Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius.
We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.
Park
Ah-Reum
AR
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea.
Kim
Hye-Jung
HJ
Lee
Jung-Kul
JK
Oh
Deok-Kun
DK
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002475:Cellobiose / Q000378:metabolism; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*; D013696:Temperature
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D002475:Cellobiose; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D007785:Lactose