双酶连续催化生物合成D-丙氨酸

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%。

Biosynthesis of D-Alanine Continuous Catalyzed by Aspartic Acid Racemase and D-Amino Acid Transaminase

A method for the preparation of D-alanine by continuous catalyzed of aspartic acid racemase and D-amino acid transaminase was established. DL-aspartic acid was obtained by racemization of L-aspartic acid with whole cell of aspartic acid racemase, and then the whole cell was removed by centrifuging, and the residual aspartic acid racemase was inactivated by heating. Then, added D-amino acid transaminase purified by Ni-column affinity chromatography, D-aspartic acid (D-Asp) and pyruvic acid (PA) were converted to D-alanine. The optimal catalytic conditions of aspartate racemase were as follows: the reaction temperature was 40 oC, 0.2 mol/L of potassium phosphate buffer solution (pH=7.0), 100 g/L of L-aspartic acid. The optimal catalytic conditions of D-amino acid transaminase were as follows: the reaction temperature was 42 oC, 0.2 mol/L of potassium phosphate buffer (pH=7.0), 4 mmol/L of pyridoxal phosphate, 50 g/L of DL-aspartic acid, the molar ratio of PA and D-Asp is 1:10. The transformation product D-alanine was obtained by the method of isoelectric point crystallization and cationic resin separation. The conversion of D-aspartic acid was 94%, the yield of D-alanine was 84%, and the ee value of D-alanine was 98%.