为了提高表达玫瑰色红球菌（Rhodococcus rhodochrousJ1）来源的高分子量腈水合酶大肠杆菌 Escherichia coli?BL21(DE3)/pET-24a(+)-nhhBrbsArbsG（BAG）的菌体酶活，建立合适的烟腈全细胞催化工艺以及重组工程菌的高密度发酵工艺，采用正交试验L9(34)对摇瓶培养基中各组分进行优化，比较烟腈底物流加和底物分批补料工艺，对重组菌进行分批补料高密度发酵培养。培养基优化后其菌体生物量提高到4.42?gDCW/L，细胞比酶活提高到30.91?U/mgDCW；与烟腈底物流加相比，烟腈分批补料工艺更适合烟酰胺的生产，通过此工艺的反应，摇瓶发酵后的BAG能够生产390?g/L烟酰胺；对BAG进行5?L发酵罐分批补料扩大培养，菌体生物量最高达到73.97?gDCW/L（OD600=200），细胞比酶活最高为42.70?U/mgDCW，单位发酵液酶活最高为2813?U/mL，用较高密度的发酵罐培养后重组菌进行烟腈催化反应，产物浓度能达到537 g/L，是已报道的大肠杆菌重组表达腈水合酶产烟酰胺所得终浓度的最高水平。

To increase the biomass and the activity ofEscherichia coliBL21(DE3) /pET-24a(+)-nhhBrbsArbsG(BAG) expressing high molecular mass nitrile hydratase fromRhodococcus rhodochrousJ1 and establish proper process of whole-cell catalysis of 3-cyanopytidine and high cell-density cultivation, we used orthogonal experiment (L9(34)) to optimize elements in culture media, comparing methods of substrate-flow and substrate fed-batchand applying technology of fed-batch to achieve high cell-density cultivation. After the optimization of culture media, the biomass and the specific activity of BAG reached4.42 gDCW/L and 30.91 U/mgDCW in the flask, respectively. The process of substrate fed-batch was more appropriate and when it was applied in the production of nicotinamide using BAG, the concentration of nicotinamide reached 390 g/L. After the high cell-density cultivation of BAG in 5 L bioreactor, the biomass of cell attained 73.97 gDCW/L(OD600=200), the specific activity and the total activity reached 42.70 U/mgDCW and 2813 U/mL, respectively. When we used high density of BAG after fermentation in the tank to catalyze 3-cyanopytidine, we obtained 537?g/L of nicotinamide, which is the highest concentration of nicotinamide that produced in recombinantE.coilexpressing nitrile hydratase.