Bovara R, Carrea G, Gioacchini A M, Riva S, Secundo F
Istituto di Chimica degli Ormoni, CNR, via Mario Bianco 9, 20131 Milano, Italy.
Biotechnol Bioeng. 1997 Apr 5;54(1):50-7. doi: 10.1002/(SICI)1097-0290(19970405)54:1<50::AID-BIT6>3.0.CO;2-X.
The transesterification activity, autolysis, thermal stability and conformation of subtilisin Carlsberg, made soluble in dioxane by covalent linking to methoxypoly(ethylene glycol) (PEG), were investigated as a function of the concentration of water in the medium. Electrospray mass spectrometry showed that the modified enzyme preparation was a mixture of proteins containing from 2 to 5 covalently linked PEG chains per subtilisin molecule. PEG-subtilisin catalyzed transesterification between vinyl butyrate and 1-hexanol was optimum at 0.55 MH(2)O, while hydrolysis prevailed above 2 MH(2)O. There was a decrease in the overall enzyme activity with increasing water concentration because of autolysis and denaturation of the enzyme. Subtilisin powder and celite-immobilized subtilisin were more stable and less susceptible to autolysis than the PEG-modified enzyme. Circular dichroism and intrinsic protein-fluorescence studies showed that the conformation of PEG-subtilisin did not change as a function of water concentrations between 0 and 9 M. The K(m,app) value of PEG-subtilisin for 1-hexanol was highly influenced by water, which behaved as a competitive inhibitor in the transesterification reaction with an affinity for the enzyme similar to that of the alcohol. The K(m,app) for the acylating agent was not significantly modified by water. Lyoprotectants such as sorbitol and free PEG did not influence the activity of PEG-subtilisin but notably increased the activity of subtilisin powder and celite-immobilized subtilisin. The addition of 1.7-5.5 M water, however, rendered enzyme preparations containing no additives as active as those containing the lyoprotectants.
通过与甲氧基聚（乙二醇）（PEG）共价连接而溶解于二氧六环中的枯草芽孢杆菌蛋白酶Carlsberg的酯交换活性、自溶作用、热稳定性和构象，作为介质中水浓度的函数进行了研究。电喷雾质谱表明，修饰后的酶制剂是一种蛋白质混合物，每个枯草芽孢杆菌蛋白酶分子含有2至5条共价连接的PEG链。PEG-枯草芽孢杆菌蛋白酶催化丁酸乙烯酯与1-己醇之间的酯交换反应在0.55 M H₂O时最佳，而在高于2 M H₂O时水解占主导。由于酶的自溶和变性，随着水浓度的增加，酶的总体活性降低。枯草芽孢杆菌蛋白酶粉末和硅藻土固定化的枯草芽孢杆菌蛋白酶比PEG修饰的酶更稳定，更不易自溶。圆二色性和蛋白质固有荧光研究表明，PEG-枯草芽孢杆菌蛋白酶的构象在0至9 M的水浓度范围内不会随水浓度变化而改变。PEG-枯草芽孢杆菌蛋白酶对1-己醇的K(m,app)值受水的影响很大水在酯交换反应中表现为竞争性抑制剂，对酶的亲和力与醇相似。酰化剂的K(m,app)值不受水的显著影响。山梨醇和游离PEG等冻干保护剂不会影响PEG-枯草芽孢杆菌蛋白酶的活性，但会显著提高枯草芽孢杆菌蛋白酶粉末和硅藻土固定化的枯草芽孢杆菌蛋白酶活性。然而，添加1.7 - 5.5 M的水会使不含添加剂的酶制剂与含有冻干保护剂的酶制剂活性相当。
