Substrate specificity and transglycosylation catalyzed by a thermostable [beta]-glucosidase from marine hyperthermophile Thermotoga neapolitana

The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were...

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Published in:Applied microbiology and biotechnology 2005-12, Vol.69 (4), p.411
Main Authors: Park, Tak-hyun, Choi, Ki-won, Park, Cheon-seok, Lee, Soo-bok, Kang, Ho-young, Shon, Kwang-jae, Park, Jang-su, Cha, Jaeho
Format: Article
Language:English
Subjects:
Bacteria
Chromatography
E coli
Enzymatic activity
Enzymes
Heat treatment
Ions
Liquid chromatography
Mass spectrometry
Microbiology
NMR
Nuclear magnetic resonance
Structural analysis
Thin layer chromatography
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Summary:The gene encoding β-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75°C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100°C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105°C, respectively. rBglA was active toward artificial (p-nitrophenyl β-d-glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl β-d-glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and β-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.[PUBLICATION ABSTRACT]
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-005-0055-1