Preparation And Research Of Cross-linked Aggregates Of Aminoacylaseby Using Dual Cross-linkers

In the present study, the cross-linked enzyme aggregate (CLEA) of aminoacylase(EC3.5.1.14) from Aspergillus melleus was prepared by precipitation with ethanoland cross-linking with glutaraldehyde and Ethylene glycol diglycidyl ether (EGDE)as cross-linkers. The operational fessibility was studied and various cross-linkedparameters were optimized. The major contents were presented as follows:(1) Three kinds of organic solvents, Ethanol, acetone and glycol dinethyl ether,were used as precipitating agents to precipite free aminoacylase. Excellentprecipitation performance was showed when the concentration of ethanol was90%V/V. Almost100%enzyme protein was remained and82%of initial enzyme wasretained. Althought acetone and glycol dinethyl ether also have great performance inprotein, the bound water on the surface of enzyme molecules was easily captured bythem, resulting in loss of enzyme activity.(2) Aminoacylase is a typical enzyme lack of amine residues. Glutaradehydemight not be the appropriate cross-linker for it and easily lead to difficulties inprocess validation and quality control. Ethylene glycol diglycidyl ether (EGDE) was anovel epoxy cross-linker. Different from glutaraldehyde, epoxide groups can reactwith amino groups, sulfhydryls and hydroxyls. The cross-linking efficiency ofaminoacylase aggregate was improved by using glutaraldehyde and Ethylene glycoldiglycidyl ether (EGDE) together as cross-linkers. Almost63.42%activity of CLEAof aminoacylase was remained by cross-linked12h at30oC. The mass ratio of enzyme,glutaraldehyde and EGDE was1:0.75:2.(3) Compared with free enzyme, the operation stability of immobilizationenzyme had been significantly improved. A large number of enzyme molecules wasgathered togather, thus the structure of enzyme was enhanced. Immobilizationenzyme optimum temperature was10oC highter than free enzyme, due to thesubatrate had to overcome more mass transfer resistance to reach the activity core ofenzyme paricles. The optimum pH of immobilization enzyme was8and free enzymewas7.(4) The reusability and storage stability of CLEA was evaluated. Aminoacylase CLEA showed72%residual activity after10cycles of repeated use. LyophilizedCLEA powder was stored one month at4oC, almost no loss of activity. There was still85%of initial activity remained by keeping CLEA in buffer solution for15days at4oC.(5) The thermal deactivation model was changed from One-exponential model(free enzyme) to two-exponential model (immobilization enzyme). The thermalinactivation mechanism of enzyme was established. The reasons for the improvementof stability of immobilized enyme was explained by thermodynamics.