建立了天冬氨酸消旋酶和D-氨基酸转氨酶双酶连续催化制备D-丙氨酸的方法.利用天冬氨酸消旋酶全细胞催化L-天冬氨酸消旋得到DL-天冬氨酸,离心去除天冬氨酸消旋酶全细胞后升温灭活残留的游离天冬氨酸消旋酶,再加入经镍柱亲和纯化的D-氨基酸转氨酶酶液,催化D-天冬氨酸(D-Asp)和丙酮酸(PA)经转氨反应生成D-丙氨酸.经单因素实验得到天冬氨酸消旋酶最佳催化条件为:反应温度40℃,0.2 mol/L磷酸钾缓冲溶液(pH=7.0),底物L-天冬氨酸质量浓度为100 g/L.D-氨基酸转氨酶最佳催化条件为:反应温度42℃,0.2 mol/L磷酸钾缓冲液(pH=7.0),4 mmol/L磷酸吡哆醛,DL-天冬氨酸质量浓度为50 g/L,底物n(PA):n(D-Asp)=1:10.转化产物经等电点结晶和阳离子树脂分离得到D-丙氨酸.在该条件下,D-天冬氨酸转化率达94%,D-丙氨酸收率为84%,对映体过量值(ee值)=98%.
A continuous catalytic method for the preparation of D-alanine by aspartic acid racemase ( AspR) and D-amino acid transaminase( DaaT) was established. Firstly,L-aspartic acid was racemized to give DL-aspartic acid by whole cells containing aspartic acid racemase. Secondly, eliminating the whole cells containing AspR by centrifuging and then inactivating the residual AspR by heating, subsequently adding DaaT purified by nickel column affinity chromatography,a transamination reaction between D-aspartic acid ( D-Asp ) and pyruvic acid ( PA ) in the above mixture led to D-alanine. Finally,the catalytic conditions of AspR were optimized via single factor experiment. The optimum conditions were found that reaction temperature was 40 ℃,concentration of potassium phosphate buffer ( pH 7. 0 ) was 0. 2 mol/L and mass concentration of L-aspartic acid was 100 g/L. And the optimal catalytic conditions of DaaT were as follows:reaction temperature 42 ℃, 0. 2 mol/L of potassium phosphate buffer (pH=7. 0),4 mmol/L of pyridoxal phosphate,50 g/L of DL-aspartic acid,and mole ratio of PA to D-Asp 1:10. Under the above conditions,treatment of the transformation product by a method of isoelectric point crystallization and cationic resin separation afforded D-alanine. The conversion of D-Asp was 94%,and the yield of D-alanine was 84%. In addition,the enantiomer excess value ( ee value) of D-alanine reached 98%.