Chitosan — Unique matrix for protease immobilisation

Bacheva, A.V.; Macquarrie, D.J.; Filippova, I.Y.

Авторы: Bacheva Anna V., Macquarrie Duncan J., Filippova Irina Yu
Журнал: Journal of Biotechnology
Том: 136
Номер: S
Год издания: 2008
Издательство: Elsevier BV
Местоположение издательства: Netherlands
Первая страница: S392
Последняя страница: S392
DOI: 10.1016/j.jbiotec.2008.07.905
Аннотация: Immobilized proteases can be used in two reversed processes: hydrolysis of peptides or proteins in water (food industry and proteomics research) and peptide bond formation in nonaqueous media (synthesis of pharmaceuticals, substrates, inhibitors, etc. of peptide nature). The main goal of this work was to create immobilized biocatalyst capable to work both in aqueous and low water media with high efficiency. Enzyme (subtilisin, chymotrypsin and papain)/chitosan biocomposite films were prepared as described in Bacheva et al. (2008). Amidase activity of subtilisin was tested using Glp-Ala-Ala-Leu-pNA ( Bacheva et al., 2008), of chymotrypsin—by Glp-Phe-pNA and of papain—by Glp-Phe-Ala-pNA ( Semashko et al., 2008). Protease activity was checked against azocasein and esterase activity—using p-nitrophenylacetate. Peptide synthesis by subtilisin/chitosan and by papain/chitosan was carried out as described in ( Bacheva et al., 2008). The distinctive feature of these biocomposites was the preparation technique providing for high loading and uniform enzyme distribution. Comparing chemical modification and physical adsorption methods it was found that biocomposites treated with glutaraldehyde (optimum 1–2%, v/v) were more effective than untreated, especially in aqueous media. Higher glutaraldehyde concentration leads to lower activity of biocatalyst. Raising of starting enzyme concentration (2–150 mg/mL) lead to increase of activity and degree of loading, but after 75 mg/mL it reached a plateau (80 mg protein/g biocatalyst) and further concentration increase had no effect. The kinetics parameters, temperature and pH-dependence of subtilisin/chitosan hydrolytic activity were studied. All the biocomposites have high storage and operational stability in aqueous and nonaqueous media. The samples possessed high synthetic activity and were capable to catalyze peptide bond formation in DMFA/CH3CN (6/4) mixture in reaction Z-Ala-Ala-Leu-OCH3 + Phe-pNA → Z-Ala-Ala-Leu-Phe-pNA for subtilisin, Z-Ala-Ala-OCH3 + Leu-pNA → Z-Ala-Ala-Leu-pNA for papain with the product yield 60–100% after 24 h of reaction. Thus biocatalytic films prepared in this work are characterized by preparative simplicity, high activity and stability in different media. This work was supported by RFBR 06-03-33056a.
Добавил в систему: Бачева Анна Владимировна

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Chitosan — Unique matrix for protease immobilisationстатья