茂源链霉菌谷氨酰胺转氨酶在毕赤酵母中的高效表达及性质

High-level expression and characterization of the transglutaminase from Streptomyces mobaraensis in Komagataella phaffii

摘要:

【背景】 谷氨酰胺转氨酶(TGase)在食品工业具有重要应用价值，但异源表达水平仍较低。【目的】 通过多组合策略实现茂源链霉菌(Streptomyces mobaraensis)谷氨酰胺转氨酶(SmTGase)在毕赤酵母(Komagataella phaffii)中高水平表达。【方法】 采用酶原区和成熟区共表达、共表达分子伴侣和过表达翻译起始因子的组合策略提高SmTGase在毕赤酵母中的表达水平，通过甘油-甲醇共补料策略进行5 L发酵罐高密度发酵实现SmTGase的高效生产，利用强阴离子交换柱对SmTGase进行纯化，测定其酶学性质。【结果】 重组菌株摇瓶发酵酶活力为5.67 U/mL。在5 L发酵罐中高密度发酵酶活力为80.5 U/mL，蛋白含量为7.68 g/L。纯化后该酶的最适催化条件为pH 7.0和55 ℃，在pH 5.5-8.0范围内及45 ℃以下具有良好稳定性。【结论】 本研究为谷氨酰胺转氨酶在毕赤酵母中高水平表达提供了重要参考。

Abstract:

[Background] Transglutaminases (TGases) play a significant role in the food industry, while their heterologous expression levels remain low. [Objective] This study aimed to achieve high-level expression of SmTGase from Streptomyces mobaraensis in Komagataella phaffii through a combinatorial strategy. [Methods] The combinatorial strategy involving co-expression of the propeptide and mature domains, co-expression of molecular chaperones, and overexpression of translation initiation factors was employed to enhance the expression level of SmTGase in K. phaffii. High-cell-density fermentation was carried out in a 5 L bioreactor with a glycerol-methanol co-feeding strategy for the efficient production of SmTGase. SmTGase was purified via a one-step strong anion-exchange column, and the enzymatic properties of SmTGase were characterized. [Results] The recombinant strain yielded an enzyme activity of 5.67 U/mL in a shake flask. Through the high-cell density fermentation in a 5 L bioreactor, the recombinant strain produced an enzyme activity of up to 80.5 U/mL with a protein titer of 7.68 g/L. The purified SmTGase exhibited the highest activity at pH 7.0 and 55 ℃, and it was stable within pH 5.5-8.0 and at temperatures below 45 ℃. [Conclusion] This study provides a valuable reference for the high-level expression of TGases in K. phaffii.