Alteration of coenzyme specificity of alkyl hydroperoxide reductase by site directed mutagenesis

• Main Author: B K Nirmalee Lakshani Goonewardena
• Other Authors: Jiang Rongrong
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/16535
• Institution: Nanyang Technological University

The ability of NADPH/NADP+ coupled enzymatic reactions to enable specific and highly efficient chemical transformations under comparatively low temperatures and pressures has made them an attractive green alternative in industrial processes. The coenzyme specificity of AhpF enzyme from Salmonella typhimurium is being investigated in this research project, which has potential to be used in industry. The gene coding for AhpF product has been successfully modified using site directed mutagenesis and cloned into two vectors pJET and pET30b for long term storage and protein expression respectively. Specific activity measurements of the mutated Ahpf[E385G] enzyme using NADPH and NADH as substrates reveal that the mutated enzyme has increased activity with NADPH cofactor whereas decreased activity with regard to NADH cofactor, compared to the wild type AhpF enzyme. These results confirm that Glutamate amino acid at the 385 position plays an important role in coenzyme specificity of AhpF enzyme.