Samiullah Khan
Quaid-i-Azam University, Pakistan
Title: Comparison of the active site mutants of Thermotoga neapolitana β-glucosidase 1A for the bioconversion of polyphenolic glucosides
Biography
Biography: Samiullah Khan
Abstract
The enzyme TnBgl1A from Thermotoga neapolitana catalyse hydrolysis of O-linked terminal β-glycosidic bonds at the non-reducing end of glycosides and oligosaccharides. Previous work has shown the enzyme to catalyse hydrolysis of flavonoid glucosides but that glucosylation at the 3-position was less efficiently hydrolysed. Hydrolysis of the flavonoid quercetin-3-glucoside could however be improved by only a single residue change in the aglycone binding site, and to further explore the effect of selected residues on activity, a set of single amino acid changes were introduced close to the aglycone binding region of the active site at positions N220(S/F), N221(S/F), F224(I), F310(L/E) and W322(A). The effect on activity was monitored using the substrates para-nitrophenyl-β-D-glucopyranoside (pNPGlc) and the flavonoid quercetin-3-glucoside (Q3). All enzyme variants were cloned and overexpressed in Escherichia coli, and purified by immobilized metal ion affinity chromatography to a purity of approximately 90%. Flavonoid-glucoside hydrolysis was monitored by developing a HPLC-assay for this purpose. The mutations N220S, N221S and F224I led to a small increase in KM compared to the wild-type using pNPGlc, while for Q3 the N220S and N221S mutations decreased KM. The turnover of both pNPGlc and Q3 was increased most by the N221S mutation. Q3 turnover was also increased by mutating N220S, while N221F and W322A led to a dramatic reduction of pNPGlc turnover. The introduced mutations did not significantly affect the thermal stability of the enzyme, which kept an apparent unfolding temperature of 101ï‚°C.