大豆过氧化物酶在毕赤酵母中功能表达

Expression of soybean peroxidase from soybean in Pichia pastoris

摘要:

【目的】大豆过氧化物酶(SBP)作用底物广泛、比活高、热稳定性好，使其在免疫检测、工业污染废水处理领域有着广泛的应用潜力。现有的生产方法主要是从大豆壳中提取，这种方法产量低，成本高，远不能满足于工业应用要求，本研究希望实现在毕赤酵母中高效表达有功能活性的大豆过氧化物酶。【方法】将大豆过氧化物酶基因以及C末端截短20个氨基酸的基因克隆pPIC-9K载体中，并在毕赤酵母X-33中诱导表达。同时还将糖基化位点的天冬酰胺突变成为谷氨酰胺，研究糖基化位点对表达的影响。【结果】全长SBP在毕赤酵母中表达是无活性的，只有截短的SBP△20在试管发酵的表达活力达23.5 U/mL，经过糖基化位点的突变表明130、144、185、197对酶活非常重要，不能突变；211和216位点去糖基化突变对酶活有所提高。【结论】经过发酵条件的优化，在5 L的发酵罐中发酵液上清最高酶活力达510 U/mL，是目前报道的最高水平。

Abstract:

[Objective] Soybean peroxidase (SBP) will be widely used in immunoassay, and wastewater treatment and so on, due to its wide substrates, high specific activity, and good thermal stability. Nowadays, it was obtained mainly by extracted from soybean hull. However, it cannot meet the requirements of industrial applications for its low yield, high cost. In this study, SBP will be expressed in Pichia pastoris. [Methods] Both the genes of SBP and truncated C-terminal 20 amino acid SBP were cloned into pPIC-9K. These constructed expression vectors were transformed into Pichia pastoris X-33, and then be used to express SBP. Furthermore, the effects of asparagine glycosylation on SBP expression were also investigated by mutating asparagine into glutamine. [Results] Full length of SBP is inactive in Pichia pastoris. But the truncated C-terminal 20 amino acid SBP showed 23.5 U/mL. Our results indicated that glycosylation site of 144, 185, 197 have a great effect on the enzyme activity. These mutants were almost inactive; Whereas 211 and 216 deglycosylation sites had little effect on activity of SBP, can not be deglycosylation. [Conclusion] The highest activity of SBP was 510 U/mL in fermentation, which is the highest level of the reported.