RRC ID 28618
Author Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T.
Title Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta.
Journal Appl. Microbiol. Biotechnol.
Abstract Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.
Volume 93(5)
Pages 1989-98
Published 2012-3
DOI 10.1007/s00253-011-3538-2
PMID 21850431
MeSH Aspergillus oryzae / enzymology Aspergillus oryzae / genetics Chromatography, Gel Chromatography, Ion Exchange Cloning, Molecular Glucans Molecular Weight Phylogeny Polysaccharides / metabolism Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism Sequence Homology, Amino Acid Tandem Mass Spectrometry Ustilago / enzymology* Ustilago / genetics beta-Glucosidase / chemistry beta-Glucosidase / genetics beta-Glucosidase / metabolism*
IF 3.34
Times Cited 13
DNA material pCR8/GW/TOPO-UeBgl3A (RDB08966)