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Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme.

Bioscience, biotechnology, and biochemistry (2019-07-03)
Yu Gao, Wataru Saburi, Yodai Taguchi, Haruhide Mori
ABSTRACT

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other α-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45°C, respectively. MalE was stable at a pH range of 4.5-10.4 and at ≤40°C. The phosphorolysis of maltose by MalE obeyed the sequential Bi-Bi mechanism. In reverse phosphorolysis, MalE utilized d-glucose, 1,5-anhydro-d-glucitol, methyl α-d-glucoside, 2-deoxy-d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, kojibiose, 3-deoxy-d-glucose, d-allose, 6-deoxy-d-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The kcat(app)/Km(app) value for d-glucosamine and 6-deoxy-d-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23-12% of that for d-glucose. MalE synthesized α-(1→3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized α-(1→4)-glucosides in the reaction with other tested acceptors.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1,5-Anhydro-D-sorbitol, crystalline
Sigma-Aldrich
D-Altrose, ≥97.0% (HPLC)
Sigma-Aldrich
D-Xylulose, ≥95% (HPLC), syrup
Sigma-Aldrich
Kojibiose, ≥98% (HPLC)
Sigma-Aldrich
D-Allose, 98%
Sigma-Aldrich
D-(−)-Lyxose, 99%
Sigma-Aldrich
Isomaltose, ~98% (TLC)
Sigma-Aldrich
L-(−)-Sorbose, for biotechnological purposes, ≥98.0% (sum of enantiomers, HPLC)