Breeding Of Aureobasidium Pullulans Produced Pullulan And Studying The Influence Of Medium Concentration On The Molecular Weight Of Pullulan

Pullulan is extracellular natural macromolecular polysaccharide,which is produced by fermentation with Aureobacidium pullulans.It is a novel kind of biodegradable material.In recent years,Pullulan was widely studied and applied in the filed of drug delivery,drug control,biological materials(such as hydrogel and artificial bone)and so on.The development of these application will directly enhance the value of pullulan,meanwhile,it also puts forward new requirements for the molecular weight size and distribution of pullulan.In particular,the demand for the high molecular weight of the pullulan(Mw>1000 kDa)existed problems.The highest molecular weight of the commercialized pullulan was 300 kDa,which was largely limited the application of pullulan in biomedicine.The aim of this subject was to breed a low pigment pullulan producing strain by the strain(ultraviolet mutagenesis and chemical mutagenesis),and study the influence of medium concentration on the molecular weight of pullulan.Finally,the transcriptional technology was firstly applied for annotating and analyzing the differential expression the function genes during the fermentation process under before and after optimization medium culture.The main results obtained were listed as follows:1.In the present paper,A.pullulans AP5 of our research term,which could produce extracellular polysaccharide,was identified though morphological and molecular biological,Meanwhile,the extracellular polysaccharide had been demonstrated that was pullulan by Fourier transfer-infrared spectrometry(FTIR)and enzymatic hydrolysate;Through ultraviolet mutagenesis and sodium nitrite mutagenesis,the low pigment and high-yielding A.pullulans SWP35 were obtained.The fermented liquid pigment content(OD640)was 0.162 through fermentation medium(g/L):sucrose 100.0,MgSO4.7H2O 0.2,K2HPO4 5.0,(NH4)2SO4 0.6,yeast powder 1.7 NaCl 1.0,pH 6.5,which decreased 21.10 times compared to the original A.pullulans AP5.The molecular weight was to 174480 Da,and the product quality was accord with national standard GB28402-2012.2.Through the Plackett-Burman fermentation optimization,it is concluded that sucrose and ammonium sulfate were positive significant effect for the molecular weight of pullulan under the 95%level of credibility,and yeast extract was negative effect;The high molecular weight of the pullulan fermentation medium was obtained by slope-climbing test and the central composite design.The optimized medium contained(g/L):sucrose 100.0,MgSO4.7H2O 0.2,K2HPO4 5.0,(NH4)2SO4 2.2,NaCI 1.0,pH 6.5.The molecular weight of pullulan reached to 845314Da under the optimized medium.Compared with pre-optimization medium culture,molecular weight increased 4.84 times.3.The 5L fermentation tanks were simultaneously carried out under the fermentation conditions of the optimization(Experiment group,EG)and the pre-optimization(Control group,CG).The molecular weight of pullulan reached respectively to 845274Da and 193349Da after 96h fermentation time.The cells under 72h fermentation time were studied as subjects in reference to the difference of the molecular weight of the pullulan during fermentation process.Based on transcriptome and enzyme activity assay,it was preliminarily demonstrated that glycosyltransferase is a key regulatory gene affecting the molecular weight of the pullulan adopting respectively the transcription level and enzyme activity level test method.In this paper,we obtained a non-pigment producing A.pullulans SWP35,and kept the strain in the patent form of the Chinese common microbial preservation center,the preservation number was CGMCC No.11602.The influence of culture medium concentration on molecular weight of the pullulan was studied,and this research would lay the foundation for further research.It was demonstrated that glycosyltransferase was a key regulatory gene affecting the molecular weight of the pullulan,it provides a theoretical basis for the construction of engineered strains that produce different molecular weight.In addition,the results of the study will guide to produce different molecular weight pullulan according to the application requirements.It will extend pullulan application range,and improve the added value of products.