Isolation of Genes Coding for Chitin-Degrading Enzymes in the Novel Chitinolytic Bacterium, Chitiniphilus Shinanonensis, and Characterization of a Gene Coding for a Family 19 Chitinase

J Biosci Bioeng. 2012 Mar;113(3):293-9. doi: 10.1016/j.jbiosc.2011.10.018. Epub 2011 Dec 16.

Abstract

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism
  • Acetylglucosaminidase / metabolism
  • Amino Acid Sequence
  • Catalytic Domain
  • Chitin / metabolism
  • Chitinases / chemistry
  • Chitinases / genetics*
  • Chitinases / metabolism
  • Escherichia coli / genetics
  • Gene Expression
  • Japan
  • Molecular Sequence Data
  • Neisseriaceae / enzymology*
  • Neisseriaceae / genetics*
  • Operon / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Sequence Alignment
  • Trichoderma / drug effects

Substances

  • Recombinant Proteins
  • Chitin
  • Chitinases
  • Acetylglucosaminidase
  • Acetylglucosamine

Associated data

  • GENBANK/AB649129
  • GENBANK/AB649130
  • GENBANK/AB649131
  • GENBANK/AB649132
  • GENBANK/AB649133
  • GENBANK/AB649134