Biochemical characterization of a novel cold-adapted agarotetraose-producing α-agarase, AgaWS5, from Catenovulum sediminis WS1-A

• Published in: Applied microbiology and biotechnology 2019-10, Vol.103 (20), p.8403-8411
• Main Authors: Lee, Choong Hyun, Lee, Chang-Ro, Hong, Soon-Kwang
• Format: Article
• Language: English
• Subjects: Agar; Agarase; Alteromonadaceae - enzymology; Amino acids; Bacteria; Binding sites; Biochemistry; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Calcium ions; Calcium-binding protein; Cold Temperature; Cold tolerance; Degradation; Enzymatic activity; Enzyme Stability; Ethylenediaminetetraacetic acids; Glycoside hydrolase; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolase; Kinetics; Life Sciences; Linkages; Microbial Genetics and Genomics; Microbiology; Molecular Weight; NMR; Nuclear magnetic resonance; Sequence Homology, Amino Acid; Temperature tolerance; Thin layer chromatography
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-019-10056-1

Although many β-agarases that hydrolyze the β-1,4 linkages of agarose have been biochemically characterized, only three α-agarases that hydrolyze the α-1,3 linkages are reported to date. In this study, a new α-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an α-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca 2+ , and was strongly inhibited by EDTA. The K m and V max of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type α-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing α-agarase was identified.