Mutation Breeding Of Strain For Biotransfor-Mation Of Iminodiacetonitrile To Iminodiacetic Acid And Optimization Of The Fermentation

Iminodiacetic acid （IDA） is an important fine chemical intermediate. Ithas wide applications and is mainly used in producing herbicide glyphosate.By now, IDA, as intermediates, mostly used in manufacturing glyphosate,and has been a growing demand. At present, the production of IDA all usechemical methods, including hydrocyanic acid method, diethanolaminemethod, hydrolysis method of iminodiacetonitrile,chloroacetic acid method.These methods lead to environment pollution, low yield and high cost.Enzymatic hydrolysis of IDA has some advantages, such as the mildcondition, environmental friendly and low cost. But enzymatic hydrolysis ofIDA faces a main problem, the low nitrilase activity. Therefore, techniquesas mutation breeding of strain and constructing genetic engineering bacteriaare required for the purpose of improving nitrilase activity. In this study, IDAwas selected as the research subject. Purification of the product, the mutation breeding of Alcaligenes faecalis ZJB09133and the optimization offermentation conditions were studied in detail.Firstly, the structure of the product, produced by biocatalysis ofiminodiacetonitrile, was confirmed as IDA using IR, ESI-MS and NMR.After discoloring by2%active carbon, under the condition of50℃and1hof treating, the discolor rate of iminodiacetic acid from bioconversion brothreached93.4%. When the volume of methanol was twice than that of IDAsolution, the crystalloid purity of IDA was98.3%and yield of IDA reachedalmost the largest. As a result, using the method of process cooling, themonoclinic crystal was obtained. Under the method mentioned above, thecrystalloid purity of IDA was98.8%, and the total yield was40.2%.An iminodiacetic acid producing strain, Alcaligenes faecalisZJB09133, isolated from soil, was treated with UV, UV-LiCl andlow-energy ion beam implantation. When the UV irradiation time and N+implantation dose was15seconds and60×2.3×10¹³ions/cm², respectively,as a result, the positive mutation rate reached the highest, which were20%and22%, respectively. Four mutant strains selected had higher relativeactivity. The three mutant strains were steady through five breeding. Amutant stain named UL144had the highest relative activity, reached58.8U/g DCW, which was77.1%higher than that of the original strain.The conditions of fermentation for mutant stain UL144was optimizedby single-factor method. The optimum medium composition was found as follow （g/L）: ammonium acetate6, yeast extract4, K₂HPO₄5, Al₂（SO₄）30.1, NaCl1, n-butyronitrile0.6%. The optimum conditions for cell growthand nitrilase formation were as follows: initial pH=8.5, medium volume80ml/500ml, inoculated volume8%. Under the above conditions,1.44g/Lbiomass and69.49U/g DCW relative activity were achieved aftercultivation of34h.