Preparation Of Chiral Amino Acids By Aminoacylase Resolution

Amino acids are the basic building blocks for proteins and nutritionally important key compounds. They play significant roles in the life. Among 20 basic amino acids, glycine is the only one not exhibiting chirality, and other 19 are all L-amino. acids. With the development of amino acids industry, the functions of D-amino acids are being discovered. 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.Methionine and Phenylalanine are essential amino acids for human development. Methionine is one of the most important feed supplements. L-phenylalanine can be used to synthesis new medicament and artificial sweetener (aspartame). D-phenylalanine has analgesic activity.Firstly, Aspergillus oryzae 3042 which produces higher aminoacylase was chosen for the production of aminoacylase in this research. The aminoacylase from Aspergillus oryzae 3042 was partially purified by ammonium sulfate fractionation, cloumn chromatography on Sephadex G50 and DEAE-Sepharose. The specific activity of aminoacylase was 647.66U/mg. The purification ratio and recovery was 54.29 and 49.53% respectively. The optimal pH of aminoacylase was 7.5-8.0, and with the increase of catalysis time, the optimal reaction temperature decreased correspondingly. The ions in the buffer lowered the activity of aminoacylase, but the Co2+ in low concentration activated aminoacylase.Optimal conditions and results of optical resolution of Met and Phe by free aminoacylase are as follows respectively:Met:Temperature 37℃; pH7.5; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate 0.3mol/L. Products: L-Met, %O.P=96.2%, yields 70.5 %; D-Met, %O.P=95.3%, yields 50.5 %.Phe:Temperature 37℃; pH7.0; Concentration of Co2+ 5 X 10'4mol/L; Concentration of initial substrate is 0.2mol/L. Products: L-Phe, %O.P=98.6%, yields 68.2%; D-Met, %O.P=97.1%, yields 60.1 %.Secondly, aminoacylase was immobilized by two methods, ionic binding to DEAE-Sepharose(DSA) and entrapping into Alginate-PMCG/PDMDAAC microcapsules.Domestic DEAE-Sepharose was used to decrease the cost of the production. The stability of DSA was much better than that of the free aminoacylase. Then continuous resolution of Met was performed by DSA column. Over 60% of the aminoacylase activity remained after being operated for 30days. Generally, total amounts of the products obtained by DSA were 10 folds more than that by free aminoacylase. It is suitable to be potentially applied in the industrial production of chiral amino acids.The leakage of aminoacylase., operation stability and properties of Alginate-PMCG microcapsules and Alginate-PDMDAAC microcapsules are quite similar. The operation stability of microcapsules was decreased as the operation times increased. But the breakage of microcapsules should be solved before application.Finally, Aspergillus oryzae 3042 was immobilized in gelatin, then crosslinked by glutaraldehyde. The enzymic characteristics of the immobilized cells were also studied. Optimal pH and temperature of the free and immobilized pellets were determined. The immobilized cells were more stable over broader temperature and pH ranges. In addition, the immobilized cells showed stable activity under operation and storage conditions.