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

Dezhi Yuan; Hua Lv; Tiantian Wang; Yulu Rao; Yibo Tang; Yiwen Chu; Xinrong Wang; Jiafu Lin; Peng Gao; Tao Song

doi:10.1016/j.fochx.2023.100915

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

Food Chem X. 2023 Sep 30:20:100915. doi: 10.1016/j.fochx.2023.100915. eCollection 2023 Dec 30.

Authors

Dezhi Yuan¹, Hua Lv², Tiantian Wang², Yulu Rao², Yibo Tang², Yiwen Chu², Xinrong Wang², Jiafu Lin², Peng Gao³, Tao Song²

Affiliations

¹ Moutai Institute, Renhuai 564500, Guizhou Province, China.
² Antibiotics Research and Re-evaluation Key Laboratory of Sichuan Province, Sichuan Industrial Institute of Antibiotics, School of Pharmacy, Chengdu University, 610106 Chengdu, China.
³ Irradiation Preservation Key Laboratory of Sichuan Province, Sichuan Institute of Atomic Energy, Chengdu 610101, China.

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 K_m 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.

Keywords: Agarase; Biochemical characterization; CBM2; Catalytic amino acid.