Cloning, purification and biochemical characterization of beta agarase from the marine bacterium Pseudoalteromonas sp. AG4.
A gene (agrP) encoding a beta-agarase from Pseudoalteromonas sp. AG4 was cloned and expressed in Escherichia coli. The agrP primary structure consists of an 870-bp open reading frame (ORF) encoding 290 amino acids (aa). The predicted molecular mass and isoelectric point were determined at 33 kDa and 5.9, respectively. The signal peptide was predicted to be 21 aa. The deduced aa sequence showed 98.6% identity to beta-agarase from Pseudoalteromonas atlantica. The recombinant protein was purified as a fusion protein and biochemically characterized. The purified beta-agarase (AgaP) had specific activity of 204.4 and 207.5 units/mg towards agar and agarose, respectively. The enzyme showed maximum activity at 55 degrees C and pH 5.5. It was stable at pH 4.5 to 8.0 and below 55 degrees C for 1 h. The enzyme produced neoagarohexaose and neoagarotetraose from agar and in addition to that neoagarobiose from the agarose. The neoagarooligosaccharides were biologically active. Hence, AgaP is a useful enzyme source for use by cosmetic and pharmaceutical industries.
Oh
Chulhong
C
Department of Marine Life Science, Jeju National University, 66 Jejudaehakno, Ara-Dong, Jeju, Republic of Korea.
Nikapitiya
Chamilani
C
Lee
Youngdeuk
Y
Whang
Ilson
I
Kim
Se-Jae
SJ
Kang
Do-Hyung
DH
Lee
Jehee
J
D000362:Agar / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D010802:Phylogeny; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D056910:Republic of Korea; D000461:Rhodophyta / Q000382:microbiology*; D012623:Seawater / Q000382:microbiology*; D012685:Sepharose / Q000378:metabolism; D016415:Sequence Alignment
D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C081798:neoagarotetraose; C068544:neoagarobiose; D000362:Agar; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase