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Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp,10.1007/s12010-010-9147-1,App

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp   (Citations: 1)
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Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H2O2 and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t 1/2 from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H2O2 (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k cat and K m values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.
Journal: Applied Biochemistry and Biotechnology - APPL BIOCHEM BIOTECH , vol. 164, no. 4, pp. 454-463, 2011
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