Productionde l'a-amylase par aspergillus oryzae cultivé sur milieu à base de déchets d'oranges. Optimisation du milieu de culture, purification partielle et étude des propriétés physico-chimiques de l'enzyme

Abstract

Orange pulp is a by-product of orange juice processing, contain a high levels of soluble sugars, was utilised as a basal medium at 2 % for a - amylase production by Aspergillus oryzae Ahlbtirg (Cohen) 1042.72. The selection of factors favoured a - amylase synthesis by the Plackett and Burman (1946) design and the determination of there optima by a central composite design of Box and Wilson (1951) allowed to retain soluble starch at 0.8 %, Yeast extract at 0.5 %, "corn steep liquor" at 1.98 %, magnesium sulphate at 0.2 g/l, manganese sulphate at 0.032 g/l, iron sulphate at 0.032 g/l and zinc sulphate at 0.009 g/l. The production of a - amylase is influenced by the type of carbon and nitrogen sources. Among the six carbon sources tested, starch is the best inducers resulted in 270% increase in the a - amylase productivity. However, among the fifth nitrogen sources tested "com steep liquor" is the best one, induce an increase in 188 % in a - amylase activity. The kinetics of growth and of the enzymatic production demonstrated that the a - amylase synthesis is of associated mycelial growth type, because the growth rate [ft = (1/x) (dx/dt)] and the production rate [e = (1/x) (dp/dt)] were positively correlated. The a - amylase was purified by sequential steps of ammonium sulphate, dialysis and Sephacryl S-200 gel filtration. By these steps, the purity of the enzyme increased by 18.50 and recovery of the enzyme activity was 43 %. The purification of the enzyme on Sephacryl S-200 allowed the separation of the enzyme into one molecular form. The purified enzyme had an optimal pH at 5, optimal temperature at 55°C. However, its half time was 90 minutes at 55°C and 40 minutes at 70°C. The Km and Vmax for starch were 4.25 mg/ml and 17.51 pmoles/min/mg of protein respectively.