Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships

International Journal of Biochemistry and Molecular Biology

Utpal Mohan¹  UC Banerjee¹  Shubhangi Kaushik¹ 

关键词: Nitrilase;    catalysis;    sequence identity;    site directed mutagenesis;    mutants;    conserved residues;   

DOI  :  

学科分类：生物化学/生物物理

来源: e-Century Publishing Corporation

【 摘 要 】

Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.

【 授权许可】

Unknown