基于肌红蛋白构筑新型生物催化剂

Constructing Novel Biocatalysts Basing on Myoglobin

利用计算机模拟与基因工程相结合的方法,对抹香鲸肌红蛋白活性中心疏水空穴进行设计,重点研究了肌红蛋白活性部位68、107位置的氨基酸(大小、疏水性)对肌红蛋白催化性能的影响。设计、制备、表达并纯化了H64D/I107V、H64D/I107A和H64D/I107G三种变体,测试了不同变体对吲哚羟基化反应的催化性能,并通过计算机模拟阐明肌红蛋白活性部位构筑与其催化性能之间的联系。研究表明:H64D/I107V、H64D/I107A和H64D/I107G三种变体均能催化吲哚的羟基化反应,kcat分别为10.6、9.2和25.2min^(-1);证明了107位置氨基酸的空间位阻效应和68位置氨基酸的疏水性能够显著影响底物在蛋白活性部位的空间构象,进而影响肌红蛋白的催化性能。研究结果为基于肌红蛋白新型生物催化剂的开发提供了新的实验依据与理论基础。

Comprehensively utilizing the methods of computer simulation and genetic engineering, we designed the active site of the sperm whale Mb. The influence of the amino acids at 68 and 107 positions on the catalytic ability was studied in detail. Three Mb mutants H64D/I107V, H64D/I107A and H64D/ I107G were designed, prepared, expressed and purified. The hydroxylation catalytic ability of these three mutants was measured using indole as a substrate. The kcat, value of these three mutants against hydroxylation reaction was 10. 6 min-1 , 9.2 min -1, and 25.2 min-1 , respectively. The computer simu- lation results indicated that the stereo-hindrance effect of the amino acid at 107 position and the hydro- phobic property of the amino acid at 68 position can influence the location of indole in the active site, and thus affect the catalytic performance of the protein.