An Application of Response Surface Methodology for Optimizing the Production of Chitin Deacetylase Enzyme by Aspergillus Flavus

Abstract

In this study, the optimum conditions for chitin deacetylase (CDA) production by Aspergillus flavus F1 in solid-state fermentation were investigated via two optimization strategies: classical optimization based on the method of one factor at a time and statistical optimization using response surface methodology. The result of classical optimization showed that corn supplemented with 2% chitin moisturized with mineral salts solution at pH=7 and five days of incubation time were the optimum conditions for increasing CDA production with approximately yield of 219.5 U/g solid substrate. Furthermore, pH, moisture level and inoculum size were systemically evaluated to improve CDA production based on a central composite design using the Design expert 7 software. Based on the enhanced regression model, a maximum predicted CDA enzyme production of 283.8 U/g could be obtained with pH 8, moisture level 1:1 (w/v) and inoculum size 3 ml/10g solid substrate, which contain 1×106 spore/ml. The verification of optimization results and determine accuracy of model showed that the actual response of CDA was 312 U/g, which approximately closed to predicted value 283.89 U/g. The crude extract of CDA was concentrated by sucrose. The results showed that 61% of CDA enzyme was yielded with a purification fold of 1.1 In addition. Then CDA was purified partially by gel filtration chromatography after concentration by sucrose with total enzyme activity and specific activity of 1476 U and 12300 (U/mg protein) respectively. Furthermore, the produced CDA enzyme showed maximum activity in pH ranged from (6-8); in which enzyme activity was 69 U/ ml. however; the enzyme stability has a wide range in acidic and alkaline pH. In addition, the enzyme was maintained its activity at temperatures from 30 to 55C˚. Whereas, the activity was declined in temperature up to 55Cº with a minimum activity (8 U/ml) observed at 80Cº.