Characterization and comparison of glyoxyl-agarose chymotrypsin and chitosan-hybrid-chymotrypsin in the hydrolysis of cheese whey proteins

ADRIANO, WELLINGTON; MENDONÇA, DANY; GALVÃO, CELIA; MAMMARELLA, ENRIQUE; GIORDANO, RAQUEL

Ciudad Autónoma de Buenos Aires, Argentina

Congreso; XXII Congreso Interamericano de Ingeniería Química; 2006

Asociación Argentina de Ingenieros Químicos

The high activity and specificity of the enzymes make them of great       interest as industrial catalysts. However, they are soluble, fragile and       expensive and their use in the industry requires easy recovery of them       from the reaction mixture and stabilization. That may be achieved through       the immobilization of the enzyme in gels of polysaccharides like       carrageen, agarose or chitosan. Cheese whey is an abundant by-product of       the dairy industry containing around 0.6% of proteins. Chymotrypsin of       bovine pancreas (EC 3.4.21.1), an endoprotease, has been immobilized and       used to hydrolyze these proteins in order to produce short-chain peptides,       a product with osmolality, allergenic potential and better flavor than       free amino acids produced by acid hydrolysis of proteins. The aim of this       work is to characterize and to compare soluble chymotrypsin with       derivatives obtained by the multipoint immobilization of chymotrypsin on       agarose (activated with glycidol and oxidized with sodium periodate) and       on a hybrid support composed by 2,5% chitosan-2,5% carrageenan-5%baker       yeast (activated with epichlorohydrin). Immobilization was carried out at       25°C, in bicarbonate buffer 100 mM pH 10.05 for 24 hours, followed by       reduction of Schiffs’ bases using NaBH4. Derivatives containing enzyme       loads from 5 to 40 mg of protein.g-1 of support were obtained. The       influence of temperature (at pH 8.0) and pH (at 25°C) on the catalytic       activity in the hydrolysis of the synthetic substrate BTEE were studied       for soluble enzyme and the two derivatives. The effectiveness of the       reaction for different loads of immobilized enzyme was determined for the       two chymotrypsin derivatives. It was also followed the cheese whey       hydrolysis degree over the reaction time, at 55°C, pH 8.0, 10 g/L of       cheese whey, using soluble chymotrypsin and the two derivatives, with the       same enzyme concentration. The maximum enzyme activities were achieved at       50°C and pH 8.0, for soluble chymotrypsin and 70°C and pH 8.0, for       glyoxyl-agarose chymotrypsin. Therefore temperatures higher than 50°C can       be used in the industrial processes if immobilized enzyme is used as       catalyst, what assure sterile conditions and lower reaction times to reach       the desired conversion. The initial rates for cheese whey proteins       hydrolysis determined using soluble and glyoxyl agarose derivatives       containing different enzyme loads showed diffusion effects only for       derivatives containing enzyme loads above 10mg of enzyme/g of support. The       determination of the hydrolysis degree over the reaction time using       soluble chymotrypsin and a glyoxyl agarose derivative containing 20 mg       enzyme/g of support showed that the diffusion limitation is significant       only in the beginning of the reaction due to the high molecular mass of       the proteins. While the reaction proceeds this effect is overcame and the       same final hydrolysis degree is achieved with soluble and immobilized       enzyme.