黑曲霉酸性蛋白酶PrA在毕赤酵母中的高效表达

Efficient expression of the Aspergillus niger acidic protease PrA in Pichia pastoris

摘要:

【目的】本研究致力于获得酸性蛋白酶PrA的高产酵母菌株，以便在食品加工、饲料添加剂等领域进行应用。【方法】构建毕赤酵母表达菌株，在摇瓶水平重组表达PrA并检测其酶学性质。通过信号肽改造、基因剂量优化、共表达分子伴侣等措施逐步提高PrA产量，并利用高密度发酵进一步提高表达水平。【结果】PrA的比酶活为3 974.00 U/mg，最适反应pH为3.0，最适反应温度为45 ℃。初始菌株的PrA产量达到738.03 U/mL。通过使用MF4I信号肽将PrA产量提高至1 206.52 U/mL，增加PrA基因拷贝数导致产量提高至2 406.47 U/mL。进一步共表达分子伴侣或分子伴侣组合将PrA产量提高至4 091.27 U/mL。经过高密度发酵，发酵168 h酶活达到43 088.00 U/mL，与初始产量相比提高58.4倍。【结论】在毕赤酵母中成功实现了酸性蛋白酶PrA的高效表达，为其未来的工业化应用奠定了基础。这些结果为进一步研究和开发该酶在食品加工和饲料添加剂领域的应用提供了重要参考。

Abstract:

[Objective] To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing, feed additives, and other related industries. [Methods] We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA. Several strategies, such as signal peptide modification, gene dosage optimization, and co-expression with molecular chaperones, were employed to enhance the production of PrA. Additionally, high-density fermentation was employed to further improve the expression level. [Results] The expressed enzyme PrA showcased the specific activity of 3 974.00 U/mg, with the optimum performance at pH 3.0 and 45 ℃. The production of PrA by the parental strain was 738.03 U/mL. The modification of the MF4I signal peptide increased the production of PrA to 1 206.52 U/mL. Moreover, an increase in the copy number of prA further increased the PrA production to 2 406.47 U/mL. Additionally, co-expression with single or combined molecular chaperones increased the PrA production to 4 091.27 U/mL. After undergoing high-density fermentation, the enzyme activity reached 43 088.00 U/mL within 168 h, representing a 58.4-fold increase compared with the initial production. [Conclusion] High-level expression of PrA was achieved in P. pastoris, which laid a foundation for the future industrial applications. The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.