The Study Of Key Factors And Mechanism Of Pullulan Biosynthesis

Pullulan, an exocellular homopolysaccharide, is produced by the strains ofAureobasidium pullulans. It is mainly made of maltotriose repeating units joined by α-（1-6）linkages. This particular linkage pattern confers pullulan with a number of remarkableproperties, such as high water solubility, good film-forming, excellent oxygen-impermeable,high plasticity, low viscosity and easy to natural degradation. This polysaccharide is widelyused in food and pharmaceutical industries. Pullulan production was under complex metabolicand environmental control. In order to understand the mechanism of pullulan biosynthesis,this paper studied the sugar metabolic pathways in pullualn formation and the proteomicsanalisis of A. pullulans under nitrogen source control. Meanwhile, the mechanism ofsurfactant enhancing pullulan production and fermentation control based on the physiologicalcharacteristics of A. pullulans were investigated. Finally, the fermentation process pullulanwas controlled and optimized by two-stage controlled pH and temperature.The highest pullulan yield was obtained when the cells were grown in the mediumcontaining sucrose. Sucrose was not simply resolved to glucose and fructose, butsyhthesised fructo-oligosaccharide by β-fructofuranosidase, which was induced by sucrose.This process could decrease the osmotic pressure of fermented liquid environment. After that,fructo-oligosaccharide was utilized by cells as carbon sourece. The activities of the keyenzymes involved in the central metabolic pathways of A. pullulans were measured incell-free extracts. We found that the EMP and HMP existed in A. pullulans, but no ED. Thepresence of iodoacetic acid could bring about the carbon flux from EMP to HMP, and reducethe pullulan production. Low concentration of iodoacetic acid could promote the cell growth.In this work, the fermentation results obtained showed that, proper concentration （0.6g/L） of （NH4）2SO4was important for pullulan biosynthesis. Excess nitrogen source wouldenhance the accumulation of biomass, but decreased the pullulan production. Nitrogenlimitation in A. pullulans seemed to influence the flux change of carbon flux flow towardexopolysaccharide accumulation. Proteins extracted from the cells of A. pullulans weresubjected to proteome analysis. Analysed through the Image Master, approximately1100visualized spots were found on the gels. After comparison and analysis the gel images,altogether19protein spots from the whole cell protein gels were excised and subjected toMALDI-TOF/TOF. Among the19protein spots cut for MALDI-TOF/TOF analysis,12protein spots were successfully identified, achieving a63%identification rate. Meanwhile, theidentified proteins were matched to known proteins from the fungus species and othermicroorganisms. Of the12protein spots identified,7were up-regulated under the nitrogenrepletion and5were up-regulated under the nitrogen limitation. The up-regulated proteins inthe nitrogen-replete sample contained nascent polypeptide-associated complex subunit beta,peroxiredoxin-6, α-glucan branching enzyme, fructose-bisphosphate aldolase, arginase,enolase, hypothetical protein ARB₀1037. In comparison,5identified proteins, includingputative cyanate hydratase protein, mitochondrial peroxiredoxin-1, hypothetical proteinSMAC₀7435, ATP synthase subunit beta, Hsp70-like protein, revealed up-regulation in the nitrogen-limited sample. These protein spots were involved energy-generating enzymes,antioxidant-related enzymes, amino acid biosynthesis, glycogen biosynthesis, glycolysis,protein transport and transcriptional regulation.The highest pullulan yield was obtained when the cells were grown in the mediumcontaining Tween80. The pullulan yield was increased by24.60%in triangle shaker and45.12%in5L fenmentor by addition of5g/L Tween80, but not enhanced biomass. The ratioof unsaturated/saturated fatty acids contained in the cells was significantly higher in5g/LTween80in comparison with the absence of Tween80. Glucosyltransferase activity involvedin pullulan synthesis was remarkably promoted with an increase of Tween80content.Meanwhile, the molecular weight of pullulan was decreased when Tween80was added.These phenomenons indicated that Tween80affected the secretion of pullulan fromintracellular to extracellular.Effect of nucleotide bases on pullulan and biomass production by A. pullulans wasinvestigated, uracil was found to have positive effect to pullulan accumulation, and theaddition time was critical for uracil to increase pullulan production. The biomass was higherwith the sonner uracil was added. The highest pullulan yield was achieved by adding5mMuracil at48h. The content of UDPG in cells was first decreased and then increased during thefermentation. The UDPG concentration was lowest at48h. Results indicated that the inceaseof pullulan yield brought by uracil was correlated with UPase activity. Metabolic kinetics ofamino acids during batch culture of A. pullulans was studied. L-Serine, L-cysteine,L-methionine, L-threonine L-tyrosine and L-lysine were selected to add to the medium, resultdemonstrated that L-serine could impove the procdtion of pullulan.The optimal temperature of cell growth was not in accordance with that of pullulanproduction. Two-stage temperature fermentation is highly superior to constant-temperaturefermentation for pullulan production. The optimal pH for cell growth is not in accordancewith that of pullulan production. Two-stage pH fermentation is highly superior to constant-pHfermentation for pullulan production. The final pH were all lower than the initial pH. Thoughthe extracellular pH was increased from pH1.5to pH9.5, the intracellular pH was slightlyincreased and maintained at a relatively stable range: pH5.7±0.7. Maximum pullulanproduction （47.83g/L） was obtained at initially higher temperature （32℃） and lower pH （3.5）,subsequently lower temperature （28℃） and higher pH （6.5）,27.45%more than the control.Meanwhile, the fermentation time reduced by12h, the ultilization of sucrose, Conversionefficiency of sucrose into pullulan and Pullulan productivity were all improved.