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.
Highlights
Chitin deacetylase (CDA) enzyme catalyzes the conversion of chitin into chitosan with the deacetylation of N-acetyl-D-glucosamine residues, it was originally identified, partially purified and extracted from the culture of the fungus Mucor rouxii, [1]
According to the localization of CDA enzyme in fungi, it has been classified into two subgroups: extracellular CDA and intracellular CDA, extracellular chitin deacetylase (CDA) is secreted into the external medium while Intracellular CDA is secreted into the periplasm
Optimization experiments The optimum conditions for CDA enzyme production by the selected isolate was investigated by two optimization strategies: classical optimization based on the method of one factor at a time and statistical optimization using response surface methodology
Summary
Chitin deacetylase (CDA) enzyme catalyzes the conversion of chitin into chitosan with the deacetylation of N-acetyl-D-glucosamine residues, it was originally identified, partially purified and extracted from the culture of the fungus Mucor rouxii, [1]. Response surface methodology (RSM) is considered as an accurate, effective and simple approach for optimization of the experimental process [12] and has been successfully used in agriculture, biology, food, chemistry and other fields [13] In this work, both classical and statistical methods were used to to optimize the fermentation parameters in order to maximize the production of CDA in solid-state fermentation from A.flavus. Specific PCR primer was used in this experiment as shown in Table-1
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