Immobilization of Carboxypeptidase A into Modified Chitosan Matrixes by Covalent Attachment.

Carboxypeptidase A (CPA) is a metalloexopeptidase that catalyzes the hydrolysis of the peptide bonds that are adjacent to the C-terminal end of a polypeptide chain. The enzyme preferentially cleaves over C-terminal L-amino acids with aromatic or branched side chains. This is of main importance for food industry because it can be employed for manufacturing functional foods from different protein sources with reduced hydrophobic amino acid content for patients with deficiencies in the absorption or digestion of the corresponding amino acids. In that way, strategies for effective multipoint covalent immobilization of CPA metalloenzyme on chitosan beads have been developed. The study of the ability to produce several chemical modifications on chitosan molecules before, during and after its coagulation to form carrier beads lead in a protective effect of the polymer matrix. The chemical modification of chitosan through the use of an N-alkylation strategy produced the best derivatives. N-alkyl chitosan derivative beads with D-fructose presented values of 0.86 for immobilization yield, 314.6 IU g-1 bead for initial activity of biocatalyst and were 5675.64-fold more stable than the free enzyme at 55 °C. Results have shown that these derivatives would present a potential technological application in hydrolytic processes due to both their physical properties, such as low swelling capacity, reduced metal chelation ability and bulk mesoporosity, and increased operational stability when compared with soluble enzyme.

Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts.

This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HA(app)) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g(-1) of gel for GLU, 7.76 mg g(-1) of gel for GLY, and 7.65 mg g(-1) of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g(-1) of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.

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

Changing gel structure and immobilization conditions led to a significant improvement in the covalent multipoint attachment of chymotrypsin on chitosan. The use of sodium alginate, gelatin, or kappa-carrageenan, activation with glutaraldehyde, glycidol, or epichlorohydrin, and addition of microorganisms followed by cellular lysis allowed the modification of the gel structure. Immobilization yields, recovered activities, and stabilization factors at 55 and 65 degrees C were evaluated. Enzyme immobilization for 72 h at pH 10.05, 25 degrees C and reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with addition of S. cerevisiae 5% and activation with epichlorohydrin led to the best derivative, which was 9900-fold more stable than the soluble enzyme. This support allowed an enzyme load up to 40 mg chymotrypsin x g gel (-1). The number of covalent bonds, formed by active groups in the support and lysine residues of the enzyme, can explain the obtained results. SEM images of the gel structures corroborate these conclusions.

Optimization of penicillin G acylase multipoint immobilization on to glutaraldehyde-chitosan beads.

The objective of this work was to study the immobilization of penicillin G acylase from Escherichia coli on to chitosan-glutaraldehyde beads by multipoint covalent binding. This process was optimized using a 2(3) experimental design. The parameters selected for the present study were the concentrations of glutaraldehyde, phenylacetic acid and sodium borohydride. Three responses were chosen, namely immobilization yield and stabilization factors of enzyme derivatives at high temperature and at alkaline pH. All the runs at the maximum (+1) and minimum (-1) levels were performed at random. Three experiments were performed at the centre point, coded as zero, for experimental-error estimation. With respect to immobilization yield, the main effectors were the concentrations of glutaraldehyde and phenylacetic acid. For stabilization factors at 50 degrees C and at alkaline pH, the main effectors were the concentrations of glutaraldehyde and sodium borohydride and the interaction between them.