Effects of Different Substrates on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and

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1 Original Paper 2016 The Author(s) Published The Author(s) by S. Karger AG, Basel Published online: September 09, 08, Published by S. Karger AG, Basel Accepted: August 01, 2016 This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) ( Usage and distribution Effects of Different on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and Pleurotus Eryngii Chunliang Xie Li Yan Wenbing Gong Zuohua Zhu Senwei Tan Du Chen Zhenxiu Hu Yuande Peng Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, P.R.China Key Words Lignocellulosic enzyme Secretome Pleurotus eryngii LC-MS/MS TMT Abstract Background/Aims: Pleurotus eryngii is one of the most valued and delicious mushrooms which are commercially cultivated on various agro-wastes. How different substrates affect lignocellulosic biomass degradation, lignocellulosic enzyme production and biological Pleurotus eryngii was unclear. Methods and Results: In this report, Pleurotus eryngii was cultivated in substrates including ramie stalks, kenaf stalks, cottonseed hulls and bulrush stalks. The results showed that ramie stalks and kenaf stalks were found to best suitable to cultivate Pleurotus eryngii respectively. In order to establish correlations between different substrates and lignocellulosic cellulases, hemicellulases and lignin depolymerization enzymes were highly up-regulated when ramie stalks and kenaf stalks were used as carbon sources. The enzyme activities results induced by ramie stalks and kenaf stalks. Conclusion: The lignocelluloses degradation, most of the lignocellulosic enzymes expressions and activities of Pleurotus eryngii had positive Pleurotus eryngii growth in different substrates were obtained. The present study suggested that most of the lignocellulosic enzymes expressions and activities can be used as tools for selecting better performing substrates for commercial mushroom cultivation The Author(s) Published by S. Karger AG, Basel Yuande Peng Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha (P.R.China) Tel , Fax ,

2 Introduction 1480 Pleurotus eryngii (P. eryngii) is an edible and medicinal white-rot fungi which has been planted extensive in the Mediterranean, central Europe, central Asia, and north Africa due to its remarkable lavor, high nutritional value and numerous medicinal features n present, P. eryngii has been cultivated on several lignocellulosic substrates including cotton seed shells, sawdust, sugarcane bagasse and corn cobs arge uantities of agro-wastes are one of the largest global carbon sources which are considered to be a potential feedstock for the production of useful industrial products nl a small portion of lignocellulosic waste is rec cled and most of the non-rec cled waste is disposed of b burning or bur ing, which is harmful to the environment he lignocellulosic substrate is composed of cellulose, hemicellulose, and lignin suall, white-rot fungi including P. eryngii are the onl microorganisms known to degrade cellulose, hemicellulose and lignin completel and simultaneousl, he conversion of lignocellulosic biomass into soluble sugars is depends mainl on the production of various ef icient lignocellulosic enmes According to previous studies, lignocellulosic enmes involved in i) cellulose degradation (endo, beta glucanase, exo, -beta glucanase and beta glucosidase), ii) hemicellulase degradation (endo, -beta x lanase, beta x losidase, alpha- -arabinofuranosidase, alpha glucuronidase and acet l x lan esterase) and iii) lignin degradation (lingnin peroxidase ( i ), manganese peroxidase (Mn ) and laccase) have been described, t is suggested that the biological e cienc of a mushroom species is mainl attributed to its h drol tic enmes s stem and the activities of h drol tic enmes are responsible for m celial coloni ation (cellulases) and sporophore formation (laccase) owever, most recent research has focused on the evaluation of individual lignocellulosic enme production and puri ication of the main components such as laccase, but ver few studies could be documented on the identi ication and uanti ication of the lignocellulosic enme pro ile of this species n the present research, more h drol tic enmes associated with biological e cienc need to be discovered Because lignocellulose is insoluble, its degradation should occur in the extracellular environment of the white rot fungi t is likel that the extracellular enmes of the fungi pla an important part in lignocellulose degradation ecretome refers to the collection of all proteins exported to the extracellular space o obtain more information about the effect of fungal ligninol tic enmes on lignocelluloses, it is necessar to characteri e the secretome of the white-rot fungi ith the aid of increasing number of available fungal genomes and advances in software, algorithms, and experimental methods, description of white-rot fungi secretomes b proteomic methods was reali ed he secretomes of Phanerochaete chrysosporium (P. chrysosporium), Ganoderma lucidum (G. lucidum), Pleurotus sapidus (P. sapidus), Pleurotus ostreatus (P. ostreatus) Phanerochaete carnosa (P. carnosa), Irpex lacteus (I. lacteus) and so on were elucidated b proteomic methods which providing excellent details about the composition of extracellular enmes, biological mechanisms of lignocellulose degradation, coordination of steps in lignocellulose degradation and its regulation - n hina, large amounts of ramie and kenaf stalk are produced ever ear from bers industrial activities, in addition, also large uantities of agro-wastes are disposed of b burning or bur ing hese lignocellulosic wastes are one of the largest global carbon sources which are considered to be a potential substrates for the production of P. eryngii owever, not all of the lignocellulosic waste is suitable for P. eryngii cultivation, for example, the biological e cienc is ver low in rice straw padd straw mushroom in comparison to saw dust and Burma eed here is no clear understanding how different substrates content affects lignocellulose utili ation vis-a-vis enme production and mushrooms ield in P. eryngii he aims of the current work are to get a deeper understanding on the various degradation patterns of P. eryngii cultivated in different lignocellulose substrates n this research, different carbon sources, such as ramie stalks (), kenaf stalks (), cottonseed hulls () and bulrush stalks (B ) derived from agricultural wastes were used

3 1481 P. eryngii to culture substrates were evaluated P. eryngii P. eryngii P. eryngii on different carbon sources can be a useful to determine the best cultivation substrates for P. eryngii Materials and Methods P. eryngii cultivation conditions P. eryngii P. eryngii cultivation, and moisture content of P. eryngii Cellulose, hemicellulose, and lignin contents determination Protein extraction, digestion and peptide tandem mass tag (TMT) labeling Mass spectrometric data search and analysis

4 1482 Enzyme assay Results P. eryngii was cultivated P. eryngii originated P. eryngii to degrade

5 Fig Table 1. P. eryngii when cultivated in different lignocellulosic substrates Table 2.P. eryngii cultivation P. eryngii consumes cellulose, hemicellulose and lignin, but prefers lignin for its development P. eryngii in

6 Fig. 2. secretome of P. eryngii in the presence of diffe- on the comparison of in the different carbon colors represent different composition of pro- proteins according to ponding region indicate the percentages of the 1484 proteins of P. eryngii were up-regulated when P. eryngii

7 1485 Table 3. denti ication and functional classi ication of the differentiall expressed proteins in P. eryngii in different treatments a Assignment of proteins to those in the B nr database (httpwww ncbi nlm nih gov ) was determined b -MM ames or predicted functions of conserved domains in the identi ied proteins were retrieved from the B nr database (httpwww ncbi nlm nih gov ) to which signi icant peptide matches () were made using the Mascot search engine b ignal peptide prediction b ignal (httpwww cbs dtu dk servicesignal), es, no c All the identi ied proteins were annotated from the db A database (httpcsbi bmb uga edu db A) l coside drolase, l cos l ransferase, ol saccharide ase, Earboh drate Esterase and AA Auxiliar Activit continued

8 1486 Fig glyco

9 1487 Fig. 4. Fig. 5.- P. eryngii P. eryngii in the P. eryngii P. eryngii when P. eryngii when subjected

10 1488 Fig. 6. P. eryngii P. eryngii in the presence of different carbon sources is descri-

11 1489 Determination of enzyme activities on different lignocellulosic biomass P. eryngii had positive correlation with

12 1490 Discussion The special and unique enzyme systems of P. eryngii compare with that of other white-rot fungi P.chrysosporium, G.lucidum, P.ostreatus and I. lacteus P. chrysosporium I.lacteus P. eryngii, P.chrysosporium and I. lacteus should also be pointed out, since proteins from this group have been implicated in the initial P. eryngii and I. lacteus cultures was similar, P. eryngii As mentioned above, P. eryngii had been reported in P. eryngii, participates in the bioconversion of exogenous aromatic I. lacteus and P. chrysosporium from that in P. eryngii I. lacteus but

13 1491 not in P. eryngii P. chrysosporium released s, gl oxal oxidases, lignin peroxidases ( i s), p ranose -oxidase, and M oxidoreductases (both producing ) t is worth to emphasi e that laccase was detected in the secretome of P. eryngii and P. ostreatus, but not in P. chrysosporium n our research, versatile peroxidases and i s were absent in P. eryngii secretome when cultured with the lignocellulosic biomass of,, and B n summar, P. eryngii has the special and uni ue enme s stems and multiple strategies of lignocellulose degrading might exist in white-rot fungi The composition of P. eryngii secretome varied in different culture media n natural environments, white-rot fungi are continuousl challenged with rapidl changing conditions that have a considerable impact on their lifest le he lignocellulosic enme pro ile secreted b fungi is known to be dependent on the t pe and composition of the culture substrate t is suggested that b using a diverse set of substrates for fungal cultivation, it would disclose a broader view of the lignocellulose-degrading capacit herefore, four t pes of substrates including,, and B were used to cultivate P. eryngii e expected to reveal the largest possible number of secreted proteins of P. eryngii he secretome composition of P. eryngii was different markedl among the four media irstl, the expression level of cellulases and hemicellulases except several proteins (gi, gi, gi and gi ) basicall increased remarkabl in the and medium compared with those in the and B medium econdl, two ke lignin-degrading enmes, accase and Mn, were highl abundant in P. eryngii growing both on and medium his is consistent with the results of the enme activit data ( ig ), suggesting and medium were more suitable carbon resources to induce the expressions of lignin-degrading enmes hirdl, tuned expression of proteases and peptidases in response to different media was observed, with serine aminopeptidase (gi ) and serine proteinase (gi ) mainl expressed in the and medium eptid l-prol l cis-trans isomerase ( ragment) speciall expressed in the medium inall, calcium-transporting Aase (gi ) and phospholipidtransporting Aase (gi ) were highl detected in the secretome in the but completel absent in the, and B medium, suggesting a different regulator mechanism for A metabolism in P. eryngii n summar, P. eryngii was able to ef icientl express partial (but not all) lignocellulosic enmes induced b lignocellulose in the medium, while expressions of other secreted proteins were regulated b diverse factors urthermore, it did not show a massive difference in an analed categor of proteins according to a comparison of the four substrates his observation suggests that the signals for,, and B to induce the formation of cellulases and hemicellulase were conserved and these four agricultural b products still remains strong candidates as natural inducer he results present in this research showed that among the different lignocellulosic substrates for the cultivation of P. eryngii, and substrates were found to the best support growth of the fungus, with the BE achieved at and, respectivel he in uence of moisture content and aeration of the substrates on lignocelluloses degrading b white-rot fungi has been emphasi ed in recent studies or example, hi et al reported that cultivation of b P. chrysosporium, lignin degradation at moisture content in the substrate was approximatel higher than that at moisture content he highest ligninase activit was obtained at moisture content when P. chrysosporium was cultivated on corn cobs with a moisture content ranging from to n the framework of the present stud, moisture contents determined in the and medium were higher than that in the and B n addition, P. eryngii cultivated with and medium exhibited a higher laccase and Mn activit compared with medium while activities of these two enmes tend to decrease when cultivated in B medium his can be attributed to the greater water holding capacit of and compared with and B medium

14 1492 Conclusion P. eryngii secretome will shed P. eryngii determine the best cultivation substrates for P. eryngii Abbreviations Acknowledgments Disclosure Statement References Pleurotus eryngii

15 1493 volvariella volvacea pleurotus eryngii on agroindustrial irpex lacteus and other white-rot fungi during wheat phanerochaete chrysosporium phanerochaete carnosa phanerochaete chrysosporium ganoderma lucidum cultivated in phanerochaete chrysosporium pleurotus sapidus. transcription factors in ramie (boehmeria nivea l. Gaud) in response to peg treatment, using illumina mushroom (pleurotus eryngii pleurotus eryngii placental plasma membrane between normal and preeclampsia pregnancies with high-resolution mass

16 1494 trametes versicolor trichoderma reesei phanerochaete chrysosporium strain cirm- peroxidase, and versatile peroxidase activities of the genus pleurotus in media with some raw plant phanerochaete chrysosporium white-rot fungi pleurotus ostreatus and phanerochaete chrysosporium