Biochemical characterization of a novel cold-adapted GH39 β-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7.
Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative β-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted β-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The Kmand Vmaxof dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10-6M) and 17.2 U/mg, respectively, whereas the monomeric form had a Kmof 1.43 mg/ml (1.2 × 10-5M) and Vmaxof 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type β-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 β-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.
Department of Bioscience and Bioinformatics, Myongji University, Yongin, Gyeonggido, 17058, Republic of Korea.
Subin
S
Jung
Department of Bioscience and Bioinformatics, Myongji University, Yongin, Gyeonggido, 17058, Republic of Korea.
Chang-Ro
CR
Lee
Biological and Genetic Resource Assessment Division, National Institute of Biological Resource, Incheon, 17058, Republic of Korea.
Won-Jae
WJ
Chi
Biological and Genetic Resource Assessment Division, National Institute of Biological Resource, Incheon, 17058, Republic of Korea.
Chang-Hwan
CH
Bae
Department of Bioscience and Bioinformatics, Myongji University, Yongin, Gyeonggido, 17058, Republic of Korea. skhong@mju.ac.kr.
Soon-Kwang
SK
Hong
D000362:Agar / Q000378:metabolism*; D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D059001:Aquatic Organisms / Q000201:enzymology / Q000378:metabolism; D003080:Cold Temperature; D004187:Disaccharides / Q000378:metabolism; D005697:Galactosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012685:Sepharose / Q000378:metabolism*
D004187:Disaccharides; D005697:Galactosides; D009844:Oligosaccharides; D012336:RNA, Ribosomal, 16S; C081798:neoagarotetraose; C068544:neoagarobiose; D000362:Agar; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase
Agar; Cold-adapted enzyme; GH39; Gayadomonas joobiniege; β-Agarase