Purification Of Glyphosate Degradation Enzyme From Aspergillus Oryzae And Its Spatial Structure Effected By High Concentration NaCl

The Aspergillus oryzae FUJX 001,a degradating glyphosate strain screened out by research group,was chose as the experimental strain.The strain was cultivated and induced to produce glyphosate degradation enzyme.Protein concentration and enzyme activity of the extracted crude enzyme were determined after treated by three extracting methods.The extracted enzyme obtained with the optimal extracting method was precipitated by different saturation of ammonium sulfate The glyphosate enzyme was separated from the crude enzyme by saturation of ammonium sulfate and column chromatography.And its enzyme characterization,effects of the substrate and NaCl on the enzyme spatial structure.The main results of this paper:?1?The hypha of Aspergillus oryzae FUJX 001 was treated with ice bath ultrasonic,liquid nitrogen grounding,and the combination of liquid nitrogen grounding and ice bath ultrasonic respectively.Results showed that the protein content of crude extracted enzyme obtained with ice bath ultrasonic was 0.006mg/mL and the enzyme activity was zero.The protein content and enzyme activity of crude extracted enzyme obtained with the second and third methods were 0.049 and0.052 mg/mL and the specific enzyme activity was 9.53 and 9.29 U/mg respectively.?2?The precipitation of ammonium sulfate and purification of column chromatography were studied.The extracted crude enzyme was precipitated by different saturation of ammonium sulfate.The recovery rates of protein concertration and enzyme activity were 86.0%and 84.03%when the saturation of ammonium sulfate was 80%.Protein precipitation efficiency was the best and the specific enzyme activity was 10.54 U/mg.By the further purification of Sephadex G-150,Sephadex G-50,DEAE Sephadex A-50 column,the recovery rate of protein were31.65%,17.98%,13.12%,and the recovery rate of enzyme activity were 51.84%,43.40%,36.21%,and the specific enzyme activity were 17.66 U/mg,26.03 U/mg,29.76 U/mg,and the purification fold were 1.64,2.42,2.76 respectively.The electrophoresis purity enzyme was detected by SDS-PAGE,and molecular weight of glyphosate degradation enzyme was about 34.7 kDa.?3?The optimal reaction temperature,thermal stability,the optimal pH and pH stability of glyphosate degradation enzyme were studied.The optimal reaction temperature of purified enzyme was 37°C and its thermal stability was good in the range 20³0°C,which reserved more than 90%specific enzyme activity.When the temperature exceeded 40°C,the enzyme activity declined rapidly.The optimal reaction pH value of glyphosate degradation enzyme was 7.0,its stability was perfect and specific enzyme activity were all over 85%with pH value of 6.0⁸.0.The substrate specificity study explained the optimal reaction substract concentration was1 g/L.?4?Space structure properties of glyphosate degradation enzyme effected by pH,temperature and NaCl were studied.Temperature and pH had different effects on glyphosate degradation enzyme structure.The alpha helix and beta fold structure proportion of enzyme were maximum,and the enzyme activity was maximum when the pH was 7.0.As the acidic and alkaline were enhanced,the enzyme activity declined,while the alpha helix and beta fold structure proportion presented the decreased tendency.The enzyme activity and beta fold structure proportion were decreased with the temperature raising during high temperature range,and it was suggested that hydrogen bond force was weakened,and the structure of enzyme was changed from order to disorder.So enzyme activity was effected in different extent by destorying hydrogen bond force and the secondary structure.The results showed the maximum intensity of the glyphosate degradation enzyme combined with substrate in all wave absorption had a decrease tendency,and?_max appeared blue shift,the largest fluorescence intensity appeared sharply declining trend and?_max appeared slight red shift phenomenon.The enzyme secondary structure had obvious changes.There were the increased proportion of beta fold structure and the decreased proportion of no rules crimp structure.And these changes tended to be stable in a short period of time,It explained that the combination of enzyme and substrate can be instantly in a steady state.High concentration of NaCl made the absorption intensity of glyphosate degradation enzyme decreased,and the fluorescence intensity increased,and?_maxax appeared blue shift,the proportion of beta-fold decreased,random curl conformation increased.It was possible that high concentration of NaCl effected on the microenvironment and fluorescence groups of molecule,as well as the introduction of NaCl ion increased the repulsion between the parts of protein secondary structure and enhanced the protein denaturation degree.In addition,the substrate and NaCl had effected on the extension way of some chemical bonds,resulting the absorption wavelenghth of FTIR appeared shift.