The Extraction And Purification Of The Cell Wall And Extracellular Polysaccharides Of Aspergillus

[Objective]Galactomanan is an important composition among the cell wall of aspergillus and it can bereleased to the extracellular during the phase of growth. Although the kit based on GM has beenused for the diagnosing of invasive aspergillosis for many years, there are much limitationswhich need to be improved. So we intended to extracted GM from aspergillus with high purityand homogeneous in molecular weight in order to develop new ways that can detect aspergillusmore efficiently.[Methods]1. The orthogonal experimental design was applied to optimize the culture conditions ofAspergillus. The temperature, ventilation and inoculation rate were determined, and theamount of the mycelium and the concentration of extracellular polysaccharides were usedas indexes.2. The crude polysaccharides were extracted through enrichment、precipitation and dialysiedof fementation fluids.Then the protein was removed by the sevage methods. Finally, thepolysaccharide P1was purified by collecting the unbound fraction using Q-anion exchangecolumn; The polysaccharides from the mycelium were obtained by alkaline disruption.Then the polysaccharide P2was purified with conA sepharose4B beads and by glucanasedigestion.3. The TOSOH TSK gel3000PWxl column were adopted to analyze the purity and calculatethe molecular weight of the polysaccharide.4. The species of monosaccharides released by Trifluoroacetic acid was estimated bythin-layer chromatography. Then the monosaccharides were dealed with bytrimethylsilylimidazole. Gas chromatography of the trimethylsilylether derivatives werecarried out to determine the proportion of the monosaccharides. [Results]1. The best conditions were temperature28℃, ventilation150milliliter and inoculation rate5%. The yield of extracellular polysaccharides and the amount of mycelium were1.151times and1.273times of the initial conditions respectively.2. We got1.58gram of the polysaccharide P1and162.7millgram of the P2, the purity were84.7%and96.0%respectively.3. The components of the P1and P2were mannan and galactose. The ratios were1:1.18.[Conclusions]we extracted polysaccharides with high purity and homogeneous in molecular weight fromthe fermentation fluids of aspergillus. According to its molecular weight and monosaccharidesproportion, it is our target galactomannan(GM) which is in consistency with the literature. Itcould be applyed to explore new detection methods since the limitation of the present ELISAmeans. However,the polysaccharides from the mycelium were more complex which demandedfor being studyed in depth.