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N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase

Paper ID Volume ID Publish Year Pages File Format Full-Text
23996 43488 2012 6 PDF Available
Title
N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase
Abstract

To study the influence of N-linked carbohydrate moiety on the catalytic and biochemical properties of glycosylated enzyme, a recombinant β-d-glucuronidase (PGUS-P) from Penicillium purpurogenum as a model glycoprotein, was deglycosylated with peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic deglycosylation procedure resulted in the complete removal of carbohydrate moiety. Compared with the glycosylated PGUS-P, the deglycosylated PGUS-P exhibited 20–70% higher activity (p < 0.05) within pH 6–9, but 15–45% lower activity (p < 0.05) at 45–70 °C. The apparent decrease in the thermal stability of the deglycosylated enzyme was reflected by a decrease in the denaturation temperature (Td) values determined by differential scanning calorimetry (DSC). The removal of N-linked glycans also reduced enzyme's sensitivity to certain metal ions. The deglycosylated PGUS-P displayed lower Km vaules, but higher kcat/Km ratios than the glycosylated isoform towards glycyrrhizin. The consequent conformational changes were also determined by circular dichroism (CD) and fluorescence spectroscopy which revealed no significant difference in the secondary but a slight dissimilarity between the tertiary structures of both isoforms of PGUS-P.

► We describe an effective enzymatic deglycosylation process of β-D-glucuronidase. ► N-glycosylation increases thermal stability and enzymatic sensitive to metal ions. ► N-glycosylation influences the tertiary structure of β-D-glucuronidase. ► N-glycosylation might be used for functional enhancement of industrial enzymes.

Keywords
β-d-Glucuronidase; Penicillium purpurogenum; N-glycosylation; Thermal stability; Conformation
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N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum β-d-glucuronidase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 157, Issue 3, 10 February 2012, Pages 399–404
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us