Abstract
β-glucosidase from Anoxybacillus ayderensis A9 (BglA9) is a potent enzyme for enzymatic hydrolysis of polydatin to resveratrol. Based on structural and bioinformatics analysis an area near + 2 subsite of the active site pocket of BglA9 was selected and single point mutations were introduced with the aim to enhance the catalytic efficiency of the enzyme towards pNPG and polydatin. The active site region selected for mutations is non-conserved between different glycoside hydrolase family 1 (GH1) enzymes and is located at the end of β-strand 6. The changes introduced in the active site residues were L221S, N222S and G226Q. The E. coli BL21 (DE3) cells were used for the expression of mutant proteins and purification was achieved by Ni-NTA column chromatography. The thermal and pH stability was retained in all the mutants. The proteins with mutated residue resulted in variations in K(m) and k(cat)/K(m) (catalytic efficiency) values. The K(m) values of mutants for pNPG and polydatin were lowered, indicating a better enzyme-substrate complex, while variations in k(cat)/K(m) values were observed for both substrates. The docking analysis supported these observations and by comparing binding energies; the mutant N222S showed the best docked complex. This investigation suggests that the + 2 subsite of BglA9 is an interesting area to be mutated and changes in amino acid residues at this site can influence both K(m) and catalytic efficiency. The deglycosylated derivates were also compared for their antioxidant activities and showed enhanced antioxidant potential as compared to glycoside measured by DPPH assay.