Chitinases (EC 3.2.1.14) cleave the -1,4-glycosidic bonds of chitin, a -1,4-linked, unbranched polymer of N-acetylglucosamine (GlcNAc), which is a major component of insect exoskeletons, shells of crustaceans, and fungal cell walls. These enzymes have been detected in a variety of organisms, including organisms that do not contain chitin as a structural component, such as bacteria, plants, and animals. The production of chitinases by plants is thought to be involved in defense reactions against chitin-containing pathogens. Bacteria utilize chitinases for assimilation of chitin as a carbon and nitrogen source, and these enzymes play an important ecological role in the degradation of chitin.Numerous chitinases have been characterized, and the corresponding genes have been analyzed. On the basis of the primary and secondary structures of the catalytic domains, chitinases are grouped into two distinct families (families 18 and 19) in the classification of glycosyl hydrolase (12, 13). The bacterial chitinases, except for Streptomyces griseus HUT 6037 ChiC (20), belong to glycosyl hydrolase family 18. In addition to the catalytic domain, many bacterial chitinases, like many polysaccharidases, such as cellulases and amylases, have a discrete binding domain that mediates adsorption to the substrate. In the past decade, studies on the molecular structure and function of substrate-binding domains have focused mainly on cellulose-binding domains (CBDs); however, relatively little is known about the chitin-binding domains (ChBDs). Most of the knowledge about bacterial ChBDs has been accumulated from studies on Bacillus circulans ChiA1 (11, 39), Clostridium paraputrificum ChiB (18), S. olivaceoviridis exoChiO1 (2), Alteromonas sp. strain O-7 ChiC (36), Serratia marcescens ChiC (31), and Pyrococcus kodakaraensis KOD1 ChiA (33). The above studies have shown that the chitinase lacking the ChBD lost much of its binding capacity and hydrolytic activity toward insoluble chitin. Thus, it has been suggested that the ChBD potentiates the catalytic activity against insoluble-chitin substrates.Although several studies have revealed that deletion of the ChBD from chitinases reduces the capacity of the enzymes to bind and hydrolyze insoluble chitin, it is unclear by which mechanism the domain elicits its effect. In a previous study (6), the chitinase gene from Aeromonas hydrophila JP101 was cloned and expressed in Escherichia coli. In this paper, we describe the purification, biochemical properties, and primary structure of A. hydrophila JP101 Chi92. Furthermore, in order to investigate the molecular basis for the capacity of bacterial chitinases to bind chitin, we also carried out biochemical studies of C-terminally truncated derivatives and glutathione Stransferase (GST) fusion proteins of ChBD CI and ChBD CII . MATERIALS AND METHODSBacterial strains, plasmids, and cultivation conditions. A. hydrophila JP101 was used in this study. It was initially isolated in our laboratory from shrimp shell-enriched soil (6). E. coli JM109 (41), JA...
A gene encoding the extracellular a-amylase of Aevomonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Eschevichia coli JA221 carrying pCAlOl revealed that approximately 60 YO of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an a-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete a-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several a-amylases were also present in A . hydrophila a-amylase at the corresponding positions.
An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose. Various kinds of bacteria, fungi, plants, and some vertebrates (22) produce chitinase, which catalyzes the hydrolysis
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.
customersupport@researchsolutions.com
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.