Identification, purification and functional characterization of a thermostable marine chitinase for potential fungal control via chitin degradation mechanism
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Date
2025
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Elsevier Ltd
Abstract
The growing prevalence of treatment-resistant Candida species highlights an urgent need for innovative antifungal therapies. The current range of antifungals, limited to polyenes, azoles, and echinocandins, are becoming insufficient due to the rise of resistance, including cross-resistance among fungal strains. Marine environment is an underexplored reservoir of unique enzymes which can be extremophilic. This study presents the cloning and expression of a chitinase gene from the bacterium Bacillus thuringiensis (BtChi), expressed in an E. coli system, yielding a protein with a molecular weight of approximately 71 kDa. Disc diffusion and MIC experiments indicated that 5 ?g/mL chitinase efficiently suppressed the growth of Candida albicans. Initial characterization identified the optimal activity at 40 °C and pH 7.0. The enzyme retained over 75 % activity across a pH range of 4–8 and a temperature range of 30–70 °C after 120 min. Activity was further enhanced by 24 % with 100 mM Na+. Kinetic parameters with colloidal chitin revealed K<inf>m</inf> and V<inf>max</inf> values to be 0.05 mg/mL and 1.37 U/mL respectively. This study holds the potential of developing a potent natural anti-fungal against the present day chemical counterparts. © 2025 The Authors
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Keywords
Bacteriology, Chitin, Cloning, Degradation, Enzymes, Escherichia coli, Gene expression, Yeast, Antifungals, Candida species, Chitin degradation, Chitinases, Degradation mechanism, Exo-chitinase, Functional characterization, Gibson cloning, Marine, Thermostable, Candida, Purification, aluminum, amphotericin, antifungal agent, bacterial enzyme, calcium ion, chitin, chitinase, cobalt, cuprous ion, genomic DNA, iron, magnesium ion, manganese, natural product, nickel, potassium ion, RNA 16S, single stranded DNA, sodium ion, zinc ion, affinity chromatography, amino terminal sequence, antifungal activity, Arthrobacter, Article, Bacillus thuringiensis, bacterial membrane, Botrytis cinerea, Candida albicans, candidiasis, cell membrane permeability, chemical structure, controlled study, disk diffusion, drug identification, drug potency, drug purification, enzyme activity, enzyme analysis, enzyme specificity, gene amplification, genetic code, high performance liquid chromatography, kinetic parameters, marine environment, MTT assay, nephrotoxicity, nonhuman, pH, phylogeny, polymerase chain reaction, process optimization, protein expression, protein folding, quantitative analysis, RNA sequence, spectrofluorometry, spore germination, substrate concentration, systemic mycosis, temperature, thermostability, thin layer chromatography, Western blotting, chemistry, drug effect, enzyme stability, enzymology, genetics, growth, development and aging, isolation and purification, kinetics, metabolism, microbial sensitivity test, Antifungal Agents, Enzyme Stability, Hydrogen-Ion Concentration, Kinetics, Microbial Sensitivity Tests, Temperature
Citation
Carbohydrate Research, 2025, 558, , pp. -