Preparation And Catalysis Characterisation Of Crosslinked Enzyme Aggeragation Of Aminoacylase

Cross-linked enzyme aggregates (CLEA), a new carrier-free immobilized method, has numerous advantages such as easy preparation, low preparing cost, high enzyme activity recovery, and good operational and storage stability and has recently become the hot issue in enzyme immobilization. Aminoacylase I (EC 3.5.1.14), which could catalyze the hydrolysis of L-acylamino acids to produce the corresponding L-amino acid, is one of most important enzymes involving in L-amino acid industrial production. The application of CLEA to aminoacylase immobilization expectedly improves the property of immobilized aminoacylase and reduces the enzyme usage cost dramatically. But up to now, the CLEA of aminoacylase has barely been reported.The preparation of cross-linked aminoacylase aggregates was investigated and optimized. Then several types of cross-linked aminoacylase aggregates were used as catalysts in a hollow fiber membrane reactor to hydrolyze N-actyl-DL-methionine for L- methionine production. Major contents were presented as follows:(1) Out of tert butyl alcohol, ethylene glycol dimethyl ether (DME) and polyethylene glycol 6000 (PEG 6000), tert butyl alcohol presented best ability to precipitate aminoacylase. The entire enzyme was precipitated with no loss of activity in the present of 80% (V/V) tert butyl alcohol concentration. Ineffective cross-linking took place when aminoacylase was directly cross-linked by glutaraldehyde due to low lysine residues content in aminoacylase. This problem could be solved through the addition of BSA before precipitation to form the co-aggregation of the enzyme and inert protein. After optimization, when the mass ratio between glutaraldehyde (GA) and enzyme was 0.75:1 and the addition of BSA was 10 mg/100 mg enzyme, 82% of the starting enzyme activity retained in the resulting CLEA (named GA-E-BSA).(2) The cross-linking performances of three kinds of epoxy cross-linker were investigated and optimized. With poly-ethylene glycol diglycidyl ether (PGDE) as a cross-linker, the obtained CLEA of aminoacylase provided the best activity recovery of 92% (named PGDE-E). The reaction mechanism of epoxy cross-linker with the enzyme differed from that of glutaraldehyde, PGDE could react with not only amine but thiol residues in the enzyme.(3) Compared to free aminoacylase, cross-linked aminoacylase aggregates had enhanced pH, storage and thermal stability. They fitted a two-exponential thermal deactivation model better than, a one-exponential model which free aminoacylase conformed to. The thermal deactivation mechanism of the cross-linked aminoacylase aggregates was proposed and the thermal deactivation model was established. The high thermal stability of the cross-linked aminoacylase aggregates was explained from the thermodynamic and dynamic point view.(4) Experiments were carried out to determine the catalysis properties of the cross-linked aminoacylase aggregates for hydrolyzing N-Ac-DL-methionine. The michaelis constants (Km and kcat) for free aminoacylase, GA-E-BSA and PGDE-E at 37℃were 0.964 mmol/L and 0.687 mmol/g?min, 1.50 mmol/L and 0.803 mmol/g?min as well as 1.02 mmol/L and 1.023 mmol/g?min, respectively. The result showed that there were substrate inhibition and competitive inhibition of L-methionine and acetic acid. The substrate and product inhibition constants (Kir and Kip) for free aminoacylase, GA-E-BSA and PGDE-E at 37℃were 699 mmol/L and 3.429 mmol/L, 725 mmol/L and 3.091 mmol/L as well as 671 mmol/L and 3.448 mmol/L, respectively.(5) The continuous resolution of N-Ac-DL-methionine by free aminoacylase, GA-E-BSA and PGDE-E was carried out in hollow fiber enzymatic membrane reactor. The effects of substrate concentration and residence time on the overall conversion were determined. A mathematical model was put forward to depict the reactor behavior after reaching reactor balance. Based on the model, the predicted results coincided well with the experimental data.