Site-directed mutagenesis of a family 42 β-galactosidase from an antarctic bacterium

International Journal of Biochemistry and Molecular Biology

Matthew V Shumway¹  Peter P Sheridan¹ 

关键词: Directed evolution;    cold active;    beta-galactosidase;    site directed mutagenesis;   

DOI  :  

学科分类：生物化学/生物物理

来源: e-Century Publishing Corporation

【 摘 要 】

Site directed mutagenesis was used to modify the active site of a cold active beta-galactosidase taken from an Antarctic psychrotolerant Planococcus Bacterial isolate. The goal was to modify the active site such that there would be an increase in activity on certain substrates which showed little to no activity with the wild type enzyme. A total of 5 mutant enzymes were constructed with amino acid changes based on an analysis done via homology modeling. All 5 modified enzymes were assayed using 14 different nitrophenol substrates. In most cases there was a loss of activity on substrates that showed activity with the wild type enzymes. None of the expected activity was observed with any of the mutants, possibly in part due to a decrease in hydrogen bonding between the active site and the substrates. With the substrates p-nitrophenyl-β-d-galacturonide and p-nitrophenyl-α-d-glucopyranoside we saw increased activity. With one of the mutants we measured a 320% increase in activity on p-nitrophenyl-β-d-galacturonide. Two other mutants showed activity on p-nitrophenyl-α-d-glucopyranoside, which showed no activity at all with the wild type enzyme.

【 授权许可】

Unknown