Production optimization, purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 x 10(5) g(-1) dry koji, 31 A degrees C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg(-1). Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % alpha-helix, 64.1 % beta-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35-55 A degrees C with optimum temperature of 40 A degrees C and was stable in the pH range of 2.5-6.5 with optimum pH of 3.5. Mn2+ enhanced its activity while Co2+ showed inhibitory effect. With casein as substrate, the kinetic parameters of K (m), V (max), energy of activation (E (a)), and attenuation index of inactivation velocity by heat inducing (lambda) were 0.96 mg mL(-1), 135.14 mu mol min(-1) mg(-1), 64.11 kJ mol(-1), and 0.59, respectively.