The Substrate Optimization And Secretome Analysis Of Pleurotus Eryngii Cultivated With Hemp Byproducts

In this study, Pleurotus eryngii(P. eryngii) "Xing-1" was used as research object.The substrate optimization of P. eryngii cultivated by hemp byproducts was conducted.The extraction methods of eight extracellular enzymes of P. eryngii mycelium were optimized.The activities of eight extracellular enzymes in both cottonseed hulls mediums and different hemp byproducts content media were comparatively analysed,then proteomics techniques were used to study secreted proteins of P. eryngii induced by hemp byproducts and cottonseed hull. The results showed that:(1)The optimal medium formulations of hemp byproducts to cultivate P. eryngii are: hemp byproducts 60%(Mass ratio,the same below), cotton seed hull 11%, wheat bran21%, corn flour 4%, sugar2%, calcium carbonate2%, pH 6.5, water content 65%,and biological efficiency reached 69.1%, which is 8 percentage points higher than the cottonseed hull medium.(2) The activities of acid cellulase, pectinase, xylanase, laccase, manganese peroxidase and lignin peroxidase with acid extraction are followed by 0.248 IU/ml,0.341 IU/ml,0.174 IU/ml,328 IU/ml,554 IU/ml,1264 IU/ml,while that of water extraction are followed by 0.085 IU/ml,0.131 IU/ml,0.111 IU/ml,294 IU/ml,496 IU/ml,1039 IU/ml. The conclusion can be got that the six enzymes after acid extraction are different with water extraction. Specifically,these extracellular enzyme activities with acid extraction are higher than that of water extraction, in which the activity of acid cellulase, pectinase, xylanase activity measured after acid extraction are twice than water exception.Thus,acid extraction is better than water extraction for these six extracellular enzymes. Amylase and protease activities with acid extraction are followed by 0.076 IU/ml,9.2IU/ml,while water extraction are 0.129IU/ml,10.8IU/ml. Accordingly, amylase and protease activity with acid extraction is less than that of water extraction. Therefore, It would be better to use acid-treated mode to extract extracellular enzymes of P. eryngii mycelium including acid cellulase, pectinase, xylanase, laccase, manganese peroxidase and lignin peroxidase, while the amylase and protease could use water way. The above values are obtained by the average of several tests.The activities of extracellular enzymes such as Laccase,Manganese peroxidase, lignin peroxidase, acid cellulase, pectinase, xylanase, amylase and protease in 60% hemp byproducts medium were higher than other groups,and the actiities optimized in the best way above are followed by 659IU/ml, 661IU/ml,1552IU/ml,0.830IU/ml,0.743IU/ml,0.662IU/ml,0.159IU/ml,11.7IU/ml. There are differences compared with other experimental groups, in which laccase, manganese peroxidase, xylanase and protease are significant differences compared with the other experimental groups. This demonstrates it is feasible to cultivate P. eryngii with hemp byproducts in the extracellular enzymes aspect.(3) SDS-PAGE showed the acid exception has more protein bags than water in the same experimental group with two different extractions.219 extracellular proteins of P. eryngii mycelium induced by cottonseed hull medium and 221 proteins induced by hemp byproducts medium were identified, and all of the physicochemical properties, such as isoelectric point and molecular weight, were analyzed: isoelectric point range 3-13, the molecular weight range 5-170 kD.。Protein functions and subcellular localization were analyzed according to report in literature and G0 annotation: 23% of the proteins are cellulase; 3% proteins are hemicellulase; 7% are pectinase proteins; 26% proteins are lignin depolymerizing proteins; 6% are protease; 16 percent are metabolism-related proteins; 19 percent are transcription and translation-related proteins. A total of 32 differentially expressed proteins were identified by dimethyl marked, of which 14 proteins are up-regulated in hemp byproducts medium, and another 18 proteins are down-regulated in hemp byproducts medium. The functional differences of these proteins wre analysed,and we found that these differentially expressed proteins are mainly cellulase, pectinase, lignin-degraded enzymes, proteases.