| Among 20 basic amino acids, it is economical to produce amino acids in large scale by chemical synthesis and chiral resolution as both L- and D- amino acids are valuable. And resolution by aminoacylase is one of the best methods in the resolution of amino acids. It is a high efficient, green and safe method. Aminoacylase exists widely in animals, plants, microorganism, It can hydrolysis of N - acyl-L or D-amino acid amide bond, produces L or the D-amino acid, Currently mainly used in the industrialized L-or D-amino acid production. In this paper, firstly, we used ammonium sulfate and acetone fractionation methods in order to purify the aminoacylase from fresh sheep kidney. Secondly, a new synthetic method,in which N-acetylation of L-threonine was proceeded in alkaline solution using acetic anhydride as acetylation agent,was described.thirdly, minoacylase was immobilized on chitosan micropheres with glutaraldehyde by cross-link reaction, the immobilization condition and characterization of the immobilized enzyme were studied. These will offer theoretic measure of amino acids production, especially threonine production by aminoacylase.The results show, purifying aminoacylase by the methods of ammonium sulfate and acetone fractionation from fresh sheep kidney, the specific activity of the aminoacylase was 226U/mg, the purification ratio and recovery was 18.8 and 52% respectively, its km is 4.228×10-2mol/L ,the optimal pH of aminoacylase was 7.5～8.0, role in the extension of to reduce, and with the increase of catalysis time, the optimal reaction temperature decreased correspondingly. The optimum conditions for the synthesis of N-acetylation of L-threonine were that the molar ratio of L-threonine and acetic anhydride was 1:1.0～1:1.05,the pH of the solution must be within 7～9,the reaction temperature was controlled at room temperature,the reaction time was within 1 hour after the solution became turbid. Optimal conditions of Thr by free aminoacylase are :temperature 37℃,pH 7.0,concentration of initial substrate 0.2mol/L.yield of L-Thr is 70.9%. Optimal conditions of Met by free aminoacylase are :temperature 37℃,pH 7.5,concentration of initial substrate 0.3mol/L. yield of L-Met is 74.3%.The optimal condition for immobilization were as follow: at 30℃and pH is 6.0, 0.1g chitosan micropheres was treature and with 5ml1% solution of glutaraldehyde, then 0.8mg aminoacylase was immobilization were carrier. Optimal temperature and pH for the immobilized enzyme were 50℃and 7.0 respectively, while for the free enzyme were 40℃and 7.5 respectively , the immobilized enzyme kept high activity between 50℃and 70℃,the temperature stability of the immobilized enzyme was better than free enzyme, its km is 11.796×10-2mol/L,which is more than free enzyme,and the immobilized enzyme also showed good operation and storage stability.
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