Extraction And Purification Of The Key Enzyme Of Tartaric Acid Degrading From Aspergillus Niger F1and Study On The Properties Of The Enzyme

Tartaric acid is the theme acid of the Amur grape, because of the high content, it has a great impaction on the quality of Amur grape wine,such as the colour flavor taste and so on. So it is tartaric acid which becomes the bottleneck of the Amur grape wine industry development.the degradation of Physical and chemical have many disadvantage,the microbial degradation, compared with the Physical and chemicai, has obvious superiority. Therefore, domestic and foreign scholars have been dedicated to seeking one microbe which can disintegrate the tartaric acid.The lab of agricultural products processing and storage project of JiLin Agricultural University get a microbial strains by extraction and purification, which can degradation the tartaric acid existing in the amur grape wine. It belongs to Aspergillus Niger after identification by the method of morphology and molecular biology. Optimize the degradation process further, the degradation ability is above90%, but the mechanism is unclear so far as to now.The mechanism of Tartaric acid degradation is as follows:put the Aspergillus niger strain which can degradate the tartaric acid into the environment which take tartaric acid as the only carbon source to grow to induced to produce the enzymes which can break down the tartaric acid, then get the key enzyme of tartaric acid metastasis by separation and purification, find out the gene which control the compose of the key enzymes by sequencing of mass spectrometry, comparison and analysis. Further, turn it to yeast and degradate the tartaric acid directly after get the train which has get the gene successfully.This paper take strain F1as the research object, which was obtained by Agricultural Products Processing and Storage Project of Food Science and Engineering College of Jilin Agricultural University in2008. Extraction and purification the enzyme which can degradation tartaric acid existing in the microbial strains after induce by tartaric acid and study the properties of it, which can provides theoretical guidance for the further study of the mechanism of tartaric acid degradation, molecular cloning,genetically modified and the application of industrial.Induce F1strains taking tartaric acid as the only carbon source, producing tartaric acid degradation enzymes cultivating four days by shaking, the cell wall of F1strains was destroyed by liquid nitrogen freezing and grinding to withdraw the intracellular tartaric acid degradation enzyme with buffer solution assisted with utrasonic, get the liquid of the enzymes by centrifuge to remove the mycelium, heating to remove the miscellaneous protein, concentrate by polyethyleneglycol.Tartaric acid degradation enzymes was purified by using DEAE-cellulose ion exchange chromatography and Sephadex G-75gel filtration chromatograpy, get the single components of the enzymes of tartaric acid degradation. On this basis, study the properties of it, the results are as follows: Most miscellaneous protein had been removed by controlling the detail conditions of ion-exchange chromatography and molecular sieve chromatography. The puritied tartaric acid degradation enzymes showed a single band in PAGE electrophoresis. Indicate it reaches the electrophoretically pure. The specific activity reached289.69U/mg, which was14.32folds higher than the crude fermenting liquor and the yield was28.63%The molecular weight of the subunit protein was70000Dalton measured by SDS-PAGE electrophoresis, conjecture the enzymes content two same subunit protein, so the molecular weight of the tartaric acid degradation enzymes was140000Dalton.The test of substrate specificity show that:the tartaric acid degradation enzymes has altitudinalsubstrate specificity, it only can degradate L(+) tartaric acid, malic acid and citric acid are inactive as either subatrates or inhibitors. For L(+) tartaric acid the Km is36.61mmol/L, Vmax is37.88μmol/min, which was tested by dual-reciprocal graph; the optimum temperature and pH value were48℃and8.5respectively. The range of pH value stability was narrow, and it appears a curve which has two peaks when maintain48h at48℃, the activity was almost unchanged at pH=7.0, presenting the most tolerance, the activity was dropped to61%at the pH=5.0, and all the activity is less than50%besides two of the pH,indicate that the enzyme is sensitive to the pH.Also,the enzyme is sensitive to the temperature. the activity dropped to64%maintaining60mins at20℃, the activity maintain80%when keep60min at30℃, the activity dropped to55%maintained60min at40℃, there is only a little activity at50℃, and when the temperature reaches60℃, there is no activity left.Metallic ions had influence on the tartaric acid degradation enzymes. The enzyme was inhibited strongly by Ag+、Pb2+、Hg2+and SDS and was inhibited a little by Cu2+and Al3+, but it can be actived by Na~+、K+NH4+、Mg2+、Zn2+、Ca2+and Fe2+in some extent.This paper has done some basic studies on the tartaric acid degradation enzymes including the extraction, purification and the properties of the enzymes and this will offer a theoretical direction for the study of its catalysed mechanism, clone and industrial application.