Isolement, identification et criblage d’actinomycètes à activité chitinolytique à partir du sol de la région de Laghouat.

Abstract

With at least 10 gigatons synthesized and degraded each year in the biosphere, chitin is the second most represented polysaccharide in nature after cellulose. It is a crystalline polysaccharide consisting of long linear chains containing more than 1000 units of N-acetyl-β-D-glucosamine linked by β-1,4 glycoside bonds. Chitin degradation is performed by chitinases (E.C. 3.2.1.14), that hydrolyze ß-1,4 bonds releasing compounds easily metabolized by many microbes. Production of microbial chitinases receives worldwide attention in both industrial and scientific communities, because of its wide applications spectrum. Chitinases are used in biochemistry, food processing, and various chemical industries due their antimicrobial, anticholesterol, and antitumor activities. They are also used in waste-water treatment, dietary fiber and in agriculture to inhibit plant pathogens. The physiological function of chitinases depends on their origin. In bacteria, chitinases play a trophic role by degrading chitin, as source of both carbon and nitrogen. Microorganisms particularly Streptomyces are efficient chitin hydrolysers. Chitinases have been identified in several Streptomyces spp. It is induced by chitin and repressed by readily utilizable carbon sources such as N-acetyl-glucosamine and glucose. In this thesis, we chose to study actinobacteria isolated from a semi-arid soil of the Algerian desert in order to identify strains with a high chitinolytic and antifungal potential. We isolated 40 phenotypically different actinobacteria. First, we carried out a functional study by screening these isolates on a minimal medium supplemented with colloidal chitin (CCM) to characterize their chitinolytic activities under extreme temperature conditions (30, 35, 40, 45 and 50 °C) and pH (5, 7 and 9). In addition, a taxonomic study was conducted by combining a BOX-PCR approach and sequencing 16S rRNA of 20 strains. This identification showed that 10 of these isolates belonged to the genus of Streptomyces, two to the genus of Micromonospora and 08 potential new bacterial species. Also, the search for antimicrobial activity against 06 test bacteria and two phytopathogenic fungi was carried out, using a novel glucose-inducing / repressing chitinase approach in the antifungal test, to prove that only chitinases can inhibit these phytopathogenic fungi. One strain (C31) was able to reduce fungal growth only after induction of chitinase by colloidal chitin. The second part of this work aimed to use statistical Plackett–Burman and Box–Wilson response surface methodology to optimize the medium components and, thus, improve chitinase production by Streptomyces griseorubens C9. First, syrup of date, colloidal chitin, yeast extract and K2HPO4, KH2PO4 were proved to have significant effects on chitinase activity using the Plackett–Burman design. Then, an optimal medium was obtained by a Box–Wilson factorial design of response surface methodology in liquid culture. Maximum chitinase production (0.902 U / ml) was predicted in medium containing 2% colloidal chitin, 0.47% syrup of date, 0.25 g/l yeast extract and 1.81 g/l K2HPO4, KH2PO4.This was confirmed by Erlenmeyer cultures where the Production of chitinase by S. griseorubens C9 reached 1.53 U/ml, the yield of enzyme production was increased 26.38% after optimization by the response surface methodology. Finally, purification and partial characterization of the enzyme produced in the optimized medium was performed. 3 major purification steps were done and the enzyme reached a purification fold of 23.24; a yield of 29.7% and a specific activity equal to 2.393 U/mg. The purified chitinase activity had optimum catalytic activity at pH 5.0 and 50 °C. It retained more than 60% stability at pH range between 4.0 and 7.0 and remained stable at temperatures between 30 °C and 60 °C. Thus, S. griseorubens C9 could be a potent strain for industrial chitinase production, as well as in biotechnology and bio-control fields.