and to improve the enzyme by molecular modification . The phytase gene Nphy was cloned from the strain pUC-Nphy/TOP10 stored in our laboratory, the recombinant expression plasmid PIC-Nphy was constructed and expressed in Pichia pastoris, the enzymatic properties of the recombinant enzyme were studied in detail.To further improve the enzymatic activity and thermal stability of wild enzyme Nphy，it was modeled by Alphafold2, Molecular modification of the wild enzyme Nphy was performed by combining single and multi-site mutations. The results showed that the optimum reaction temperature of recombinant enzyme Nphy was 50℃, the optimum reaction pH was 6.5, and it was neutral phytase. By molecular modification of the wild enzyme, a mutant Nphy1 with 23.5% higher specific activity and 10.69% higher heat resistance than wild enzyme Nphy was obtained.The optimum reaction temperature and pH were consistent with Nphy, The enzyme activity per unit volume of 50 L liquid fermenter was 32.9% higher than Nphy. The activity and thermal stability of neutral phytase obtained in this study were improved, which enriched the neutral phytase database and provided reference for its application research.