Isolation And Idenfition Of Pseudomonas Fluorescent With Gsh Production Capability And The Optimization Of Fermentation Conditions

Glutathione (GSH) is an important bioactive peptide which is playing anincreasingly important role in medicine and foodstuff. At present, GSH is usuallyproduced by yeast fermentation. The production of GSH is painfully low by bacteriumfermentation, but bacterium has many advantages: large variety of species, simplenutritional requirements, short growth cycle, modify by genetically engineering easily.Therefore, we carry out the research that the production of GSH by bacteriumfermentation, which may solve the problem of single variety and may got the stablehigh-yielding strain.The gshB gene is the key gene in the production of GSH. By using Genebank,Pseudomonas fluorescens containing gshB gene was chosen to produce GSH. We carriedout experiment for the purpose of Screen Pseudomonas fluorescens with high-yieldingGSH.1. Samples were collected at the root of grape, banana and other plant at Shatangfarm Liuzhou Guangxi. Pseudomonas fluorescens could grow at4℃and fluoresce inultraviolet light. So we put Samples at4℃for enrichment and then streak cultivationwas used in KB medium. Colonies with luorescence in ultraviolet light were chosen tostreak cultivation.7positive isolates of12isolates were got by using specific primerPCR.7positive isolates were screened by using specific primer PCR based on gshB gene.Then DTNB method was used to analysis the GSH production. One isolate, namelyBJYG12, with high-yielding GSH was obtained and identified as Pseudomonasazotoformans by biochemical assays and16S rDNA sequencing.2. The optimal fermentation conditions of BJYG12were confirmed as follows:fermentation temperature28℃, pH7.5, inoculum5%. By orthogonal experiment: thebest carbon source is glucose； the best nitrogen source is yeast extract; glucose20g/L,yeast extract25g/L, phosphate1.5g/L, magnesium sulfate2.5g/L. The optimized mediumincreased18%in production than KB medium. Feeding fed-batch culture method wasused for the higher production of GSH. Change the culture at the end of logarithmicgrowth for the first time. Then change the culture at at the middle or end of stabilizationgrowth. Through this method, the production of GSH increased30%than before.3. The content of GSH was determined by High Performance LiquidChromatography (HPLC). Analytical conditions for HPLC was as follows; Symmetryconlumns C18temperature30℃, flow velocity0.8mL/min, determine wavelength210nm,carbinol and30mmol/L phosphate (containing0.1%Sodium octanesulfonate) in aproportion of7.5:92.5. This method is efficient and feasible which confirmed by precision test, stability test and recovery test. At the same condition, DTNB test got alittle higher result than HPLC test, but they are almost the same. So DTNB test is simpleand could be used at preliminary assay. HPLC test could be used at accurately andprecisely assay.