Process Biochemistry 47 (2012) 2032–2039 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization Islem Younes a,∗ , Olfa Ghorbel-Bellaaj a , Rim Nasri a , Moncef Chaabouni b , Marguerite Rinaudo c,d , Moncef Nasri a a Laboratory of Enzyme Engineering and Microbiology, National School of Engineering, P.O. Box 1173-3038 Sfax, Tunisia b Laboratory of Industrial Chemistry, National School of Engineering, BP W 3038 Sfax, Tunisia c European Synchrotron Radiation Facility (ESRF), BP 220, 38043 Grenoble Cedex 9, France d Research Centre of Natural Macromolecules (CERMAV-CNRS) affiliated with Joseph Fourier University, BP53, 38041 Grenoble Cedex 9, France a r t i c l e i n f o Article history: Received 21 January 2012 Received in revised form 11 May 2012 Accepted 14 July 2012 Available online 1 September 2012 Keywords: Shrimp shells Chitin Chitosan Enzymatic deproteinization Bacillus mojavensis A21 Response surface methodology a b s t r a c t Different crude microbial proteases were applied for chitin extraction from shrimp shells. 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. Exper- imentally, 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 dis- solved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Chitin, the second most abundant biopolymer next to cellu- lose, and its derivatives like chitosan are widely recognized to have immense applications in many fields [1]. They are widely used in the food industry, medicinal fields, chemical industries, textiles, wastewater treatment plants, etc. [2]. The main sources of raw material for the production of chitin are cuticles of various crustaceans, principally crabs and shrimps. However, crustacean shells consist of compact matrices of chitin fibers interlaced with proteins. These matrices are reinforced through the deposition of mineral salts, mainly those of calcium [3]. They have to be quantitatively removed to achieve good accessibility and the high purity necessary for biological applications. Although many methods can be found in the literature for the removal of proteins and minerals, effects on the molecular weight and acetylation degree cannot be avoided with any of these extraction processes [4]. Therefore, a great interest still exists for the optimization of the extraction to minimize the degradation of chitin, while, at the same time, bringing the impurity levels down to a satisfactory ∗ Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail address: issslem@hotmail.com (I. Younes). level for specific applications. Conventionally, preparation of chitin from such shellfish wastes involves deproteinization and demineralization with strong bases and acids. However, the use of these chemicals may cause a partial deacetylation of the chitin and hydrolysis of the polymer, resulting in final inconsistent phys- iological properties [5]. The chemical treatments also create waste disposal problems, because neutralization and detoxification of the discharged wastewater are necessary. Furthermore, the interest of the protein hydrolysate is reduced due to the presence of sodium hydroxide [6]. To overcome the defects of chemical treatments, some efforts have been directed toward its substitution by more eco-friendly processes such as bacterial fermentation and treat- ment by proteolytic enzymes which have been applied for the deproteinization of crustacean wastes [7,8]. The aim of this work is to investigate the influence of several operating parameters such as, enzyme/substrate ratio, temperature and incubation time on the deproteinization degree of shrimp shells by non-commercial Bacillus mojavensis A21 crude enzyme. Response surface methodology (RSM) is useful for designing experiments, building models and analysing the effects of sev- eral independent variables [9,10]. The main advantage of RSM is the reduced number of experimental trials needed to evaluate the effect of multiple factors on the response. In order to deter- mine a suitable polynomial equation that describes the response 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.017