Biotransformation Of Iminodiacetonitrile To Iminodiacetic Acid By Nitrilase-Producing Strain

The nitrile compounds and its hydralysis product are widely used in the fine chemical industry. Enzymatic hydrolysis of nitrile meets on the“green chemistry”because of the mild condition, high yields and ecofriendly nature. The desired product of this research is iminodiacetic acid which is an important intermediate during the production of glyphosate, and it is widely used in other aspect of light industry. Glyphosate herbicides are the most commonly used herbicides in the world. About 30% consumption of the herbicides is glyphosate herbicides, and it is believed that the quantity demanded for glyphosate in 2010 will be 1,000,000t. But now the mostly used manufacturing process for glyphosate utilises IDA as intermediate, that toxicity ruins the environment. The objective of the present study was to screen for a strain to transform the iminodiacetonitrile to iminodiacetic acid which should have high yield and specific nitrilase activity. The activity of the wild strain was increased through optimize culture conditions and the bio-transformation conditions of strains. The results could be useful for bio-transformation preparation of iminodiacetic acid.In this study, a high throughput screening model was established employing iminodiacetonitrile as the only nitrogen source through enrichment and then utilize the discoloration of bromocresol purple during the plate culture. At last, according to the HPLC method, strain ZJB09133 was selected for further study because of its high activity. According to the physiological and biochemical characteristics and its 16S rDNA gene sequence analysis, the strain was identified as a strain of Alcaligenes faecalis, and named Alcaligenes faecalis ZJB-09133.The conditions of fermentation for nitrilase production of A. faecalis ZJB-09133 were further optimized by single-factor method. The optimal conditions of fermentation for A. faecalis ZJB-09133 nitrilase production were as follows(g/L):ammonium acetate 7.0, yeast extract 4.0, K2HPO4 5, MgSO4 0.3, NaCl 1, n-butyronitrile 0.3%, initial pH 8.0, iooculum volume 6%, temperature 35℃。Under these conditions a biomass of 0.47g/Land specific activity 38.5U/g were achieved respectively after cultivation for 36 h.The effect of bioconversion conditions on specific activity was investigated. The optimal substrate concentration and cell concentration were 20g/L and 1.5mg/ml separately. Moreover, the characteristics of nitrilase were studied and the detailed process of biotransformation of iminodiacetonitrile to iminodiacetic acid was also investigated. The conversion rate could reach 70% under the condition of substrate 2%, conversion time course 12 h, and reaction temperature 35°C in phosphate buffer pH 8.0. This studies paved the foundation for the industrial production of iminodiacetic acid by using biotransformation method constructed in this paper.