Creating a novel source of microbial enzymes by mutation has played an important role in enzyme production industry developments. The potential of hydrolytic enzymes of twenty-four different isolates of Trichoderma was investigated, and the best isolate was exposed to gamma irradiation for induced mutation. Trichoderma mutants were isolated from an optimum dose of 250 Gy and subjected to a process of screening for eva‎luating their extracellular enzyme activity. The amount of extracellular protein (µg/ml), xylanase, and cellulase activity (Exo-glucanase, endo-glucanase, β-glucosidase, and total cellulase, U/mL) were assayed in the Trichoderma fermentation medium containing corn bran as a substrate. The purity and composition of enzyme-rich proteins were also eva‎luated using a polyacrylamide gel electrophoresis (SDS-PAGE) test. The internal transcribed spacer’s rDNA region and the translation elongation factor 1-α encoding gene were selected as candidate DNA barcodes to identify the best Trichoderma isolates. The highest hydrolysis capacity of arabinoxylanase was observed in Trichoderma afroharzianum NAS107. Of the 118 mutant isolates, 19 isolates were screened as the best mutant isolate and the highest cellulase and xylanase activity were observed in superior mutant isolates of NAS107-M44 and NAS107-M82, which is approximately 1.6-2.5 times higher than their wild-type strain, respectively. The optimization of other growth factors and composition of fermentation medium was done by the superior mutant isolate NAS107-M82. The produced enzyme powder had a specific xylanase enzyme activity of 60,000 U/g, and the optimum temperature 45 ℃ and the optimum pH of 5.0. This enzyme included a complex of xylanase enzymes consisting of Xyl I, Cel12A, and Xyl IV enzymes with molecular weights of 18, 26, and 43 kilodaltons, respectively. Also, this enzyme has a cellulase enzyme specific activity of 65,500 U/g, and the optimal activity temperature of the cellulase enzyme complex is 50°C and the optimal pH of its activity is 4.8. The complex of cellulase enzymes produced by the superior mutant organism consists of CBH I, CBH II, EG II, BGL I, BGL II, Xyl I, Xyl IV and Cel 12A (or EG III) enzymes, which act synergistically on lignocellulosic compounds. The results of the eva‎luation of the produced enzyme on the quality of bulk bread indicated that the applying of 300 units per kilogram of flour can improve all the rheological factors of the dough and the bulk bread produced from it in terms of quality indicators and eva‎luations organoleptic. The produced xylanase enzyme led to an increase in the water absorption (%), the degree of softening of the dough, mixing tolerance index, and the elasticity of the dough after 90 minutes of fermentation, and also led to decrease in the dough stability time and the development time of dough. Also, the specific volume of the produced bread and the porosity of the tissue were 2.2 times and 1.85 times higher than the control treatment, respectively. Xylanase enzyme led to increasing the softness of the bread texture and maintaining its quality even for 7 days of storage and improving the chewability of the bread and improving the quality characteristics of the texture. In general, sensory eva‎luations including bread volume, crust color and baking uniformity, crumb texture, crumb color, and its flavor in the treatments containing xylanase enzyme (especially the treatment containing 300 units of xylanase enzyme per kilogram of flour) by the eva‎luators are the most earned the points. The results showed that mutation caused by gamma radiation can be a useful method to access superior mutant isolates of industrial organisms such as Trichoderma fungus and produce useful enzyme metabolites such as xylanase enzyme of bakery industry. This technique can also be used in other organisms to create potential mutations with various industrial applications.

صبيحه سليمانيان زاد

مهدي كديور , سميرا شهبازي

محمد حجتي , بهرام شريف نبي , مريم فنائي كهراني