Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp.
Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.
School of Environmental Science and Engineering, Kochi University of Technology, Kami City, Kochi, Japan. ariga.osamu@kochi-tech.ac.jp
Osamu
O
Ariga
Takayoshi
T
Inoue
Hajime
H
Kubo
Kimi
K
Minami
Mitsuteru
M
Nakamura
Michi
M
Iwai
Hironori
H
Moriyama
Mitsunori
M
Yanagisawa
Kiyohiko
K
Nakasaki
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D020633:Cellvibrio / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004187:Disaccharides / Q000032:analysis / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005697:Galactosides / Q000032:analysis / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000032:analysis / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C081798:neoagarotetraose; C068544:neoagarobiose; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase