Design of New Immobilized-Stabilized Carboxypeptidase A Derivative for Production of Aromatic Free Hydrolysates of Proteins

摘要

This paper presents stable stable carboxypeptidase A (CPA)-glyoxyl derivatives to be used in the controlled hydrolysis of proteins.They were produced after immobilizing-stabilizing CPA on sross-linded 6% agarose beass activated with low ane hihg concentrations of aldehyde groups and different immobilzation times.The CPA-glyoxyl derivatives were compared to other agarose derivatives prepared using glutaraldehyde as activation reactant The most stabilized CPA-gloxyl derivatiuve was produced using 48 h of immobilizsation time and high activation grode of the support.This derivative was approximately 260-fold more stable than te soluble enzyme and presented approximately 42% of the small substrate N-benzoylalycyl-L-phenyladlaine (hippuryl-L-phe) These results were much better than those achieved usign the onventional suport glutaraldehyde-agarose.Amino acid analysis ot he products of the acid hydrolysis of CPA(both soluble and immobilized) showed that approximately four lysin resibues were kinked on the glyoxyl agarose based,usggesting the exsistence of an intense multipiont covalent attachment between the enzyme and the support The maximum temperature of hydrolysis was increased from 50 deg C (solubhle enzyme) to 70 det C (most stable CPA-glyodyl derivative).The most atable CPA-glyoxyl derivative could hge efficiently used in the hydfrolysis of l,ong-chain peptides at high temperature (e.g.,60deg C) being able to release-2-fold more aromaticf amion acids (Tyr,Phe and Trp) than the soluble enzyme under the same operational conditons this new CPA derivative greatly increased the feasibility fo using this protease in the production of protein hydrolystates that must be free of aromstic amino acids.