title: Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar.
abstract: In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (D-galactose and 3,6-anhydro-L-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476-Zg4663 (SZ) and Zg4663-Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (Kcat/KM) from 14.8 (mg/mL)-1s-1to 15.8 (mg/mL)-1s-1. Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.
authors.affiliation: Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk, 28644, Republic of Korea.
authors.forename: Bassam
authors.initials: B
authors.lastname: Alkotaini
authors.affiliation: Department of Food Science and Biotechnology, Chungbuk National University, Cheongju, Chungbuk, 28644, Republic of Korea.
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Han
authors.affiliation: Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk, 28644, Republic of Korea. bskim@chungbuk.ac.kr.
authors.forename: Beom Soo
authors.initials: BS
authors.lastname: Kim
mesh_terms: D000362:Agar / Q000378:metabolism*; D004186:Disaccharidases / Q000235:genetics / Q000378:metabolism*; D004187:Disaccharides / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*
chemical_list: D004187:Disaccharides; C068544:neoagarobiose; D000362:Agar; D004186:Disaccharidases; D006026:Glycoside Hydrolases; C039308:neoagarobiose hydrolase; C019624:agarase; D005690:Galactose
keywords: Agar saccharification; Bifunctional agarase; Fusion enzyme; Neoagarobiose hydrolase; Red algae