Tsuchiyama Shotaro, Doukyu Noriyuki, Yasuda Masahiro, Ishimi Kosaku, Ogino Hiroyasu
Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Biotechnol Prog. 2007 Jul-Aug;23(4):820-3. doi: 10.1021/bp060382y. Epub 2007 May 5.
The PST-01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water-soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Cbz-Asp-Phe-OMe) synthesis from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester (Phe-OMe) in the presence of water-soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz-Asp-Phe-OMe were attained by the PST-01 protease when 30 mM Cbz-Asp and 500 mM Phe-OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 degrees C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz-Asp-Phe-OMe using the PST-01 protease attained 83% in the presence of 50% (v/v) DMSO.
PST - 01蛋白酶是一种金属蛋白酶，其活性中心含有锌离子，在水溶性有机溶剂存在下非常稳定。研究了在各种条件下，由N - 苄氧羰基 - L - 天冬氨酸（Cbz - Asp）和L - 苯丙氨酸甲酯（Phe - OMe）在水溶性有机溶剂存在下合成阿斯巴甜前体N - 苄氧羰基 - L - 天冬氨酰 - L - 苯丙氨酸甲酯（Cbz - Asp - Phe - OMe）的反应速率和平衡产率。当使用30 mM Cbz - Asp和500 mM Phe - OMe时，PST - 01蛋白酶能实现更高的Cbz - Asp - Phe - OMe反应速率和产率。在pH 8.0和37℃时获得最大反应速率。在二甲基亚砜（DMSO）、甘油、甲醇和乙二醇存在下，能获得更高的反应速率。在DMSO存在下平衡产率最高。在50%（v/v）DMSO存在下，使用PST - 01蛋白酶时Cbz - Asp - Phe - OMe的平衡产率达到83%。
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