Mutation Breeding Of High-yielding Fibrinolytic Enzyme Strains XY-1and Study On Its Enzyme Production Conditions

Objective:The aim is to obtain higher fibrinolytic enzyme producing strains throughmutation breeding method, then to obtain the best fermentation condition of mutantstrain by single factor experiment and orthogonal test.Methods:UV mutagenesis was used to breed the fibrinolytic enzyme producing strainXY-1; the method subculture for ten passages was used to selecting stable mutantstains. Fibrinolytic enzyme gene was amplified by PCR method from mutant strainand starting strain; then they were compared with the PubMed BLAST to see whetherthere are differences in their genes. The method shake-flask experiments andmeasurements of OD600were applied to research the difference of growth curvebetween mutant strain and starting strain. The shake-flask culturing was used infermentation of mutant strain and starting strain to research the difference ofproducing enzyme curve between them. Mutant strain and starting strain wasfermented with shaking flask fermentation method obtaining two kinds of enzymes.To research thermal stability of enzyme, two kinds of crude enzymes were kept at thetemperature of-20℃,4℃,37℃, room temperature,40℃,50℃,60℃and70℃forsome time, then the activity of enzymes ware measured.The optimal carbon and nitrogen source was selected from glucose, xylose,sucrose, maltose, lactose and peptone, ammonium sulfate, yeast extract, peptone, beefrespectively, using single factor method. Orthogonal test of4factors and3levels wasused to optimize the fermentation medium components. The fermentation conditions including temperature, inoculate volume and primary pH were determined by singlefactor method.Results:Two fibrinolytic enzyme producing mutant strains were screened that couldsteadily inherited10generation, the two mutant strains were named WY-1and WY-2.Both of their yield of enzyme reached3times of the starting strain. The similarity inenzyme gene of starting strain and mutant strain was reached99%; there was nosignificant difference between them. The culture time to the logarithmic growth phaseand maintaining the logarithmic growth phase was similar between starting strain andmutant strain, but the growth quantity of the mutant strain is larger than the startingstrain, at the same time. The enzyme production rate of strain and the mutant strainwas different: The starting strain reached the peak of enzyme production in17h, itsyield of enzyme was13.8FU/ml, and its enzyme production was not declined aftercultured for ninth days; Mutant strain reached the peak of enzyme production in3days, the enzyme amount was43.67FU/ml, after6days the yield of enzyme showeda downward trend, at9days down to35FU/ml. The enzyme production of mutantstrain was3.15times of that of the starting strain. The thermal stability of the twokinds of enzymes was similar. Two kinds of crude enzymes can save about80%in16days at the temperature of-20℃,4℃, room temperature; in37℃and40℃theenzymes could save80%in5.5h, when the temperature was higher than50℃, itsstability decreased significantly.The optimal carbon and nitrogen source was maltose and peptone. The optimalfermentation medium components were: carbon source was2%, nitrogen content is3%, and K2HPO4/KH2PO4is0.2%/0.1%, magnesium sulfate0.1%. The fermentationconditions were as follows: the incubation time was72h; culturing temperature was37℃, primary pH was7,0.5%for inoculation.Conclusion:Obtained a high yield mutant strain by UV mutagenesis, its fibrinolytic enzyme yield was43.67FU/ml, was3.15times of that of the starting strain. The expected goalof mutagenesis was achieved. When optimized the culture conditions of the mutantstrain, the fibrinolytic enzyme in production reached53FU/ml, which was1.65timesbefore optimization.