Large Scale Production and Crystallization of Acid Carboxypeptidase from Submerged Culture ofPenicillium janthinellum, and Stability of the Crystalline Enzyme

Abstract

Acid carboxypeptidase of Penicillium janthinellum IFO–8070 was produced effectively in submerged culture on a medium of 4 ~ 5% rice bran. The enzyme production was enlarged to volume cultivation of 150-liters in a 200-liters jar fermentor, and the yield of acid carboxypeptidase per milliliter filtrate reached to the maximum 3 days after inoculation. Acid carboxypeptidase of low molecular weight (M.W. = 51,000) produced in the liquid culture broth was purified and crystallized in a large scale. Purification steps include Amberlite CG–50 treatment, ammonium sulfate precipitation, dialysis using “Diaflow,” activated charcoal treatment, and condensation using collodion-bag, or condensation and dialysis using “Diaflow.” The crystals of the acid carboxypeptidase suspended in 50 mm acetate buffer (pH 3.7) were completely stable for six months at 5°C. On the other hand, at low enzyme concentration (0.01 U/ml) in 50 mm acetate buffer (pH 3.7), crystallized enzyme was somewhat labile, whereas, this inactivation was completely depressed by covering enzyme solution with toluene.