An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.
Chitin, a water insoluble β (1,4) linked polymer of N acetyl D glucosamine (GlcNAc), is one of the most abundant renewable forms of biomass. In the natural environment, chitin is decomposed mainly by microorganisms that can utilize it as a nutrient source. Various hydrolases are involved in chitin degradation [i.e., chitinase (EC 3.2.1.14), β N acetylhexosaminidase (EC 3.2.1.52), chitin deacetylase (EC 3.5.1.41) and chitin oligosaccharide deacetylase (COD, EC 3.1.1)]. Chitinivorous microorganisms produce water soluble mono or oligosaccharides from chitin using one or several of these chitinolytic enzymes, and then transport these soluble saccharides into the cell for further degradation. In a previous paper, we reported that Vibrio parahaemolyticus KN1699 produces the heterodisaccharide β N acetyl D glucosaminyl (1,4) D glucosamine (GlcNAc GlcN) as the primary chitin degradation product using following two types of extracellular enzymes: glycoside hydrolase family 18 chitinase, which produces N,N diacetylchitobiose, (GlcNAc)2, from chitin, and carbohydrate esterase (CE) family 4 COD, which hydrolyzes the N acetyl group at the reducing end GlcNAc residue of (GlcNAc)2. Recently, we clarified that GlcNAc GlcN is not only a nutrient for strain KN1699, but also functions as an inducer of chitinase production by this bacterium.4) Moreover, effect of this heterodisaccharide on the chitinase induction was confirmed also in other chitin decomposing Vibrio strains harboring CE family 4 COD genes. These findings suggest that CODs involved in the synthesis of this signal compound are key enzymes for chitin catabolism in some species of Vibrio.To date, aside from COD of strain KN1699, only two CODs, one from Vibrio alginolyticus H 8 5,6) and one from Vibrio cholerae EI Tor N16961, 7) have been purified and characterized. Although these three enzymes have high amino acid sequence homologies (80 99%), their specificities for chitin oligosaccharides are different, indicating the importance of characterizing other microbial CODs. We therefore screened natural sources for bacteria that secrete enzymes that can convert (GlcNAc)2 to GlcNAc GlcN. As a result, we isolated a target bacterium (strain SN184) from the bacteria that adhered on the surface of α chitin flakes (wrapped with tea bag filter paper) sunk in the sea near Tsumekizaki (Shimoda City, Shizuoka Prefecture, Japan). Gram stain results indicated that strain SN 184 is Gram negative. The genotype of strain SN184 was investigated by comparing the nucleotide sequence of its 16S rDNA (GenBank accession no. AB469367) to the sequence database BLASTN. The results confirmed that the isolate is most closely related to many strains of the genus Vibrio (about 97% identity). Although we tried to determine the species of this isolate by investigating its physiological characteristics, it was not possible to determine the species of this bacterium using the methodology outlined in Bergey s Manual of Systematic Bacteriology. 8)The bacterium was therefore named Vibrio sp. SN184.To clone ...
Oligosaccharides can exhibit physiologically useful functions. Many of these oligosaccharides are prepared by enzymatic degradation of biomass polysaccharides, or by enzymatic conversion of oligosaccharides produced by higher plants. New oligosaccharides with potential therapeutic activities are currently being developed; the physiological properties of oligosaccharides obtained by the hydrolysis of chitin, a β (1,4) polymer of N acetyl D glucos amine (GlcNAc), are of particular interest. Studies have reported that hexa N acetylchitohexaose, (GlcNAc)6, exhibits antitumor 1 3) and antimicrobial activity 4,5) in mice by enhancing the immunological defense system. These findings have focused attention on GlcNAc and (GlcNAc)6 as potential agents for arthritis therapy and immunotherapy, respectively, and have raised the possibility that chitin oligosaccharides could have physiologically useful functions. Generally, GlcNAc and chitin oligosaccharides are obtained by hydrolysis of chitin using strong acid. Chitin, one of the most abundant of all biomass polysaccharides, is the major component of the shells of crustaceans such as crab and shrimp, the exoskeletons of insects, and the cell walls of fungi. The hydrolysis of this biomass polysaccharide by enzymes possessing high product specificity would be more efficient than acid hydrolysis for the production of specific oligosaccharides. Various enzymes involved in chitin hydrolysis [e.g., chitinase (EC 3.2.1.14), β N acetylhexosaminidase (EC 3.2.1.52), chitin deacetylase (EC 3.5.1.41), and chitin oligosaccharide deacetylase (EC 3.5.1. )] have been identified. Chitinase catalyzes the degradation of water insoluble chitin into water sol uble chitin oligosaccharides through hydrolysis. A number of chitinases have been isolated from bacteria and their properties investigated. 6) In addition, genes encoding a variety of chitinases have been cloned. Based on their amino acid sequences, these chitinases are classified as belonging to either glycoside hydrolase (GH) family 18 or 19 (http: afmb.cnrs mrs.fr CAZY ). β N Acetylhexosa minidase catalyzes hydrolysis of chitin oligosaccharides to release the monosaccharide GlcNAc. Enzymes from various sources have been classified in three GH families: 3, 20 (the main family), and 84. Microbial β N acetylhexo saminidases have been reviewed. 7) Chitin deacetylase and chitin oligosaccharide deacetylase are involved in hydrolysis of the acetamide group of the GlcNAc residue of chitin and chitin oligosaccharides. 8) Both enzymes, isolated from various sources, are classified as belonging to carbohydrate esterase (CE) family 4 (http: afmb.cnrs mrs.fr CAZY ). Several microbial chitin deacetylases and chitin oligosaccharide deacetylases have been isolated and characterized.9 13) Previously, high yield production of di N acetylchitobiose, (GlcNAc)2, had been accomplished by cultivating the exo type chitinase producing marine bacterium, Vibrio anguillarum E 383a, in a medium containing colloidal chitin. 14)To obtain the enzymes that produce spec...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.