Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.
The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.
Lee
Dong-Geun
DG
Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University, Busan, Korea.
Park
Geun-Tae
GT
Kim
Nam Young
NY
Lee
Eo-Jin
EJ
Jang
Min Kyung
MK
Shin
Young Gyun
YG
Park
Gwang-Seok
GS
Kim
Tae-Min
TM
Lee
Jae-Hwa
JH
Lee
Jung-Hyun
JH
Kim
Sang-Jin
SJ
Lee
Sang-Hyeon
SH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020560:Proteobacteria / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019624:agarase