Biochemical characterization and key catalytic residue identification of a novel alpha-agarase with CBM2 domain.

ABSTRACT

Agarooligosaccharides have great potential in food industry because of their various bio-activities, while the limited availability and diversity of α-agarases hinder agarooligosaccharides' broader application. To overcome this limitation, a computer-assisted method was used to screen and identify novel agarases. Firstly, one novel α-agarase, AgaB, with an N-terminal CBM2 domain (the first report of this domain in agarases), was discovered. Purified agarases only exhibited activity against agarose, with optimum activity at 40℃ and pH 8.0. Analysis of hydrolysis products indicated that AgaB is an endo-type α-agarase, producing agarotetraose and agarohexaose. Secondly, AgaB truncated CBM2 showed increased Km values, suggesting that CBM2 aids in substrate binding. Thirdly, E468 and D333 are possibly catalytic amino acids, which was supported by molecular docking results and mutants. Biochemical characterization of first reported CBM2-containing agarase and catalytic mechanism study lay the foundation for the exploration and development of α-agarases in the future.