Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

Shariza Binti Jamek, Christian Nyffenegger, Jan Muschiol, Jesper Holck, Anne S. Meyer, Jørn Dalgaard Mikkelsen

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume101
Issue number11
Pages (from-to) 4533–4546
ISSN0175-7598
DOIs
Publication statusPublished - 2017

Keywords

  • C-terminal CBM
  • Chitinase
  • Ferrimonas balearica
  • Microbulbifer variabilis

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