Screening Of Becteria Producing Nicotinamide In Microbial Method And Optimization Of Enzymatic Reacting Conditions

Nicotinamide is a kind of Vitamin B and it’s very important. In the body, It’s one part of the formation of two kinds of coenzyme. Nicotinamide is mainly used in feed industry as nutrition additives, it also has a very broad application prospect in medical industry, food industry and cosmetics industry. In the industrial production of nicotinamide,we mainly use chemical method and biological method. The biological method has a big superiority that chemical method hasn’t it does little harm to the environment and has a lower production cost, and it’s very simple in process.Under the conditions of laboratory, we mainly carried out our research in several aspects and achieved some results as follows:(1) Started from the strain Norcardia sp.HD1220 we had in the laboratory, we used the method of ARTP mutagenesis, microwave mutagenesis and EMS mutagenesis to make mutagenesis of the strain. The three methods were all used alone. Then we made the composite mutagenesis under best conditions of the three method of mutagenesis. The best conditions of composite mutagenesis were: the time of ARTP mutagenesis was 240 s; the time and intensity of microwave were 90 s and 60 %; the concentration of EMS mutagenesis was 0.8 %. By the mutagenesis we screened one stain whose nitrile hydratase had a very high enzyme activity of 1764 U/mL and the stain had stable heredity. Compared to the stain Norcardia sp.HD1220, the enzyme activity increased by 33.13 %, and we named the stain as NHD-1.(2)In the detection of Nicotinamide, we used the HPLC method. By optimizing the wavelength of UV detection, the flow rate of mobile phase and the proportion of mobile phase in the HPLC assay, we got the best conditions of HPLC method : the wavelength of UV detection was 220 nm; the flow rate of mobile phase was methanol: water=1:3; the proportion of mobile phase was 0.4 mL/min. under these conditions, the peak time of niacinamide was 5.306 min. Compared to the method before optimization we used, the peak time significantly shortened.(3) By optimizing the conditions of catalytic reaction of the nitrile hydratase, we got the best conditions: the concentration of bacteria producing niacinamide was 1.8 %; the fungus age was 48 h; the concentration of substrate was 100 g/L; the temperature was 29 ℃; the PH was 7; the reaction time is 180 min, and we should separate nicotinamide late in the actual fermentation. By Plackett-Burman experiment we confirmed that conditions of concentration of substrate, temperature, PH had significant influences on the enzyme activity of nitrile hydratase. By response surface test we optimized these three factors: the concentration of substrate was 98.88 g/L; the temperature was 29.10 ℃; the PH was 7.14. Under these conditions, we analyzed the enzyme activity is 1784 U/mL. Through verification test, we confirmed the enzyme activity of nitrile hydratase was 1775 U/mL. It was very close to the theoretical value. It showed that the response surface test was a very effective method in optimization of enzyme catalysis.