Characterization of Chitinase from the Africancatfish, Clarias Gariepinus (Burchell, 1822) (original) (raw)
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Advances in Microbiology, 2015
A chitinase was identified in extracellular products of a virulent Aeromonas hydrophila isolated from diseased channel catfish (Ictalurus punctatus). Recombinant chitinase (rChi-Ah) was produced in Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42˚C and pH 6.5. The affinity (Km) for chitosan was 4.18 mg•ml −1 with Vmax of 202.5 mg•min −1 •mg −1. With colloidal chitin as substrate, rChi-Ah generated N,N'-diacetyl-glucosamine predominantly. Conversion of chitosan (≥75% deacetylated) by rChi-Ah revealed five major products: 2 to 4 units of glucosamine, all of which had at least one acetyl group. It was determined that N-acetylated glucosamine was the recognition and cleavage site of rChi-Ah; the minimal and maximal cleavages were two and four glucosamine units, respectively. Functional analysis of rChi-Ah suggests that A. hydrophilachitinase is a bioactive chitinolytic enzyme, which may benefit the pathogen for survival and/or infection.
Microbiology Indonesia, 2008
Chitinases (EC 3.2.1.14) are enzyme that catalyze the degradation of chitin into the monomer N-acetyl-Dglucosamine (Park et al. 1997; Yi-Wang et al. 2001). While chitosanases (EC 3.2.1.132) are glycosyl hydrolase that catalyze the degradation of chitosan into α-D-glucosamine monomers. Chitin is a linear polymer of N-acetyl-Dglucosamine units linked through α (1-4) glycosidic bonds and distributed widely in nature as the skeletal materials of crustaceans and insects (Minoru et al. 2002), and also as a cell wall component of bacteria and fungi. Chitosan is a partially or fully deacetylated chitin. The α 1.4 glycosidic bond at linear polymer of N-acetyl-D-glucosamine of chitin is very strong and the chitinase or a specific chitosanase can catalyze degradation of the bond into a simple monomer. Chitin combined with protein and (organic salt) CaCO 3 form the skeletal material of crustacean and insects and this structure is involved in self defence mechanism against pathogenic bacteria and evaporation (Yamasaki 1993). The use of protein of skeletal crustacean as a protein source in poultry feed is inhibited by chitin compounds, because the poultry's digestive tract does not produce chitinase to hydrolyze chitin. Therefore, before adding to poultry feed, the crustacean skeleton should be hydrolyzed by chitinase into simple monomers, so that poultry can then digest. Generally, bacteria use their chitinase for degrading chitin as their carbon source, but some of them use chitinases for their self defence mechanism against pathogenic microorganisms. The characterization of chitinase from some bacteria has been undertaken, for example from Bacillus
2021
This study aimed at investigating the chitinase enzyme activity produced by chitinolytic bacteria from the skin of blue swimmer crab (Portunus pelagicus) and identification of the genus isolate. This study consists of two stages: firstly, the qualitative and quantitative activity of the chitinase enzyme; and secondly, biochemical identification of the bacteria. The quantitative chitinase enzyme activity is measured using the UV-Vis spectrophotometer UV-Vis at the wavelength at 660 nm. The chitinase enzyme is obtained from the isolation of chitinolytic bacteria cultured within a media to grow solid chitin, which contains colloidal chitin substrate as chitinase inductor at the temperature of 30°C. The highest chitinolytic activity is obtained from the 24 h supernatant culture, with a value of enzyme activity at 0.149 U/mL. Macroscopic and microscopic identification showed that the chitinolytic bacteria isolate R1, whereas the biochemical cell shows the characteristics of the genus Pse...
2011
Article History In the present investigation Bacillus sp. strain was isolated and screened from the red soil collected from Doiwala region of Dehradun (U.K), India. Serial dilution technique was adopted to isolate the organism and was screened for its chitinolytic activity. The biochemical tests were performed to prove its validity. The microorganism was also screened by inoculating a loop full of the isolated strain in basic cresol red dye and incubated for about 18-24 h. The conversion of colour of the red dye into purple (pH, 6.5-8.8) was taken as an indication for the presence of Bacillus sp. Amylase production by the organism was also screened by introduction of iodine in the broth/agar culture having starch. The broth/agar medium having starch but no bacterial strain was used as the control. The disappearance of color confirmed the presence of Bacillus strain producing amylase which degrades the starch. The chitinous wastes were collected from fresh water crustaceans viz. fresh water crab (Potamon sp.) and fresh water prawn (Palaemon sp.) and the chitin extracted was used as the substrate for chitinase. The yield of chitin extracted from fresh water prawn (Palaemon sp.) was found to be comparatively higher than that of chitin extracted from fresh water crab (Potamon sp.). Standard colloidal chitin was used as the reference control. The enzyme activity of chitinase for degradation of chitin extracted from crab and prawn was compared. The results confirmed that chitinase activity for degradation of crab chitin was comparatively higher than that of degradation of prawn chitin. The enzyme activities were found to be 0.11 µg/ml/minute and 0.09 µg/ml/minute for degradation of crab and prawn chitin respectively. The antimicrobial activity of chitinase extracted was determined against the bacterial and fungal cultures. Potent antibacterial activity of chitinase was observed against the bacterial cultures but no antifungal activity was observed. The chitinase produced by the species was able to degrade the chitin and chito-oligosaccharides produced was separated by TLC and purified by HPLC.
Chitinase Isolated from Water and Soil Bacteria in Shrimp farming Ponds
2012
Chitinases have received attention because of their wide applications in the medicine, biotechnology, agriculture, waste management and industrial applications such as food quality enhancer and biopesticide. Excessive use of insecticides has led to several problems related to pollution and environmental degradation. In this study, isolation and identification of native bacterial strains with chitin hydrolysis activity, took place from water and soil of shrimp culture ponds in Bushehr and Abadan. To investigate the capacity of our chitinase for using in insecticide, biochemical properties of selected chitinase obtained in this research were compared to that of produced by Bacillus cereus p-1, isolated from an insecticide tablet. In this research, three mesophilic strains containing: Serratia marcescens B4A, Citrobacter freundii B1A and Bacillus cereus B3R were isolated. Results showed a 1600 bp band corresponding to chitinase gene. The similarity between temperature and pH profile an...
Chitinases as Antibacterial Proteins: A Systematic Review
Journal of Young Pharmacists
Chitinases are hydrolases that catalyze the cleavage of the β-1,4-Oglycosidic linkages in chitin, a polysaccharide abundantly found in nature. Chitin is an important structural component of the cell wall of most fungi and the exoskeleton of arthropods, including insects and crustaceans. These enzymes are widespread in the living world, being found in organisms from all three domains of life. Due to their hydrolytic activity on chitin, chitinases have great biotechnological potential in different areas, such as human health, agriculture and food technology. The antifungal, insecticidal and nematicidal effects of many chitinases have been intensively investigated in the scientific literature, aiming to exploit these properties to protect crops against phytopathogenic fungi and insect pests and parasitic nematodes. On the other hand, the effects of chitinases on bacteria have been underexploited, possibly because chitin is not present in bacterial cell walls. The aim of this study was to search the scientific literature for works describing chitinases with antibacterial activity. Three bibliographic databases were searched using the keywords "chitinase" and "antibacterial" as descriptors and the chosen articles were selected according to specific inclusion and exclusion criteria. As a result, we identified only 5 reports wherein 6 purified chitinases have been shown experimentally to have antibacterial activity. Three out of these 6 antibacterial chitinases were shown to be bifunctional enzymes, which have chitinase and lysozyme activity. The possible mechanism of action of these antibacterial chitinases is discussed, highlighting their potential as antibacterial agents.
Bioscience, Biotechnology, and Biochemistry, 1997
A ftake-chitin degrading marine bacterium was isolated and identified as A eromonas hydrophila. This strain secreted five chitinases and an JI-N-acetylglucosaminidase. The main chitinase (Chi-A) was purified and characterized. The optimum pH of Chi-A was 5-8, and the activity was inhibited by Hg2 + and Fe 3 +. Chi-A was different from chitinases of other Aeromonas species with respect to molecular weight (62,000) and insensitivity to monoiodoacetate. The amino-terminal amino acid sequence showed extensive similarity with chitinases from Gram-negative bacteria.
Chitin (C8H13O5N)n has been estimated as the second most abundant biomass in the world after cellulose forming structural component of many fish food organisms, including fungi, crustaceans, coelenterates, protozoan and green algae (Rinaudo, 2006; Khoushab and Yamabhai, 2010). Chitin has been reported to make up 3.6% (wet weight) of the stomach contents of juvenile black sea bream, Acanthopagrus schlegeli (Om et al., 2003) that indicates feeding of chitin rich organisms in fish. The ability to degrade chitin is considered to involve principally the action of the enzyme chitinase (EC 3.2.11.14) that hydrolyzes insoluble chitin to its oligo and monomeric components. Chitinases are present in a wide range of organisms including viruses, bacteria, fungi, insects, higher plants and animals playing important physiological and ecological roles (Cody et al., 1990). To the authors' knowledge, the first investigation on chitin degradation by bacteria was made by Benecke (1905), who reported chitinolytic Bacillus chitinovorus from the polluted waters of Kiel harbour. Freshwater carps cultured in India mostly feed on plankton, algae, aquatic organisms and detritus representing omnivorous feeding aptitude (Jhingran, 1997). The chitin content of various copepods (e.g., Cyclops, Diaptomous etc.) comprising natural food for the carp fry and fingerlings has been reported to range from 21 to 95 mg g -1 by dry weight (Båmstedt, 1986). Being rich in nutrients, the micro-environment of fish gut confers a favourable niche for the microorganisms (Kar and Ghosh, 2008), and the gut microbiota in fish is closely related to the food that they use to consume . These distinct microbial communities may contribute uniquely to the nutrient cycling in the system (Ringø et al., 2012). Therefore, feeding on chitin rich components might suggest likely occurrence of the chitinase-producing gut microorganisms in fish. However, in comparison to the comprehensive work conducted on different enzyme-producing gut microorganisms in fish, information on the chitinolytic gut microorganisms are scarce . Studies have indicated that fish feeding on chitin rich diets have higher chitinase activity . Apart from such sporadic information, likely occurrence of chitinolytic bacteria in fish gut and their significance in feed utilization of the host species are inconclusive and contradictory. Previous studies conducted with Indian major carps indicated beneficial aspects of gut associated enzyme-producing microbiota in the host fish with regard to nutrition . Application of autochthonous chitinase-producing gut bacteria as probiotics or supplementation of bacterial chitinase as feed additive might be assumed as a strategy for effective utilization of the chitin rich natural feedstuffs in fish. However, screening and characterization of chitinase-producing autochthonus fish gut microorganisms can be viewed as a prerequisite for their likely application in fish. Microbial production of chitinase has drawn global attention not only because of its extensive application, but also for the need of effective producer organisms. Therefore, the present study aimed at (1) isolation and enumeration of chitinaseproducing gut microorganisms in 3 Indian Major Carps and 3 exotic carps, (2) identification of the most promising chitinase-producing micro-organisms by 16S rRNA partial gene sequence analysis, and finally (3) optimization of the various process parameters that influence chitinase production by the promising bacterial strains, Bacillus pumilus HMH1 (KF454036) and Bacillus flexus CMF2 (KF454035).