分子改造提高谷氨酰胺转氨酶的催化活性

Improved catalytic activity of transglutaminase through molecular modification

谷氨酰胺转氨酶(EC2. 3. 2. 13,Transglutaminase,TGase)是一种重要的食品酶。为提高其催化活性,通过Discovery Studio 2017预测了Streptomyces mobaraense TGase中影响其与底物α-N-CBZ-GLN-GLY结合自由能的氨基酸位点,构建得到结合自由能下降的TGase突变体:Y24W、E300W和Y302R。与野生TGase相比,E300W的比酶活提高了31%; Km、kcat和kcat/Km值分别提高了10%、42%和29%,说明E300W比酶活的提高主要是由于酶转换数的增加。Y24W、E300W和Y302R的热稳定性均有不同程度下降。作用力分析发现,Y24W、E300W和Y302R主链-主链氢键分别减少1、2和4个。上述结果表明,基于蛋白质结合自由能分析的策略能迅速鉴定影响TGase催化活性关键氨基酸,进一步突变能有效提高其催化活性。

Transglutaminase(EC2.3.2.13,TGase)is an important food enzyme.In order to enhance its catalytic activity,the amino acid sites that could affect the binding energy of S.mobaraense TGase and its substrateα-N-CBZ-GLN-GLY were predicted through Discovery Studio 2017.Then mutants(Y24W,E300W and Y302R)with reduced binding energy were constructed.Compared with wild type of TGase,the specific activity of E300W was increased by 31%.K m,k cat and k cat/K m vaule of E300W were increased by 10%,42%and 29%respectively.The results showed that the increase of specific enzyme activity was mainly due to the increase of enzymatic conversion number.While the thermal stability of all the mutants decreased in varying degrees.And the structure analysis indicated that the main-chain hydrogen bonds of Y24W,E300W and Y302R decreased by 1,2 and 4,respectively,compared with original TGase.These results suggested that the strategy based on the analysis of binding free energy could rapidly identify the key amino acids that affect the catalytic activity of TGase,and further mutation might effectively improve its catalytic activity.