A Box–Behnken
design with three variables and three levels was applied in order to approach the prediction of optimal
enzyme/substrate ratio, temperature and incubation time on the deproteinization degree with Bacillus
mojavensis A21 crude protease. These optimal conditions were: an enzyme/substrate ratio of 7.75 U/mg,
a temperature of 60 ◦C and an incubation time of 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, in these optimized conditions, a deproteinization degree of 88 ± 5% was obtained in good
agreement with the prediction and larger than values generally given in literature. The deproteinized
shells were then demineralized to obtain chitin which was converted to chitosan by deacetylation and
its antibacterial activity against different bacteria was investigated. Results showed that chitosan dissolved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria
tested.
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