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  • As a support chitosan presents interesting characteristics


    As a support, chitosan presents interesting characteristics that reinforce its great potential for enzyme immobilization [18,19]. Firstly, it is produced from renewable natural sources in addition to being biodegradable and water insoluble [20,21]. Also, the main peculiarity in the chitosan structure is the presence of amino groups, such as glucosamine units that have been generated after the deacylation of chitin. 2-Glucose amino has a moderate pK (7.8) [22] and this makes chitosan a support with moderate anion exchange capacity, especially at pH values lower than 8.0 [23,24]. On the other hand, the modification of its chemical structure is convenient to obtain a more chemically-resistant material in acidic media, for instance, and also to reduce its water retention capacity [25]. Moreover, chemical modification is required if the enzyme is to be covalently immobilized to the support [[26], [27], [28]]. In most cases, activation is carried out using glutaraldehyde [[29], [30], [31]]. However, although this reagent is very versatile [32,33], it must be used at moderate pH values due to its poor stability at more alkaline pHs [32]. That way, the possibilities of achieving intense multipoint covalent attachments are reduced considerably. The heterofunctionality of this support [34] is the main source to its versatility: it has an amino group below the glutaraldehyde moiety. Thus being, it would be interesting to develop a new strategy to produce a chitosan-derived heterofunctional support with higher prospects of producing an intense multipoint covalent attachment than when using glutaraldehyde-based chitosan matrices. In this context, supports activated with divinyl sulfone (DVS) have been considered highly appropriate for multipoint immobilization of Ac-IETD-pNA [35,36], sometimes even outperforming the results achieved using glyoxyl supports, which had been previously reported as the best matrix to this end [37]. DVS-activated supports are capable of reacting with primary and secondary amines, hydroxyl, phenyl, imidazol or thiol groups within a wide range of pH values [35]. DVS-activated supports have been tested for enzyme immobilization in several papers in recent years [[38], [39], [40], [41], [42], [43], [44], [45], [46]], although their real potential to provide an intense multipoint covalent attachment has only recently been reported [35,36]. The properties of this support enable the achievement of a very intense multipoint attachment [35,36] and, during the last reaction stages, to alter the enzyme environment and even its catalytic properties in some instances [[47], [48], [49]]. DVS has also been used for obtaining some heterofunctional supports [34]. For example, octyl-DVS supports allowed the immobilization of lipases via interfacial activation, and subsequently, their stabilization through multipoint covalent attachment [50]. Other examples of DVS-based heterofunctional supports are those containing amino-vinylsulfone groups, which enabled to elucidation of some problems encountered in standard vinyl-sulfone-activated supports for galactosidase immobilization by promoting a prior ionic exchange of the enzyme taking place on the support [51]. Chitosan modification with vinyl sulfone groups aiming at enzyme immobilization has only been reported once in the literature [52], and yet the study did not explore the potential of DVS-activated chitosan. In fact, the results using transaminases, the enzymes utilized in the aforementioned article, presented inferior performance when using DVS when compared to that using glutaraldehyde [52]. Therefore, we are convinced that a systematic study considering all the steps of enzyme immobilization on DVS-activated supports, from chitosan activation to the immobilization protocol (including the first immobilization, incubation under conditions favorable to multipoint covalent attachment and the blocking step) should enable researchers to take full advantage of the DVS potential [35].