| Nicotinic acid, also named pyridine-3-dicarboxylic acid, is a necessary chemical toanimals. Nicotinic acid is widely used as oxidation and reduction in daily life. At present,nicotinic acid is far beyond the domestic demand and most depend on input. The traditionalmethod of production is chemosynthesis, and many shortages are still in existence such assevere pollution, and more byproduct and rigorous reaction condition. By contrast of thechemosynthesis, the microbial conversion has many advantages, such as environmentalfriendly process, less byproduct and mild reaction condition. The new manufacture method ofnicotinic acid accords with the direction of future production.This topic focused on the study and development of synthesis nicotinic acid by microbialconversion of3-cyanopyridine. The paper was divided into three parts to the process, and thedetails were as follows:1. The studies of catalytic process of Pseudomonas putida XY4resting cellsWe studied the effects of pH, temperature, metal ion, substrate concentration, productconcentration, organic solvent, enzyme modifier and cell concentration on enzyme activity,the pH and temperature stability, the enzyme’s substrate specificity and biotransformationprocesses of nicotinic acid were also been studied. The results showed that, the optimalcatalytic conditions were: pH7.2,30℃and the substrate concentration was50mM. Theenzyme activity was strongly inhibited by Ag+, Cu2+and Fe3+. It showed that there was anecessary sulfhydryl group in the enzyme structure. Urea and glycerol can improve theenzyme activity by10%and EDTA can reduce70%of the enzyme activity. The enzyme had ahigher tolerance to the high concentration of hexane and xylene. The nitrilase had a highspecificity on aromatic heterocyclic nitrile, such as3-cyanopyridine and4-cyanopyridine,followed by glycididazole nitrile and acrylonitrile. The nitrilase had no specificity on amidecompounds. It indicated that the bacteria did not produce nitrile hydratase and amide, butaromatic nitrilase. The converting rate of the substrate was5.9g/h/one gram biomass.2. The studies of continuous production of nicotinic acid We studied the effects of resting cell biomass, reaction system and feeding strategy on thetransformation ability. The results showed that, it was disproportionately on increasedamplitude caused by the enzyme inhibition of substrate or product in the resting cellstransformation reaction system. The growing cell system is superior to the resting cell systemin the choice of reaction system. The variable-speed feeding mode using high substrateconcentrations was better than others. The highest conversion datas we achieved were asfollows: transformation of3-cyanopyridine156.2g, generation of nicotinic acid185g,conversion time7h, conversion rate100%, molar conversion rate118%. The results wereobtained in1L system of variable-speed feeding mode using high substrate concentrations.Besides, we determined the best seed age of fermentation, the result was20h and the bestinoculation amount was3%.3. Extraction and identification of the productThe optimum extraction of nicotinic acid in the conversion solution was:（1）The pre-processing stage of conversion solution: heat treated30min on70℃,centrifuged20min on8000rpm.（2）The extraction stage of crude product:Acid precipitation-crystallization, adjusted pHto3.7by slowly added acid to sapple, centrifugued and dried crystal. The yield and purity canbe achieved to90%. The residual nicotinic acid in the supernatant can be recoveried by rotaryevaporator afer centrifugation.（3）The refining stage of product:Decolorization and Recrystallization can been done at the same time. The optimalparameters of decolorization by adsorbent was as follows: activated carbon3%, temperature80℃, reaction time40min.The identification results of refined product showed: purity99.9%. All the spectroscopiccharacteristics were in line with the requirements and the product can be applied to food andmedicine and other fields.The results of the research can not only promote industrialization of nicotinic acidproduction with bio-catalyzing method, but also be useful for producing other similar organicacid from cyanic compound by microbial bioconversion. |
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