重组谷氨酰胺转胺酶突变体的表达、分离纯化及其催化应用  

Purification and application of a transglutaminase mutant

作　　者：宋小平[1] 沈何放 余银 马若彤 潘李娜 SONG Xiaoping;SHENG Hefang;YU Yin;MA Ruotong;PAN Lina(Department of Pharmacy,Anhui Medical College,Hefei 230061,China;Anhui Engineering Research Center of Recombinant Protein Pharmaceutical Biotechnology,Hefei 230022,China)

机构地区：[1]安徽医学高等专科学校药学系,安徽合肥230061 [2]安徽省重组蛋白药物工程技术研究中心,安徽合肥230022

出　　处：《食品与发酵工业》2021年第23期46-51,共6页Food and Fermentation Industries

基　　金：安徽省自然科学基金项目(1808085MC86);安徽医学高等专科学校科研创新团队项目(2019zrtd01)。

摘　　要：为了研究重组谷氨酰胺转胺酶突变体(microbial transglutaminase mutant,MTG-MUT2)的分离纯化、糖基化和催化应用,将毕赤酵母(Pichia pastoris)GS115用于高密度发酵表达MTG-MUT2。首先,通过毕赤酵母高密度发酵获得粗酶液,组氨酸标记亲和层析法分离纯化得到高纯度的MTG-MUT2,酶活力为12.3 U/mL。与粗酶比较,蛋白纯化2.7倍,回收率为87.4%。接着,采取分子筛分离MTG-MUT2的3种不同成分,研究糖基化对酶活性的影响。研究发现MTG-MUT2的酶活性主要由其糖基化蛋白决定,N282位点的糖基化对酶的活性起很大作用。最后,将该酶用于L-赖氨酸(660 mmol/L)和L-谷氨酰胺(600 mmol/L)的催化反应,反应产物(谷氨酰胺二肽)的拉曼散射光谱的特征峰比野生酶高20%。结果表明MTG-MUT2具有比野生酶更高的热稳定性和催化效率。To study the purification,glycosylation and catalytic application of recombinant Microbial Transglutaminase Mutant(MTG-MUT2),the high-density fermentation of Pichia pastoris GS115 was used for the expression of the MTG-MUT2.Firstly,the crude enzyme was obtained by high density fermentation of Pichia pastoris,and the MTG-MUT2 was purified by histidine labeled affinity chromatography with the enzyme activity of 12.3 U/mL.Compared with the crude enzyme,the protein was purified 2.7 times with a recovery of 87.4%.Then,three different components of MTG-MUT2 were separated by molecular sieve,and the effect of glycosylation on enzyme activity was studied.It was found that the enzyme activity of MTG-MUT2 was mainly determined by its glycosylated protein,and the glycosylation of N282 site played a significant role in the enzyme activity.Finally,the MTG-MUT2 was applied for catalytic reaction of L-lysine(660 mmol/L)and L-glutamine(600 mmol/L).The characteristic peak of Raman scattering spectrum for the reaction product(glutamine dipeptide)catalyzed by MTG-MUT2 was 20%higher than that of the wild enzyme.These results indicate that the MTG-MUT2 has higher thermal stability and catalytic efficiency than the wild enzyme.

关 键 词：谷氨酰胺转胺酶 谷赖二肽 表达条件 催化条件 表面增强拉曼散射光谱 糖基化 

分 类 号：TQ925[轻工技术与工程—发酵工程]