Optimization of acid protease production by Aspergillus niger I1 on shrimp peptone using statistical experimental design

Abstract

Medium composition and culture conditions for the acid protease production by Aspergillus niger I1 were optimized by response surface methodology (RSM). A significant influence of temperature, KH(2)PO(4), and initial pH on the protease production was evaluated by Plackett-Burman design (PBD). These factors were further optimized using Box-Behnken design and RSM. Under the proposed optimized conditions, the experimental protease production (183.13 U mL(-1)) closely matched the yield predicted by the statistical model (172.57 U mL(-1)) with R(2) = 0.914. Compared with the initial M1 medium on which protease production was 43.13 U mL(-1), a successful and significant improvement by 4.25 folds was achieved in the optimized medium containing (g/L): hulled grain of wheat (HGW) 5.0; KH(2)PO(4) 1.0; NaCl 0.3; MgSO(4)(7H(2)O) 0.5; CaCl(2) (7H(2)O) 0.4; ZnSO(4) 0.1; Na(2)HPO(4) 1.6; shrimp peptone (SP) 1.0. The pH was adjusted at 5 and the temperature at 30°C. More interestingly, the optimization was accomplished using two cheap and local fermentation substrates, HGW and SP, which may result in a significant reduction in the cost of medium constituents.

Figure 1

Effects of carbon sources at 10 g/L (a); nitrogen sources at 5 g/L (b) on the production of proteolytic activity by A. niger I1. HGW: hulled grain of wheat, MJTP: Mirabilis jalapa tuber powder, SF: shrimp flower, CHVSP: combined heads and viscera sardinelle powder, MP: meat peptone, SP: shrimp peptone, AC: ammonium chloride, YE: yeast extract, AS: ammonium sulphate, CP: casein peptone.

Figure 2

Response surface plot of proteolytic enzyme production showing the interactive effects of the temperature and KH₂PO₄ concentrations (a, b), and initial pH, temperature, and KH₂PO₄ concentrations (c).

Figure 3

SDS-PAGE and zymography of A. niger I1 crude enzyme preparation. Lane 1: zymography on casein; lane 2: SDS-PAGE after staining with Coomassie blue R250; lane 3: molecular mass markers.

Figure 4

Effect of pH on the activity (a) and stability (b) of the extracellular proteases of A. niger I1 strain. pH optima were determined by incubating the crude enzyme with the substrate at different pH values at 60°C. The maximum activity obtained at pH 3.0 with hemoglobin as substrates was considered to be 100%. The pH stability was determined by incubating the crude enzyme in different buffers for 1 h at 4°C, and the residual activity was measured at pH 3.0 and 60°C with hemoglobin as a substrate. The activity of the enzyme before incubation was taken as 100%.

Figure 5

Effect of temperature on the activity (a) and stability (b) of the extracellular proteinases of A. niger I1. The temperature profile was determined by assaying proteolytic activity at temperatures between 30°C and 70°C. The activity of the enzyme at 50°C and pH 4.0 using hemoglobin as a substrate was taken as 100%. The temperature stability was determined by incubating the crude enzyme in different temperature for 1 h, and the residual activity was measured at pH 4.0 and 50°C with hemoglobin as a substrate. The nonheated enzyme was considered as 100% control.