A Structurally Novel Chitinase from the Chitin-Degrading Hyperthermophilic Archaeon Thermococcus chitonophagus

Abstract

A structurally novel chitinase, Tc-ChiD, was identified from the hyperthermophilic archaeon Thermococcus chitonophagus, which can grow on chitin as the sole organic carbon source. The gene encoding Tc-ChiD contains regions corresponding to a signal sequence, two chitin-binding domains, and a putative catalytic domain. This catalytic domain shows no similarity with previously characterized chitinases but resembles an uncharacterized protein found in the mesophilic anaerobic bacterium Clostridium botulinum. Two recombinant Tc-ChiD proteins were produced in Escherichia coli, one without the signal sequence [TcChiD(⌬S)] and the other corresponding only to the putative catalytic domain [Tc-ChiD(⌬BD)]. Enzyme assays using N-acetylglucosamine (GlcNAc) oligomers indicated that both proteins hydrolyze GlcNAc oligomers longer than (GlcNAc) 4 . Chitinase assays using colloidal chitin suggested that Tc-ChiD is an exo-type chitinase that releases (GlcNAc) 2 or (GlcNAc) 3 . Analysis with GlcNAc oligomers modified with p-nitrophenol suggested that Tc-ChiD recognizes the reducing end of chitin chains. While TcChiD(⌬BD) displayed a higher initial velocity than that of Tc-ChiD(⌬S), we found that the presence of the two chitin-binding domains significantly enhanced the thermostability of the catalytic domain. In T. chitonophagus, another chitinase ortholog that is similar to the Thermococcus kodakarensis chitinase ChiA is present and can degrade chitin from the nonreducing ends. Therefore, the presence of multiple chitinases in T. chitonophagus with different modes of cleavage may contribute to its unique ability to efficiently degrade chitin. IMPORTANCEA structurally novel chitinase, Tc-ChiD, was identified from Thermococcus chitonophagus, a hyperthermophilic archaeon. The protein contains a signal peptide for secretion, two chitin-binding domains, and a catalytic domain that shows no similarity with previously characterized chitinases. Tc-ChiD thus represents a new family of chitinases. Tc-ChiD is an exo-type chitinase that recognizes the reducing end of chitin chains and releases (GlcNAc) 2 or (GlcNAc) 3 . As a thermostable chitinase that recognizes the reducing end of chitin chains was not previously known, Tc-ChiD may be useful in a wide range of enzyme-based technologies to degrade and utilize chitin. Chitin is a ␤-1,4-linked insoluble linear polymer of N-acetylglucosamine (GlcNAc) and is the main component of the exoskeleton of crustaceans and insects, as well as the cell walls of fungi. Chitin is the second most abundant natural polysaccharide after cellulose, and its annual formation rate is in the order of 10 10 to 10 11 tons (1). At present, the majority of chitin remains unused, and thus the development of effective methods to convert chitin into useful biomaterials and/or bioenergy is important to maintain our supplies of edible polysaccharides, such as starch.Chitinases are enzymes responsible for the hydrolysis of chitin polymer, and they produce GlcNAc and/or its oligomers as products. Chitinases are found ...

A novel thermostable chitinolytic machinery of Streptomyces sp. F-3 consisting of chitinases with different action modes

“…The product profiles for Ss Chi18A and Ss Chi19A toward colloidal chitin were mainly G2 and G1, with visible amounts of G3 (Additional file 1: Fig. S2), a common feature for GH18 chitinases [22, 23]. In the product profiles of Ss Chi18B and Ss Chi18C, besides the major products G2 and G1, G4 and G5 were also accumulated, indicating that the functions of Ss Chi18B and Ss Chi18C may be different from that of Ss Chi18A.…”

Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production

“…chitonophagus is known to possess multiple chitinases (40)(41)(42). Recently, two groups of investigators have conducted genome analyses of this archaeon, and both predicted the presence of three chitinase genes (43,44). Reclassification of the organism to the genus Pyrococcus has also been proposed (species name Pyrococcus chitonophagus) (43).…”

“…Reclassification of the organism to the genus Pyrococcus has also been proposed (species name Pyrococcus chitonophagus) (43). Biochemical characterization of one of the T. chitonophagus chitinases, ChiD, has revealed that the enzyme is a structurally novel chitinase exhibiting exo-type activities and mainly releases (GlcNAc) 2 units from the reducing ends of chitin chains (44). As T. kodakarensis ChiA contains an endo-chitinase domain and an exochitinase domain that recognizes only the nonreducing ends of chitin chains, introduction of the ChiD gene into T. kodakarensis KC04ΔtM1 may further improve its chitin degradation capabilities.…”

In silico characterization of bacterial chitinase: illuminating its relationship with archaeal and eukaryotic cousins

“…Also, some archaea like Pyrococcus furiosus [4] or Thermococcus kodakarensis [5] can produce chitinase under hyperthermophilic conditions. Furthermore, just recently, a new family of chitinase from Thermococcus chitonophagus has been reported by Horiuchi et al [6] which contains two additional chitin-binding domains along with catalytic domains.…”