Cloning and Purification of a Glycerol Specific Alditol Oxidase for Biosensor Construction

Abstract

Biosensors have been used to detect the presence of carbohydrates in serum and solution. Alditol oxidase (AldO) is a redox enzyme which catalyzes the oxidation of longer-chain polyols but has a low affinity for glycerol, despite the similarity in structure. Glycerol is an undesirable byproduct of wine fermentation, negatively affecting quality. Glycerol is also a product of adipose tissue metabolism during fasting, indicating abnormal blood glucose. In both situations, glycerol concentrations must be closely monitored. Using site-directed mutagenesis, a quadruple mutant of AldO was found to have increased specificity towards glycerol (AldOG). Glycerol oxidases are rare in nature, and the small, monomeric characteristics of AldOG suggest compatibility for biosensor incorporation. Previous research found recombinant AldOG with a GST tag to be insoluble. The synthetic AldOG gene was cloned into E. coli competent cells with an N-terminal hexahistidine tag aiming to improve solubility. Protein isolation and solubilization was attempted using native and denaturing NiNTA affinity chromatography. Functional AldOG optimization procedures will be used in collaboration with Dr. Samuel Mugo for biosensor incorporation. Functional AldOG is also to be used in a 300-level biochemistry laboratory course developed by Dr. Nina Bernstein.

Faculty Mentor: Dr. Nina Bernstein