Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105.
A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na(+), K(+), Mg(2+) and Ca(2+). The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K (m), V (max), K (cat), and K (cat)/K (m) values toward agarose of agarase rHZ2 were 5.9 mg ml(-1), 235 U mg(-1), 401 s(-1) and 6.8 × 10(5) M(-1) s(-1), respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.
Department of Biology, Shantou University, Shantou, 515063, People's Republic of China.
Bokun
B
Lin
Guoyong
G
Lu
Yandan
Y
Zheng
Wei
W
Xie
Shengkang
S
Li
Zhong
Z
Hu
D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000378:metabolism; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005697:Galactosides / Q000378:metabolism*; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008623:Mercaptoethanol / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005697:Galactosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C081798:neoagarotetraose; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase; D004229:Dithiothreitol