| Gentiopicroside, the main secoiridoid glucoside of Gentiana macrophylla(G.macrophylla), is a compound of pharmaceutical importance. It has variousimportant pharmacological functions including anti-oxidation, anti-inflammatory,and protective and choleretic, anti-tumor. Recently, the wild resource ofG.macrophylla tended to be over-exhausted owing to the increasing commercialinterest in gentiopicroside as a medical treatment and the felling of herbexcessively. Because of the low survival rate of seedlings and the fewproductivity of gentiopicroside in the artificial cultivation so that difficult tomeet the demands of clinical application. Together with the decreasing in thequality of medicine and affecting by the seasonal variation, so it is most urgent toenlarge the resources of gentiopicroside to satisfy the growing needs of market.AS a result, it is of great significance to establish an efficient method to obtaingentiopicroside.With the development of the low energy ion biology, ion beammutation could be widely used for the breeding of microorganisms and plant, aswell as the germplasm improvement of crops with its unique mechanism andmutagenized effects.In this research, an optimal two-stage temperature control strategy wasestablished to improve the yield and the productivity of glutathione in the batchfermentation, according to the optimization of cultivation conditions abuoutHansenula polymorphaDL-1.(H.polymorphaDL-1). For another, determiningthe optimal ion implantation conditions under the study of nitrogen ions injectedin H.polymorphaDL-1, simultaneously, breeding of stain for producinggentiopicroside by the implantation induced G.macrophylla total DNAtransformation into H.polymorphaDL-1.However, it provided the theoretical and practical basis for the productionof sesquiterpene by microbial, also, it is meaningful for the development andprotection of genetic resources of medicinal plants. Moreover, it carries out a new way for the protection and reasonable use of Gentiana scabra. The resultswere as follows:(1)An optimal two-stage temperature control strategy was established toimprove the yield and the productivity of glutathione in the batch fermentation.Through single-factor experiments, the optimum growth temperature ofH.polymorphaDL-1, the optimum biosynthesis temperature of GSH and initialpH value of medium were obtained. The results showed that the effect oftemperature on glutathione production was different from that on cell growth andmaximum glutathione was obtained at37℃, but the optimum growthtemperature was at30℃. When the temperature shifted from45℃to37℃forfurther cultivation of12h, a glutathione yield of416.13mg/L and an intracellularglutathione content of103.15mg/g were achieved, which was69.6%and55.4%higher than that of the constant temperature fermentation, respectively.Subsequently, scale-up studies were conducted in a5L fermenter, a cell dryweight of42.35g/L and a glutathione content of927.68mg/L were achieved.Therefore, the temperature-shift strategy would be a valuable alternative toimprove glutathione production.(2)The conditions of transformed H.polymorphaDL-1by nitrogen ionsimplantation are that implanted ten seconds and interval of ten seconds by25keV nitrogen ions (N~+) at a dose of2.5×1016ions/cm2under the vacuum pressureof1×103Pa.Under the condition, the positive mutation rate of the wild strainH.polymorphaDL-1cells were mutated by nitrogen ion beam is38%, as thesame time, obtained an industrial strain with high-producing glutathione andZinc Chloride–resistant is named of H.Polymorpha18(HP18) which waspreserved in China Microbiological Cuture Collection Center on September29,2012, with the number of CGMCC.NO.6642. The intracellular GSH content ofHP18in the shake flask reached to337.16mg/L by ion beam implantation,1.56times more than that of the original strain when the fermentation time, a cell dryweight of6.24g/L were achieved which was9.09%higher than the originalstrain.(3)Through the analysis of the composition of G.macrophylla by chemicalreaction、layer chromatography and RP-HPLC method established the detectedmethod of gentiopicroside in the fermentation broth. a) the qualitative detectable method acid hydrolysis reaction(10%sulfuricacid), alpha naphthol and alkaline tartrate copper test;2%copper sulfate、10%vanillin and Weiggering test;20%sodium hydroxide and hydrochloricacid-magnesium powde tests;b) The content of gentiopicroside was determinated by RP-HPLC,The determination of gentiopicroside by HPLC was enforcemented with amobile phase of Methanol: Water (30:70) on Symmetry-C18column(250mm×4.6mm,5μm) under ambient condition. Also, adding20microliterssample with the speed of1ml/min detecede on the wave leght270nm with thedetector Waters2487, the maximum absorption peak appeared in the12.3minute,and the RSD of recovery test and accuracuy test were was1.16%and2.2%,respectively.(4)The purity of G.macrophylla genomic that extracted by Plant genomeextraction DNA kit was1.8(±0.02) detected by spectrophotometer and itsconcentration was about1μg/μl, which did not contain such as protein andpolysaccharide(5)Studying on nitrogen ion implantation induced the randomtransformation of G.macrophylla total DNA into H.PolymorphaDL-1of ionbeam mediated transgenic technology,30recombined yeast strains werescreened by the qualitative detectable method was accounting for3.98%of thetotal colonies that selected randomly.(6)One strain which number was DL10049with producing gentiopicrosidewas obtained by rescreening. TLC and HPLC method detected thegentiopicroside of fermentation cultivated for72hours in the shake flask was2.588mg/L and the dell dry weight was4.6g/L.(7)This recombinant strain was unstable, the gentiopicroside content ofsixth generation was decreased30.9%compared the second generation. |
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