Studies on the production of acid protease by submerged fermentation.

摘要

Proteases are enzymes that hydrolyze peptide bonds and, therefore, lead to the disassembly of proteins. Commercially these are extremely important as more than 60% of the total enzyme market is made up of proteases, out of which 40% are acid proteases. The objective of this study was to compare the available standard strains, Rhizopus oligosporus MTCC-556, Rhizomucor miehei MTCC-546 and Aspergillus awamori MTCC-548, which were examined for the production of acid protease by submerged fermentation. Aspergillus awamori showed maximum proteolytic activity and was selected for further optimization studies. During the course of study the medium was altered. Effect of different carbon sources (lactose, sucrose, and combination of these two in same ratios and glucose) on the proteolytic activity of acid protease produced by A. awamori MTCC 548 was studied and it was found that glucose showed highest proteolytic activity. The effect of various concentrations of glucose was also studied on the acid protease production and its 1% concentration was found to be optimum; it showed proteolytic activity of 0.11 U/ml. Among the different nitrogen sources, such as casein, peptone, skim-milk powder and peanut meal, the peanut meal was found better for enzyme production. Peanut meal, with a concentration of .2% in the medium, increased proteolytic activity up to 0.218 U/ml. The effect of additives, such as Tween-80 and chemicals like CaCl₂ and skim-milk powder, was also studied and it was found that 0.05% Tween-80 was effective in enzyme production and a proteolytic activity of 0.225 U/ml was obtained. The enzyme extract was separated in the form of supernatant by using centrifuge and the enzyme activity was analyzed by Anson's method using a spectrophotometer. The enzyme produced was recovered by using a saturated solution of 80% ammonium sulphate and protein content was determined by Lowery method using a spectrophotometer. It was found that the specific enzyme activity was increased from 0.155 U/mg proteins to 0.174 U/mg proteins showing a purification fold by a factor of 1.12 by using 80% saturated ammonium sulphate.