Cloning, expression and characterization of a new agarase-encoding gene from marine Pseudoalteromonas sp.
The beta-agarase gene agaA, cloned from a marine bacterium, Pseudoalteromonas sp. CY24, consists of 1,359 nucleotides encoding 453 amino acids in a sequence corresponding to a catalytic domain of glycosyl hydrolase family 16 (GH16) and a carbohydrate-binding module type 13 (CBM13). The recombinant enzyme is an endo-type agarase that hydrolyzes beta-1,4-linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the predominant products. In two cleavage patterns, AgaA digested the smallest substrate, neoagarooctaose, into neoagarobiose, neoagarotetraose and neoagarohexaose. Site directed mutation was performed to investigate the differences between AgaA and AgaD of Vibrio sp. PO-303, identifying residues V(109)VTS(112) as playing a key role in the enzyme reaction.
Lu
Xinzhi
X
Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, 266003, Qingdao, China.
Chu
Yan
Y
Wu
Qianqian
Q
Gu
Yuchao
Y
Han
Feng
F
Yu
Wengong
W
D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics; D012685:Sepharose / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000201:enzymology
D004269:DNA, Bacterial; D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; C081798:neoagarotetraose; C068544:neoagarobiose; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase