重组钝齿棒杆菌全细胞转化生产L-瓜氨酸条件优化

Production of L-citrulline by a recombinant Corynebacterium crenatum SYPA 5-5 whole‑cell biocatalyst

摘要:

实现了精氨酸脱亚胺酶 (ADI) 首次在钝齿棒杆菌Corynebacterium crenatum SYPA 5-5中的高效表达。通过Ni-NTA亲和层析纯化得到纯化ADI，经SDS-PAGE测定其分子量约为46.8 kDa，酶学性质研究发现ADI的最适催化温度为37 ℃，最适pH为6.5，ADI在最佳催化条件下作用于L-精氨酸的米氏常数为12.18 mmol/L，最大反应速率为0.36 μmol/(min·mL)。优化了重组菌全细胞转化产L-瓜氨酸的工艺条件，在最优条件下可一次转化300 g/L L-精氨酸，转化速率达8 g/(L·h)。进行重组菌5 L罐发酵并进行罐上全细胞转化300 g/L L-精氨酸，一批菌体可进行多次转化，累计产量达1 900 g以上。

Abstract:

Arginine deiminase (ADI) was first high-efficient expressed in Corynebacterium crenatum SYPA 5-5. The ADI was purified by Ni-NTA affinity chromatography and SDS-PAGE analysis showed the molecular weight (MW) was 46.8 kDa. The optimal temperature and pH of ADI were 37 ℃ and 6.5 respectively. The Michaelis constant was 12.18 mmol/L and the maximum velocity was 0.36 μmol/(min·mL). Under optimal conditions, 300 g/L of arginine was transformed and the productivity reach 8 g/(L·h). The recombinant strain was cultivated in a 5-L fermentor and used for whole-cell transformation of 300 g/L arginine, under repeated-batch bioconversion, the cumulative production reached 1 900 g/L.