茂原链霉菌XM4的TGase酶学性质分析及其高产菌株的构建

Enzymatic properties of TGase from Streptomyces mobaraensis XM4 and construction of a high-yield strain of Tgase

摘要:

【目的】系统地分析茂原链霉菌CGMCC 4.1851(菌株XM4)谷氨酰胺转氨酶(TGase)的酶学性质，随后对该菌株进行改造以构建高产菌株，实现TGase在链霉菌内高效表达并缩短发酵周期、提高TGase生产效率。【方法】通过测定发酵液pH和TGase研究菌株XM4的发酵特性，采用醇沉结合离子层析纯化菌株XM4的TGase，测定酶的适宜反应条件(pH、温度、金属离子)和酶动力学等指标，并通过结合酪蛋白交联实验评价其催化效率；然后利用基因工程技术，通过异源表达、替换核糖体结合位点(ribosome binding sites,RBS)提高改造原始菌株，测定TGase产量。【结果】菌株XM4的TGase在pH 4.0−11.0范围内具有良好的活性和稳定性，其中最适反应温度为50℃，最适pH值为10.0；在改造后的表达系统中实现了TGase在链霉菌中的高效表达，产量相较于原始菌株提升了103.3%，同时发酵时间缩短至24 h。【结论】菌株XM4的TGase具有良好的耐酸碱性和热稳定性，在食品工业特别是乳制品加工中具有广阔的应用前景。同时，改造菌株可实现TGase的高效生产，为TGase的工业化生产与应用提供了新的选择。

Abstract:

[Objective]To systematically analyze the enzymatic properties of transglutaminase (TGase) from Streptomyces mobaraensis CGMCC 4.1851(strain XM4) and subsequently develop a high-yielding strain by engineering for achieving efficient expression of TGase in Streptomyces with reduced fermentation duration and enhanced production efficiency.[Methods]The pH of the fermentation broth and TGase activity were measured to assess the fermentation characteristics of strain XM4.TGase from XM4 was purified by alcohol precipitation combined with ion-exchange chromatography.The reaction conditions (pH,temperature,metal ions) were optimized for the enzyme,and the enzymatic kinetics were tested.The catalytic efficiency was evaluated by casein cross-linking experiments.Subsequently,genetic engineering was employed to enhance the modified strain through heterologous expression and replacement of the ribosome-binding site (RBS),followed by measurement of TGase production.[Results]TGase from strain XM4 exhibited good activity and stability within the range of pH 4.0–11.0,with the highest activity at 50℃ and pH 10.0.The modification realized efficient expression of TGase in S.mobaraensis,inceasing the production by 103.3% compared with the original strain and reducing the fermentation time to 24 h.[Conclusion]TGase from strain XM4 demonstrates excellent acid-base tolerance and thermal stability,demonstrating broad application prospects in the food industry,especially dairy processing.Additionally,the engineered strain enables efficient production of TGase,providing new options for the industrial production and application of TGase.