Extraction And Analysis Of Proteins Of Extracellular Culture Fluid In White-rot Fungi By Cadmium Induction

White rot fungi can effectively adsorb heavy metals in wastewater, but need todefense the hazards of heavy metals when in adsorption. Proteins involve inmetabolism, proliferation, transcription, signal transduction and many other lifeactivities, and the proteins in extracellular culture fluid of white rot fungi are closelyassociated with the crystallization of heavy metals. Further research should be carriedout on the regulation mechanism of white rot fungi on heavy metal poisoning and theinteraction between them from the level of proteins. Until now the relationshipsbetween extracellular culture fluid proteins and heavy metals have not been wellstudied. In this article, the appropriate extraction time under Cd(Ⅱ) stress was studied.To study the functional regulation of Phanerochaete chrysosporium under Cd(Ⅱ)stress, differential expression proteins were extracted, separated by2D-DIGE,identified by MALDI-TOF/TOF MS, and analysed by database search.In the logarithmic growth phase within50~70h, the maximum Cd(Ⅱ) adsorptioncapability was77.1mg/g occurred at65h, and the protein concentration was more in60~70h than in other period. The reflection of the interaction between P.chrysosporium and Cd(Ⅱ) could be effectively and directly at the maximumadsorption capability, therefore the protein extraction was performed at65h.The proteins in range of26.0KDa~33.0KDa changed by Cd(Ⅱ) induction throughthe SDS-PAGE experiment. Some proteins might be up-expression.17differentialexpression spots were got by2D-DIGE experiment, of which a part of relatively densespots were concentrated in26.0KDa~33.0KDa. Proteins in this range might haveimportant role in regulation of heavy metal poisoning and heavy metal adsorption.Three proteins was successfully identified with MALDI-TOF/TOF MS, whichnamed malate dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and glutathione S-transferase (GST). MDH and GAPDH were down-regulated under Cd(Ⅱ) stress, while GST was up-regulated under Cd(Ⅱ) stress. Themolecular weight of three proteins was35KDa,15KDa and31KDa. Three proteinswere related to the oxidative stress in many microorganisms, which were recognizedas important enzymes in microorganism. Cadmium inhibited the growth of P.chrysosporium to some extent and reduced the outward transport of MDH andGAPDH. Although Cd(Ⅱ) is not a redox active metal ion, it can cause oxidative stress resulting in the change of antioxidant enzymes in wasterwater. The above studyshowed that the adaptive response for oxidative stress of cadmium could be adjustedby the enzymes like GST, GAPDH and MDH, and the antioxidant regulationmechanism was likely the key for P. chrysosporium against the damage of heavymetal cadmium, expecially the regulation of intracellular antioxidant. This studyprovided certain reference on understanding of the detoxification mechanism ofwhite-rot fungi with Cd(Ⅱ).