Optimization of phospholipase production condition in submerged medium for Trichoderma atroviride sp. ZB-ZH292

Phospholipases are important groups of enzyme with wide applications in various industries. In this study, two-step optimization including Plackett-Burman screening design and response surface methodology (RSM) optimization were done with the goal of higher production level of phospholipase by selected mutant strains of Trichoderma atroviride sp.ZB-ZH292. First step was done by screening and evaluation of seven factors affecting the enzyme activity and biomass production by selected mutant, using Plackett-Burman design at two levels each namely temperature, time, amount of soybean phospholipids (as a carbon source), peptone level (as nitrogen source), equal ratio of mono and di-potassium hydrogen phosphate (as phosphorus source), seed size, and seed age. According to the result of screening design, incubation time (P<0.01), incubation temperature (P<0.01), and soybean phospholipids as a carbon source (P<0.05), had significant effect on the enzyme activity, so they were selected and used as independent variables in central composite design (CCD) under response surface methodology (RSM). In the analysis of 20 experimental runs, the effects of three independent variables including incubation temperature (20-30 °C), incubation time (3-5 day), and phospholipid concentration (3-9%) were evaluated on phospholipase activity. Analysis of variance (ANOVA) showed that the optimum values of soybean phospholipid, incubation temperature and incubation time were 4.32%, 29.73°C, and 101.76 h, respectively. At this optimum point, the phospholipase activity was found to be 3.57 (U/ml) which is in good agreement with the predicted value (3.56 (U/ml)) by the model.