载体两性电解质修饰对固定化γ谷氨酰转肽酶pH耐受性和催化活性的影响

摘要

针对γ谷氨酰转肽酶（ GGT） pH耐受性差的缺陷，首先以硅烷化改性的介孔氧化钛晶须（ sMTw）为载体对重组枯草芽胞杆菌GGT进行固定化，再以pH 8．0～10．5的载体两性电解质Pharmalyte（ CA，pH 8．0～10．5）对其进行后修饰，得到固定化酶sMTw GGT CA。结果发现：sMTw GGT CA的pH耐受性较游离酶明显提高，可在pH 6．0～11．0范围内保持稳定的催化活性。同时，sMTw GGT CA的热稳定性也较游离酶有所提高，其最适作用温度为50℃左右，热失活反应活化能Ed为49．88 kJ／mol。 sMTw GGT CA对γ谷氨酰对硝基苯胺（ GpNA）的亲和力常数Km为0．579 mmol／L，与游离酶相近。%In order to improve pH tolerance of γ⁃glutamyltranspeptidase ( GGT ) , recombinant γ⁃glutamyltranspeptidase ( GGT ) from Bacillus subtilis was immobilized onto silylated mesoporous TiO2 whiskers(sMTw). Then it was modified with carrier ampholyte(Pharmalyte pH 8. 0 to 10. 5,CA). The pH tolerance of modified immobilized enzyme(sMTw⁃GGT⁃CA)was greatly improved in comparing with the free GGT, stable under pH ranging from 6. 0 to 11. 0. Furthermore, the thermal stability of sMTw⁃GGT⁃CA was also higher than that of the free GGT, with optimal temperature as high as 50 ℃. The thermal inactivation energy( Ed ) of MTwA⁃GGT⁃CA was calculated to be 49. 88 kJ/mol. Also,the Km of MTwA⁃GGT⁃CA towards GpNA was 0. 579 mmol/L, very close to that of the free GGT.