title: Design of new immobilized-stabilized carboxypeptidase a derivative for production of aromatic free hydrolysates of proteins.
abstract: This paper presents stable carboxypeptidase A (CPA)-glyoxyl derivatives, to be used in the controlled hydrolysis of proteins. They were produced after immobilizing-stabilizing CPA on cross-linked 6% agarose beads, activated with low and high concentrations of aldehyde groups, and different immobilization times. The CPA-glyoxyl derivatives were compared to other agarose derivatives, prepared using glutaraldehyde as activation reactant. The most stabilized CPA-glyoxyl derivative was produced using 48 h of immobilization time and high activation grade of the support. This derivative was approximately 260-fold more stable than the soluble enzyme and presented approximately 42% of the activity of the soluble enzyme for the hydrolysis of long-chain peptides (e.g., cheese whey proteins previously hydrolyzed with immobilized trypsin and chymotrypsin) and of the small substrate N-benzoylglycyl-l-phenylalanine (hippuryl-l-Phe). These results were much better than those achieved using the conventional support, glutaraldehyde-agarose. Amino acid analysis of the products of the acid hydrolysis of CPA (both soluble and immobilized) showed that approximately four lysine residues were linked on the glyoxyl agarose beads, suggesting the existence of an intense multipoint covalent attachment between the enzyme and the support. The maximum temperature of hydrolysis was increased from 50 degrees C (soluble enzyme) to 70 degrees C (most stable CPA-glyoxyl derivative). The most stable CPA-glyoxyl derivative could be efficiently used in the hydrolysis of long-chain peptides at high temperature (e.g., 60 degrees C), being able to release 2-fold more aromatic amino acids (Tyr, Phe, and Trp) than the soluble enzyme, under the same operational conditions. This new CPA derivative greatly increased the feasibility of using this protease in the production of protein hydrolysates that must be free of aromatic amino acids.
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luiz, km 235, 13565-905, São Carlos, SP, Brazil.
authors.forename: P W
authors.initials: PW
authors.lastname: Tardioli
authors.forename: R
authors.initials: R
authors.lastname: Fernández-Lafuente
authors.forename: J M
authors.initials: JM
authors.lastname: Guisán
authors.forename: R L C
authors.initials: RL
authors.lastname: Giordano
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D043422:Carboxypeptidases A / Q000138:chemical synthesis* / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D015195:Drug Design; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000138:chemical synthesis / Q000737:chemistry; D006841:Hydrocarbons, Aromatic / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008894:Milk Proteins / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011492:Protein Hydrolysates / Q000138:chemical synthesis*; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D004800:Enzymes, Immobilized; D006841:Hydrocarbons, Aromatic; D008894:Milk Proteins; D011492:Protein Hydrolysates; C024471:whey acidic proteins; D010649:Phenylalanine; C049123:N-(hippuryl)phenylalanine; D043422:Carboxypeptidases A; D002918:Chymotrypsin; D014357:Trypsin