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Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of α-d-glucose 1-phosphate

Paper ID Volume ID Publish Year Pages File Format Full-Text
25255 43563 2007 10 PDF Available
Title
Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of α-d-glucose 1-phosphate
Abstract

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (α-d-glucopyranosyl-1,2-β-d-fructofuranoside) and phosphate into d-fructose and α-d-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 U mg−1. LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of α-d-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 °C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 g l−1 h−1 for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using α-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. d- and l-arabitol were found to be good glucosyl acceptors.

Keywords
Glc 1-P, glucose 1-phosphate; α-d-Glc 1-P, α-d-glucose 1-phosphate; GP, glucoside phosphorylase; GH, glycoside hydrolase; GT, glycosyltransferase; LmSPase, sucrose phosphorylase from Leuconostoc mesenteroides; SPase, sucrose phosphorylase (EC 2.4.1.7); β-
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Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of α-d-glucose 1-phosphate
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 129, Issue 1, 30 March 2007, Pages 77–86
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering