| Phytases catalyze the hydrolysis of phytic acid or phytate(myo-inositol hexakisphosphate) to inositol and inorganic phosphate. Their roles in eliminating anti-nutritional effects of phytate in plant fodder or foods, in increasing the efficiencies of protein and various microelement utilizations in the animal body and the animal productivities, in reducing the amount of phosphorus in animal excretions and thus reducing environmental pollutions, etc, have been the research focuses domestically and internationally. Phytases are found mainly in plants and microorgnisms. Although microbial phytases have great developmental potentials, its producing ability in natural microbial strains is limited, impeding its further utilization as phytase-producers. In addition, some unfavorable properties of phytases such as heat instabilities can't completely meet the requirements of fodder processing and rearing industries. All these could be improved by means of gene engineering. Through the screening of phytase-producing microbial strains, cloning and modifying of phytase gene and constructing engineered strains by molecular biology methods, it is possible to increase the production of phytases, modify its properties, reduce costs and increase its efficiencies. Presently it's one of the research hot points the worldwide.This investigation aims to screen out the high-yield acidic phytase-producing microbial strains and men further to select for high yield strains by mutagenesis and still then to clone and analyze the phytase gene in the high-yield phytase-producing mutant strains. On these bases, to construct high yield phytase gene engineered microbial strains for the industrialized production of phytase.Main results are as follows. 1. The screening of high yield phytase producing microbial strains.In total 102 strains of acidic phytase producing strains were selected from soil by selective plate containing calcium phytate. Among them 32 strains with relatively large clear circle were purified and re-selected by shaking-culturing. After fermented for 5days at 28 C and shaking at 220r/min, the activity of phytase was determined by NH4VO4 -(NH4)6Mo7O24 method at 37 C and pH2.5 or pH5.5. Results show that all together three strains with relatively high production and stable phytase were selected. Strain 03214 showed the highest enzyme activity at 37 C and pH2.5, reaching 345U/mLfermentation. Strain 041 and 0324 showed the highest enzyme activity at 37'C and pH5.5, reaching 285U/mLand 250U/mLfermentation respectively. Strain 03214 was used for further characterization and as starting strains for selection. Preliminary result show that strain 03214 was Aspergillus niger, designated Aspergillus niger 03214.2.Selection of high yield phytase producing strain Aspergillus niger N14 by complex mutagenesisThe starting Aspergillus niger 03214 was induced with UV treatment at various time periods and then plating screened and shaking-culture re-screened. A high phytase activity mutant strain 03214-3 was obtained with 373U/mL fermentation, up by about 8% compared with the starting strain. And then the mutant strain 03214-3 was induced with N-methyl-N-nitro-N-nitrosoguanidine (0.8mg/mL) at various time periods; and finally a mutant strain, designated Aspergillus nigerN14, with increased phytase activity, 22.3% more than the original strain Aspergillus niger03214, reaching 422.3345U/mL fermentation, was obtained.Aspergillus nigerN14 was sub-cultured on PDA medium for 7 generations, and the activity of each generation was monitored respectively. Results show that the activity was stabilizes somewhere between 410~430U/mLfermentation, suggesting good genetic stability of the mutant Aspergillus niger N14, which could be used for further gene engineering investigation. 3. Extraction of genomic DNA and cloning of the gene phyA from Aspergillus nigerN14 The genomic DNA of the mutant strain Aspergillus nigerN14 was extracted by benzyl chloride and trace method. UV spectrophotometer determination of the preparations show the ra...
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