Gene cloning and enzymatic properties of hyperthermostable beta-glycosidase from Thermus thermophilus HJ6.
A microorganism (strain HJ6) producing extracellular beta-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4 microm) about 0.4 microm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80 degrees C. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding beta-glycosidase (TtbetaGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7 kDa. TtbetaGly was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for beta-glycosidase activity were found to be 90 degrees C and 8.5, respectively. The half-life of heat inactivation was about 30 min at 95 degrees C indicating that TtbetaGly had higher thermostability than beta-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that beta-D-fucoside and beta-D-glucoside were better substrates of TtbetaGly than beta-D-galactoside. The catalytic efficiency (k(cat)/K(m)) of TtbetaGly at 80 degrees C increased 70-fold to that at 40 degrees C, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.
Gu
Na-Yeon
NY
Department of Biomaterial Control (Brain Korea 21 program), Don-Eui University, 995 Eomgwangno, Busanjin-gu, Busan 614-714, Republic of Korea.
Kim
Jung-Lim
JL
Kim
Hyun-Jung
HJ
You
Dong-Ju
DJ
Kim
Han-Woo
HW
Jeon
Sung-Jong
SJ
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002855:Chromatography, Thin Layer / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*
D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases