Pepsin immobilization on activated carbon and functionalized with glutaraldehyde and genipin for the synthesis of antioxidant peptides of goat casein.

In this article, the synthesis of antioxidant peptides in the enzymatic hydrolysis of caprine casein was analyzed at three different time points (60 min, 90 min, and 120 min) using immobilized pepsin on activated and modified carbon (AC, ACF, ACG 50, ACG 100). The immobilization assays revealed a reduction in the biocatalysts' activity compared to the free enzyme. Among the modified ones, ACG 50 exhibited greater activity and better efficiency for reuse cycles, with superior values after 60 min and 90 min. Peptide synthesis was observed under all studied conditions. Analyses (DPPH, ß-carotene/linoleic acid, FRAP) confirmed the antioxidant potential of the peptides generated by the immobilized enzyme. However, the immobilized enzyme in ACG 50 and ACG 100, combined with longer hydrolysis times, allowed the formation of peptides with an antioxidant capacity greater than or equivalent to those generated by the free enzyme, despite reduced enzymatic activity.

Functionalized activated carbon as support for trypsin immobilization and its application in casein hydrolysis.

This study aimed to immobilize trypsin on activated carbon submitted to different surface modifications and its application in casein hydrolysis. With the aim of determining which support can promote better maintenance of the immobilized enzyme. Results showed that pH 5.0 was obtained as optimal for immobilization and pH 9.0 for the casein hydrolysis reaction for activated carbon and glutaraldehyde functionalized carbon. Among the supports used, activated carbon modified with iron ions in the presence of a chelating agent was the one that showed best results, under the conditions evaluated in this study. Presenting an immobilization yield of 95.15% and a hydrolytic activity of 4.11 U, same as soluble enzyme (3.76 U). This derivative kept its activity stable at temperatures above 40 °C for1 h and when stored for 30 days at 5 °C. Furthermore, it was effective for more than 6 reuse cycles (under the same conditions as the 1st cycle). In general, immobilization of trypsin on metallized activated carbon can be an alternative to biocatalysis, highlighting the advantages of protease immobilization.

Pepsin immobilization: Influence of carbon support functionalization.

Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that provide superficial modification by inserting new functional groups capable of binding the enzymes forming covalent bonds. In this study the effect of different modification methods of activated carbon (functionalization with genipin, metallization, metallization in the presence of chelating agent, and functionalization with glutaraldehyde) on efficiency of pepsin immobilization was evaluated. The effect of immobilization pH and the reaction medium on hydrolysis activity of bovine casein was also evaluated. The functionalization of activated carbon using iron ions allowed an immobilization capacity of 98.93 mg·g-1, with immobilization efficiency greater than 99%, and enzyme activity of 2.30 U, which was higher than the other modifications, and closer to the enzyme in the native form activity (3.32 U). In general, the carbon surface modifications were responsible for forming more stable bonds between support and enzyme, improving its proteolytic activity (from 1.84 to 2.30 U) when compared to traditional immobilization methods by adsorption and covalent binding using glutaraldehyde (from 1.04 to 1.1 U).

Hydrolysis of casein from different sources by immobilized trypsin on biochar: Effect of immobilization method.

The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.