DNA Finger-print Analysis And Fermentation Research Of A.Pullulans Mutated By High Pressure

High-pressure biology is a new cross-field of high pressure physics and biology. The effect of high pressure to thermodynamics parameters,kinetics characteristic and regulatory mechanism of biosystem and the molecular mechanism of high pressure to nucleic acid and protein have been studied and acquainted widely. The Aureobosidium Pullulans was induced under diffrent high pressure process(80MPa,160MPa,240MPa,320MPa). The flocculant activity of the mutant strain metabolites used as screen target.The flocculant activity of the mutant metabolites was improved. For the purpose of finding the best ferment condition, an orthogonality experiment was carried out in three elements and three levels to optimum combination of glucose\sucrose concentration (source of carbon), original pH and yeast extract concentration (source of nitrogen) in the medium. The main elements which affect flocculant activity are the kind and the concentration of the carbon source and initial pH.Synchronous culture the original strain and the mutant strain respectively in industrial medium and laboratory medium. Flocculant activity, amylose and pigment contents of the ferment products were measured. The results show that in the fifth day the amylase contents of the mutant strain exceeded the original strain by 8%. As the time of culture extended, the flocculant activity of the two strains were all declined as the result of the change of the fermentation. The colours of the two strain's ferment fluid were remarkable different, illustrated that high pressure treatment caused pigment secrete of the mutant strain changed. The flocculation constituent analysis demonstrated flocculant-active ingredient was the extropolysaccharides.Total genomic DNA was extracted from high-pressure treated A. pullulans strain and the untreated original strain . The genomic DNA was amplified using oligonucleotides of random sequences by polymerase chain reaction (PCR). RAPD bands specific to the two A. pullulans strains were selected from the polymorphic pattern yielded with primer S11,S15,S24,S42. The DNA sequences were submitted to GeneBank.Homology searche was performed within GenBank's database using the BLAST (Basic Local Alignment Search Tool) algorithm. To predict the structure of proteins encoded by the specific bands, 3D-PSSMfold recognition server procedure was performed. The result of analysis revealed these proteins involved in a series of life activity such as nutrition and energy metabolism of microorganism, maintain the cell shape and gene expressing regulation etc. The appearing of these specific bands proved that high pressure treatment arouse the change of physiology and biochemistry which was caused by hereditary molecular altered. The physiological implications and molecular biology analysis results, which were derived from the experimental data, are discussed, suggesting that high pressure process was an effective technology providing a theoretical evidence for discovering the metabolic network in microorganism responsive to high pressre.