一种新谷氨酰胺转氨酶的发掘、纯化、酶学性质测定及重组表达

Discovery, purification, enzymatic characterization, and recombinant expression of a novel transglutaminase

摘要:

【背景】谷氨酰胺转氨酶(transglutaminase, EC 2.3.2.13)简称TG酶，能够催化谷氨酰胺残基的γ-羧酰胺基基团与赖氨酸残基的ε-氨基基团之间交联，形成异性肽键，从而改变蛋白质的构象和功能。目前TG酶已经广泛应用到食品、生物医药、纺织、皮革加工等领域。【目的】从天然茂原链霉菌中挖掘性能优异的TG酶，并将其在工业底盘菌株中重组表达以提高产量。【方法】通过发酵测定了一株茂原链霉菌(Streptomyces mobaraensis) CGMCC 4.266的TG酶生产能力。使用Capto S阳离子交换层析柱对该TG酶(TGe)进行纯化。通过测定TGe的最适pH和pH稳定性、最适温度和温度稳定性以及交联酪蛋白的能力来分析其酶学性质。将工业茂原链霉菌内源tg基因进行敲除构建Δtg，然后将tge基因在Δtg中重组表达。【结果】TGe最适pH值为5.0，在pH 4.0−10.0范围内保持较高的活性。最适反应温度为50 ℃，与商品化酶相当，在4−40 ℃范围内TGe稳定性较好，在40−65 ℃范围内TGe活性略优于商品化酶。酪蛋白交联实验表明，在50 ℃条件下，TGe交联能力明显优于商品化酶。tge在Δtg中重组表达，TGe产量显著提高可达6.3 U/mL，相较野生菌株提高162.5%，并且TGe催化活性不受影响。【结论】对天然茂原链霉菌中的TG酶进行挖掘表征，是获取性能更加优异TG酶的有效方式。异源TG酶在成熟工业菌株中成功表达为提高茂原链霉菌TG酶的产量提供了新策略。

Abstract:

[Background] Transglutaminase EC 2.3.2.13 (TGase) catalyzes cross-linking between the γ-carboxamido group of glutamine residues and the ε-amino group of lysine residues. This process leads to the formation of an isopeptide bond, which modulates the conformation and functions of proteins. TGases play a crucial role in food, pharmaceutical, textile, and leather processing industries. [Objective] To mine a high-performance TGase from natural Streptomyces mobaraensis and enhance the titer of this enzyme by recombinant expression in the industrial chassis. [Methods] The TGase production potential of S.mobaraensis (CGMCC 4.266) was evaluated by shake-flask fermentation. The TGase (TGe) was purified by Capto S cation exchange chromatography. The enzymatic properties including optimal pH, pH stability, optimal temperature, thermal stability, and cross-linking ability with casein were evaluated. We knocked out tg from industrial S. mobaraensisand obtained Δtg, in which tge was introduced and expressed. [Results] TGe showcased the optimal pH 5.0, with high activity within the range of pH 4.0–10.0. This enzyme achieved the highest activity at approximately 50 ℃, which was comparable to that of commercially available TGases. TGe exhibited good stability within 4–40 ℃, and its activity surpassed those of commercial TGases at 40–65 ℃. In addition, TGe demonstrated higher cross-linking ability with casein at 50 ℃ than commercial TGases. The recombinant expression of tge in Δtg increased the TGe titer (6.3 U/mL) by 162.5% compared with the wild-type strain, without compromising the catalytic activity of TGe. [Conclusion] High-performance TGases can be mined from natural S. mobaraensis. The heterologous expression of TGases in mature industrial strains gives a novel insight into enhancing the TGase titer of S.mobaraensis.