L’α-Amylase d’une nouvelle souche de Rhizopus oryzae FSIS4 isolée de blé des zones arides

Abstract

The application field of enzymes is expanding increasigly. However, these enzymes show a relatively poor performance given the constraints related to industrial processes. In order to respond properly to the needs of the industry (temperature ≥ 70 °C), it becomes crucial to identify new enzymes possessing properties that enable them to catalyze reactions in desired conditions. In this context, research of enzymes from extremophiles microorganisms can provide a solution to these issues. As a result, the main challenges of commercially produced enzymes are the thermostability, production cost and performance. Therfore, the objective of this thesis focuses on thermophilic mold isolation and identification isolated from durum wheat grown in 7 arid regions in Algeria. Thus, 246 strains of thermophilic fungi were isolated from 53 samples. After purification, 8 types are identified namely: Aspergillus, Alternaria, Rhizopus, Penicillium, Fusarium, Mucor, Geotricum and Moniliella. A total of 37 isolates belonging to the genus Rhizopus are tested for the production of α-amylase, but only 25 strains are selected to test the thermostability of the enzyme at 80 °C. The FSIS4 strain proved to be the most efficient one for both production and enzyme thermostability of α-amylase. Molecular identification by PCR sequencing products from regions ITS1 and ITS2 rDNA of the FSIS4 strain allowed to get two sequences. These show a similarity of 99% with many strains of R. oryzae. The ITS1 sequence is deposited in the Genebank of NCBI with the access number (Accession No: KU726976.1). To produce the α-amylase from R. oryzae FSIS4, we decided to up grade the use of a low-cost agro-ressource, decommissioned dates. The optimization of this medium, using design of experiments as a statistical approach, permitted to select 4 factors of significant effects on the production of the enzyme namely: starch, yeast extract, CaCl2 and the MgSO4, the optima of these factors are determined by the response surface methodology are respectively 5.42 g/L; 2.30 g/L; 0.47 g/L; 0.39 g/L. The kinetics’ study in a fermenter revealed an optimum growth at 24 h of fermentation and a maximum out put of 11034 UI after 28 hours of incubation. For the enzyme purification process, we selected an easy, fast and cost-effective technique which is the three phase partitioning (TPP). The α-amylase is selcetively fractionated in one phase while the other proteins were located inanother one. This system permits to purify the α-amylase and concentrate it in the intermediate phase.The TPP system yielded 168.83% with a purification fold of 14.94. The electrophoresis under denaturing conditions and the zymogram carried out on the intermediate phase allowed to check the purity of the protein and to calculate the molecular mass of the enzyme which is 54.8 kDa. The characterization of the partially purified enzyme depicts a pH and an optimum temperature of 5 and 60°C respectively. The enzyme is stable at 80°C with a half-life of 165 min, whereas at 90°C it is rather 105 min. The α-amylase of R. oryzae FSIS4 is therefore among the most thermoactive and thermostable fungal α-amylases described. The Ca2 +, NH42 +, Mg2 + ions increase the activity of the α-amylase of R. oryzae FSIS4 whereas; Zn2 +, Fe2 +, Na+ and Cd2 +, urea and SDS moderately inhibit the enzyme. A stronger inhibitory effect is observed in the presence of Cu2 +, Hg2+ and EDTA whereas β-mercaptoethanol improves the α-amylase activity by 30.33%. The α-amylase of R. oryzae FSIS4 is stable in the presence of ethanol and methanol up to 30%, tween 20 and 80 do not affect amylase activity while X-triton increases enzymatic activity of 13%. The kinetics parameters of the enzyme calculated are a Km of 4.16 g/L whereas the Vmax is 6760 IU. The incorporation of the formulated thermostable α-amylase of R. oryzae FSIS4 in bread induced an increase of both specific volume and the the ratio (height / width) of the bread, compared to that obtained with a commercial α-amylase. These results suggest the possibility of using this enzyme to replace imported amylases and additives consisting of chemicals and emulsifiers conventionally used in bread making, in order to improve not only the textural, rheological and flavor properties of bread, but also to limit the staling speed of the bread.