Substrate specificity and transglycosylation catalyzed by a thermostable beta-glucosidase from marine hyperthermophile Thermotoga neapolitana.
The gene encoding beta-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 degrees C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100 degrees 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 degrees C, respectively. rBglA was active toward artificial (p-nitrophenyl beta-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 beta-D: -glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and beta-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.
Park
Tak-Hyun
TH
Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan 609-735, South Korea.
Choi
Ki-Won
KW
Park
Cheon-Seok
CS
Lee
Soo-Bok
SB
Kang
Ho-Young
HY
Shon
Kwang-Jae
KJ
Park
Jang-Su
JS
Cha
Jaeho
J
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D012623:Seawater / Q000382:microbiology; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000201:enzymology* / Q000235:genetics; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D001617:beta-Glucosidase