Improving the properties of chitosan as support for the covalent multipoint immobilization of chymotrypsin

ADRIANO, WELLINGTON; MENDONÇA, DANY; RODRIGUES, DASCIANA; MAMMARELLA, ENRIQUE; GIORDANO, RAQUEL

BIOMACROMOLECULES

ACS Publications

Lugar: Columbus, USA; Año: 2008 vol. 9 p. 2170 - 2179

1525-7797

In this work, several strategies were tested to improve the covalent multipoint attachment of chymotrypsin on chitosan. Hybrid gels with different internal structure were obtained using sodium alginate, gelatin or -carrageenan and changing the polymer concentration, 2.5-5.0% (m/v) and the activating reactant, glutaraldehyde, glycidol or epichlorohydrin. The addition of microorganisms to the hybrid polymer followed by cellular lysis after producing the gel beads, the increase of the immobilization reaction time and the effect of the reduction of the final derivative with sodium borohydride were also tested. The influence of these variables on the chymotrypsin immobilization parameters, immobilization yield and recovered activity, as well the stabilization factor at 55°C and 65°C, were assessed. The derivative half-live was increased from 34 min at 55°C, for pure chitosan 2.5% activated with glutaraldehyde at pH 7.0, 4°C, to 468 min at 65°C, for chymotrypsin immobilized on chitosan 2.5%-carrageenan 2.5%, with addition of 5% of S.cerevisiae, activation with epichlorohydrin, immobilization for 72 h at pH10.05, room temperature and reduction of the final derivative. This best derivative was 9900-fold more stable than the soluble enzyme. A maximum load of 40 mg chymotrypsin.gbead-1 was achieved. The number of aldehyde and oxirane groups generated in the support, and of the lysine residues of the enzyme involved in the multipoint attachment, as well SEM images of the gel structures, explain the obtained results.