The Fermentation Production Technology Research Of Nucleotide

Nucleotide has many important properties and functions and plays an important role in many aspects such as the composition of the cell,energy production and consumption,organism metabolism and function regulation.So nucleotide has wide applications which are closely related to human life.We often choose Bacillus or Brevibacterium ammoniagenes to product nucleotides in industrial production.Our laboratory preserved the Brevibacterium ammoniagenes for the wild type,whose capacity of nucleotides fermentation production was low.So,it was necessary to carry out mutation breeding of Brevibacterium ammoniagenes strain to change its inheritable character and metabolic pathway,making the bacterium synthetic ways miss some intermediate.Thus we got auxotroph strain to roll up the required product.In this paper,we preliminarily studied the fermentation production of nucleotides by Brevibacterium ammoniagenes,mainly including high yield strains breeding,screening seed and fermentation medium,optimization of fermentation conditions,establishment of determination method and mixed culture in the fermentation tank.The method of mutate breeding were ultraviolet mutagenesis,ultraviolet mutagenesis combined with microwave mutagenesis and diethyl sulfate mutagenesis.The auxotroph strains were screened and identified by culturing mutant strains in supplementary medium,which were enriched by penicillin bacterium.The results showed that 10 seconds microwave radiation induced the strains with 30 seconds ultraviolet mutagenesis could meet the demand of production.The test conditions of fundamental measurement and high performance liquid chromatography(HPLC)method were optimized and established the HPLC standard curve.The optimum extraction condition was 0.1 mol/L KH2PO4 leaching 40 minutes at 40? in view of extraction solution composition,extraction time and extraction temperature.The determination conditions for HPLC were optimized through the investigations of mobile phase composition,mobile phase flow and extraction time.The optimum determination conditions were that 0.1 mol/L KH2PO4:methanol=9:1,flow 0.5 mL/min in the detection wavelength of 260 nm and column temperature 30?.From the one-way analysis and orthogonal experiment design,the best liquid fermentation condition was optimized.The optimal seed medium for producing nucleotide was composed of soluble starch 2.0%,corn steep liquor 3.0%,urea 0.2%,MgSO4·7H2O 0.2%,NaCl 0.2%,pH 7.0.Fermentation medium for producing nucleotide was that:molasses adding amount 11%,cottonseed protein powder 8%,MnSO4 0.01%,K2HPO4 1%,KH2PO4 1%,CaCl2 0.01%,MgS04-7H20 1%,pH 8.0.When the mutant strains of Brevibacterium ammoniagenes fermented separately,the best fermentation effect was obtained with 6%inoculation quantity at pH 8.0 and fermenting 4 days by 190 r/min at 32?.If all other things were equal,the culture temperature was improved from 28? to 34? after fermentation 30 hours,shorting the training period to 78 hours.The nucleotide output by using mutant strains and bacillus subtilis at 30? for 3 days was similar to the elevated temperature test with 6%inoculation quantity.