Selection Of Nitrolase - Pruducing Strains And Optimization Of The Culture Conditions

During the process of producing acrylic acid by converting acrylonitrile using a microbial method,the nitrilase played a critical role.The efficiency of conversion frome acrylonitril into acrylic acid relies on the activity of nitrolase directly.In this study,we devote to screen nitrolase– producing strains and compare their enzyme activity to search for high nitrolase– producing strains.By establishing a rapid screening system which called rapid plates screening system to speed up the screening process, that we could screen numerous soil samples in a short time.After that,we used UV and chemical mutagenes induce the strains to mutation and used the optimization of strain growth conditions to improve enzyme production activity and enhance the stability of strains.Using gas chromatography detection of acrylic acid dencity in culture, acrylic acid can be detected within a very short time to screening a lot of samples.It is found that the optimal conditions for determinate acrylic acid was as follows: PEG-20M capillary column(30 m×0. 32 mm i. d., 0.25μm,), temperature Programming(the initial temperature of 100℃,after 1 min rise to 200℃by 70℃/min),the detector and vaporization chamber temperature were 230℃and 210℃,carrier(N2)flow rate was 7.6 mL/min.The relative standard deviation of this method (RSD)(n = 5)were 0.7% and 1.2%.The average recoveries(n = 5)were 99.83% and 100.38%.Nitrilase-producing strains were screened. Acrylonitrile is a toxic compound,for most micro-organisms in wild nature,acrylonitrile performance different toxic effects,most of them were killed in a low acrylonitrile containing environment.This toxity of acrylonitrile allows us to screening strains by using a acrylonitrile containing medium.Microorganizms in soil samples were painted on the selective medium,and stains could produce nitrolase to converting acrylonitrile into lower toxity compound acrylic acid,then they will grow on the medium and form some colonies,otherwise,they die.Acrylonitrile could be the sole nitrogen source to screen nitrolase - producing strain or use with other nitrogen source.Screening nitrilase-producing strains by enrichment culture, through add acrylonitrile into the basic medium step by step, or using acrylonitrile as the sole N source/C source,will increased to the concentration of acrylonitrile from 0.01% to 2%,increase in nitrilase-producing strains in the medium,and improve strains'toxic tolerance.Using a strain enriched and screened from the soil,0614P,was the original strain. Irradiation by ultraviolet radiation to screening mutants with higher nitrolase activity, through primary and secondary screening, the strain numbered 0614P1117 which has the highest enzyme activity.The ability of catalytic conversion of acrylonitrile into acrylic acid as target,single factor experiments were taken.The type of the carbon and nitrogen sources, temperature, pH,rotation speed, inoculum concentration,inducer concentration,substrates concentration and metal ions were studied. After orthogonal optimization,we concluded the optimize condition of fermentation as following: glucose 2%,peptone 0.9%,inoculation 6%,acrylonitrile 0.4%,Caprolactam 5%,K2HPO4 0.1%;H2PO4 0.1%;MgSO4·7H2O 0.05%,pH 7.0,temperature 28℃,rotation speed 200r/min.