Molecular cloning and characterization of two β-agarases from marine bacterium Gilvimarinus agarilyticus JEA5

Abstract

Agar is an important polysaccharide that consists of agarose and agaropectin. Agarose has a linear chain structure of alternating residues of 3-O-linked β-D-galactopyranose and 4-O-linked 3,6-anhydro-α-L-galactopyranose. Agarose is widely used in food, cosmetics and biological research. They are classified into α-agarase (EC. 3.2.1.158) and β-agarase (E.C. 2.2.1.81) according to the cleavage pattern. β-agarases have mainly classified into three GH families, i.e., GH-16, GH50, GH86 and 118. GH-16 family has most abundant members, including agarase, carrageenase, glucanase, galctosidase, laminarinase. We isolated the agar-degrading bacteria G. agarilyticus JEA5 from the coastal area of Jeju Island, Republic of Korea. The draft genome sequence (coverage 399×) of the JEA5 strain was obtained by paired-end sequencing on an Illumina High-Seq 2000 instrument at the Macrogen (Republic of Korea). Putative agarases were identified by the Basic Local Alignment Search Tool (BLAST) algorithm.We identified two GH-16 agarases and designated Gaa16A and Gaa16B. The Gaa16a has a 1323 bp of open reading frame (ORF), which encodes 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. A signal sequence is located in the N-terminal region. The conserved glycosyl hydrolase (GH) 16 and RICIN domain were identified in the poly peptide chain. Gaa16A showed optimal activity at 50°C and used in food, cosmetics and biological research. They are classified into α-agarase (EC. 3.2.1.158) and β-agarase (E.C. 2.2.1.81) according to the cleavage pattern. β-agarases have mainly classified into three GH families, i.e., GH-16, GH50, GH86 and 118. GH-16 family has most abundant members, including agarase, carrageenase, glucanase, galctosidase, laminarinase. We isolated the agar-degrading bacteria G. agarilyticus JEA5 from the coastal area of Jeju Island, Republic of Korea. The draft genome sequence (coverage 399×) of the JEA5 strain was obtained by paired-end sequencing on an Illumina High-Seq 2000 instrument at the Macrogen (Republic of Korea). Putative agarases were identified by the Basic Local Alignment Search Tool (BLAST) algorithm.We identified two GH-16 agarases and designated Gaa16A and Gaa16B. The Gaa16a has a 1323 bp of open reading frame (ORF), which encodes 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. A signal sequence is located in the N-terminal region. The conserved glycosyl hydrolase (GH) 16 and RICIN domain were identified in the poly peptide chain. Gaa16A showed optimal activity at 50°C and