title: Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules.
abstract: Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
authors.affiliation: Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia.|Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Shariza B
authors.initials: SB
authors.lastname: Jamek
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Nyffenegger
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Muschiol
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Holck
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark. am@kt.dtu.dk.
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology*; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism; D004187:Disaccharides / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020563:Gammaproteobacteria / Q000201:enzymology*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D012706:Serratia marcescens / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004187:Disaccharides; D002686:Chitin; C032438:chitobiose; D006867:Hydrolases; D002688:Chitinases
keywords: C-terminal CBM; Chitinase; Ferrimonas balearica; Microbulbifer variabilis