Proteomic Analysis of Temperature Dependent Extracellular Proteins

Article pubs.acs.org/jpr

Proteomic Analysis of Temperature Dependent Extracellular Proteins from Aspergillus f umigatus Grown under Solid-State Culture Condition Sunil S. Adav,* Anita Ravindran, and Siu Kwan Sze* School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 S Supporting Information *

ABSTRACT: Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus f umigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40−50 °C, demonstrating the thermophilic nature of the isolate A. f umigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application. KEYWORDS: Aspergillus f umigatus, thermophilic, cellulases, thermostable enzymes, bioenergy and biorefinery

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INTRODUCTION

enzymes upon their application in conversion of biomass into various chemicals and biofuel. Biorefinery, in which biomass is converted to various chemicals by highly efficient biocatalysts/enzymes, will eventually replace the petroleum-based refinery process. This demands intensive research on lignocellulolytic enzymes for lignocellulose hydrolysis. Although various methods like acid or alkali hydrolysis of lignocellulosic biomass have been tested, these methods generate process inhibitory products that affect further processes and also increase production cost. On the contrary, enzymatic treatment is reported as efficient, advantageous due to low investment, low polluting or nonpolluting of the bioprocess, and environmentally friendly. In nature, biomass composting places are prosperous with diverse microbial consortium of different species of bacteria and fungi. Such microbial consortium has potential to produce a vast diversity of cellulolytic, hemicellulolytic, and lignindegrading enzymes to degrade complex biomass.6,7 The fungal strains contribute significantly in recycling lignocellulosic biomass owing to their capacities to produce a wide array of highly active lignocellulolytic enzymes.8 Hence, fungal strains like Trichoderma reesei, T. viride, Fusarium oxysporium,

Lignocellulosic biomass comprises mainly cellulose, hemicellulose, and lignin; and its content varies with plant species. Biomass such as agriculture wastes, crop residues, and forest wastes constitute potential source of feedstock that can be converted into biofuel, feed, specialty chemicals, and other value added products.1 Cellulose and hemicellulose can be hydrolyzed into monomeric sugars and subsequently fermented into organic acids, chemicals, or biofuel, while the aromatic compounds, vanillin and gallic acids, can be produced from lignin.2 Vanillin is used as an intermediate in the chemical and pharmaceutical industries for the production of herbicides, antifoaming agents, or drugs such as papaverine, L-dopa, antimicrobial agent trimethoprim, and in household products such as air-fresheners and floor polishes.2 Moreover hemicelluloses are sources of xylose from which xylitol and furfural can be derived.3 The value added products derived from lignocellulosic biomass have been reviewed.4 According to Wilson,5 cellulases are the third largest industrial enzyme sold worldwide, and have applications in a number of industries. Their demand is increasing significantly because of the emerging advanced biofuel and biorefinery industries which require tremendous amounts of various enzymes in their processes, and thus cellulases will soon become the largest sold © XXXX American Chemical Society

Received: January 23, 2013

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dx.doi.org/10.1021/pr4000762 | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

Piptoporus betulinus, Penicillium echinulatum, Penicillium purpurogenum, Phanerochaete chrysosporium, Aspergillus niger, A. f umigatus, and many more have been intensively studied.9−15 Enzymes are considered and accepted as nature’s catalysts. Prominently, microbial enzymes are better choices than enzymes derived from plants or animals due to their immensely wide catalytic activities. The other advantages of microbial enzymes over plant or animal derived enzymes are their high yield, ease in genetic manipulation, and regular supply which is independent of seasonal fluctuations.16 Moreover, rapid growth of microbes, stability of microbial enzymes over their corresponding plant or animal enzymes, and safety are key advantages. Yet, enzyme production cost, process cost, and process stability at industrial elevated temperature are major concerns in their industrial applications. To limit process costs and increase the process efficiencies, it is advisable to use thermostable enzymes in the industrial processes. But most cellulases are not stable at high temperature, and hence several efforts are being made to obtain thermostable cellulases.17 The second major problem is enzyme cost, which can be resolved through microorganism selection and improving cellulase production conditions and processes. This can be achieved by screening for novel enzymes or novel thermostable strains that produce high quantities of thermostable enzymes. To search potential lignocellulolytic fungi, lignocellulose composting sites are potential sources of such strains. Thus, this work isolated biomass degrading fungal species from the biomass decomposing matter, identified them by 18S rRNA sequencing, and analyzed potent strain for its extracellular secreted proteins at different temperatures using sawdust as a major carbon source by a highly sensitive proteomics technique. The results revealed thermophilic nature of the isolated fungal strain and thermostability of their biomass degrading enzymes.

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database for homologous sequences using BLAST (Basic Local Alignment Search Tool). Although we identified several other fungal strains, fungi belonging to Aspergillus sp. were selected for further study. The fungal isolates belonging to Aspergillus sp. were labeled as A. f umigatus LF1 to A. f umigatus LF9 and their sequences deposited in GenBank under accession numbers JF815065 to JF815073 respectively. Based on preliminary experiments, isolate A. f umigatus LF9 was used in this study for detailed characterization. Bio-Edit was used to align the homologous sequences while MEGA4 software19(Molecular Evolutionary Genetics Analysis software version 4.0) was used for phylogenic analysis. Microorganism Cultivation and Secretome Extraction

The working stock of isolated and identified fungal isolate was prepared and maintained at 4 °C on potato-dextrose agar (Sigma, St. Louis, MO, USA). A starter culture was cultivated in potato dextrose medium composed of 20 g L−1 D-glucose and 4 g L−1 potato extract (Sigma, St. Louis, MO, USA) by inoculating A. f umigatus LF9 from the stock and incubating at 30 °C, 100 rpm. Aspergillus f umigatus is pathogenic and the most common Aspergillus species that cause disease in individuals with an immunodeficiency, and hence biosafety guidelines were strictly followed while working with this fungal strain. The fungal biomass was collected by centrifugation at 7000g at 4 °C (Beckman Coulter, Brea, CA, USA), washed with sterilized Milli-Q water, and used for further experiments. Lignocellulosic biomass (sawdust, 10 g L−1) was moistened using mineral medium (ammonium sulfate 3.1 g L−1, sodium chloride 1.5 g L−1, dipotassium phosphate 1.2 g L−1, monopotassium phosphate 0.9 g L−1, and magnesium sulfate 0.3 g L−1) and sterilized, and the experiment was started by inoculating A. f umigatus LF9. Sawdust was collected from a hard wood cutting sawmill in India that cut wood for construction, furniture, flooring, and utensils. The preliminary solid-state fermentation (SSF) experimental design contained two flasks for each test condition. The flasks were incubated at 20, 30, 40, 50, 60, and 70 °C. The growth was monitored by analyzing total protein content in the extracted secretome. Based on the preliminary experimental data, we fixed secretome harvesting at exponential phase (i.e., 5 days) and temperature range of 20−50 °C for further study. To obtain secretome for proteomics analysis, we used three flasks for each test temperature, and mean data with standard deviation were reported. To extract secretome, 100 mL of 50 mM sodium acetate buffer (Sigma, St. Louis, MO) pH 6.0 ± 2.0 was added into the flasks and shaken in a shaking incubator at 200 rpm for 10 min. Secretome was collected by centrifugation at 7500g at 4 °C (Beckman Coulter, Brea, CA, USA) for 7 min, by removing the solid mass such as sawdust and fungus. The supernatant was further clarified by filtration through 0.25 μm filter. The supernatant (10−15 mL) was retained and stored at −80 °C for enzyme assay and characterization. The remaining secretome was concentrated by lyophilization, proteins were precipitated by ice-cold acetone, and protein content was determined by the Bradford method.

MATERIALS AND METHODS

Isolation and Identification of Fungal Strains

The fungal strains were isolated from biofertilizer compost from Singapore. The biofertilizer compost consists of organic material wastes such as chopped/crushed wooden furniture and forest wastes like weeds, stalks, stems, cut tree branches (road side), and dead branches mixed with pig-wastes and composted for three months. The compost samples were serially diluted with sterile water and pour-plated on Rose Bengal chloramphenicol agar medium containing (g L−1) mycological peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0; magnesium sulfate 0.5; rose bengal 0.05; chloramphenicol 0.1; agar−agar 15.0, pH 7.2 ± 0.2 at 25 °C. The plates were incubated at 30 °C for 72 h. Based on the morphological differences, single fungal colonies were selected, grown separately, and purified by several repetitions of culture plating. Frozen stocks of the cultures were prepared with sterile glycerol and stored at −80 °C in multiple aliquots. For identification, freshly cultured mycelia were collected and ground in a mortar with liquid nitrogen. Total DNA was extracted via enzymatic lysis using extraction buffer (100 mM Tris-HCl at pH 8.0, 100 mM EDTA at pH 8.0, and 1.5 M NaCl) containing Proteinase K (10 mg mL−1), and ITS regions of the isolates were amplified by polymerase chain reaction as described earlier.18 The PCR products were resolved on a 1% agarose gel, purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and sequenced using the ABI Prism model 3730 (version 3.2) DNA sequencer. The sequences were compared with the NCBI

Protein Separation, Protein Digestion, and Peptide Extraction

Proteins (20 μg) from each test sample were separated on 10% SDS-PAGE at 100 V, and protein bands were visualized by staining with Coomassie Brillant blue G-250. Each sample lane was separately sliced into five portions, further cut into pieces (approximately 1 mm2), washed with 75% acetonitrile B



Article

where Nobserved is the number of experimentally observed peptides and Nobservable is the calculated number of observable peptides for each protein. The false discovery rate (FDR) was calculated using in-house build program as 2 × Md/(Md + Mt), where Md represents the number of decoy matches, and Mt is the number of target matches. Then, mean values with standard deviations of the proteins that were identified in at least two biological replicates were reported in this study. The existence of signal peptide sequences was checked using the signal peptide prediction program SignalP23,24 version 3.0 (http://www.cbs.dtu.dk/services/SignalP), SecretomeP database25 (http://www.cbs.dtu.dk/ services/SecretomeP/), and Fungal Secretome KnowledgeBase26 (http://proteomics.ysu. edu/secretomes/fungi.php). Hierarchical clustering of the proteins was performed by choosing “Pearson correlation” using Gene Pattern.27

containing 25 mM ammonium bicarbonate buffer (Sigma, St. Louis, MO, USA), and destained. The destained gel pieces were reduced with dithiothreitol (10 mM) and then alkylated using iodoacetamide (55 mM) as described earlier.11,20 The dehydrated gel pieces were subjected to sequencing grade modified trypsin (Promega, Madison, WI) digestion at 37 °C overnight. The peptides were extracted using 50% acetonitrile/ 5% acetic acid, vacuum centrifuged to dryness, and analyzed by LTQFT Ultra mass spectrometer. Mass Spectrometric Data Analysis

Each tryptic digested fraction was reconstituted in 0.1% formic acid (FA) containing 3% acetonitrile (ACN) and analyzed using LTQFT Ultra mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with an online UHPLC (Dionex, USA). For each analysis, a 4 μL sample was injected from an autosampler (Dionex, USA) and concentrated in a Zorbax peptide trap (Agilent, USA). Mobile phase A (0.1% FA in H2O) and B (0.1% FA in ACN) were used to establish 60 min gradient which consisted 40 min 5−35% B; followed by 10 min 35−60% B; maintained at 80% B for 5 min and finally reequilibrated at 5% B for 5 min. The flow rate of the HPLC was kept at a constant flow rate of 0.3 μL min−1. The samples were injected into the LTQFT through an ADVANCE CaptiveSpray Source (Michrom BioResources, Auburn, CA, USA). The LTQFT was tuned to the following parameters: ion transfer tube temperature180 °C; collision gas pressure 0.85 mTorr, and positive ion mode for data acquisition. A full MS scan (350−1600 m/z) was acquired in the FT-ICR cell at a resolution of 1,000,000 and a maximum ion accumulation time of 1000 ms. Protein fragments generated by collision-activated dissociation (CAD) were captured and measured by the linear ion trap with a maximum ion accumulation time of 100 ms, isolation width of 2 Da. The 10 most intense ions above a 500 count threshold were selected for fragmentation in CAD.

Zymogram Development and Enzyme Assays

The zymographic analysis using 10% SDS-PAGE gel containing 1% carboxymethyl cellulose was performed as described elsewhere.18 In brief, SDS-PAGE gel was loaded with 10 μg of protein and electrophoresis was performed at 90 V for 120 min. The gels were washed overnight with 50 mM Tris-HCl containing 25% isopropanol and incubated at 50 °C for 1 h. Then, gels were stained with 0.1% (w/v) Congo red for 15 min and washed with 1 M NaCl until bands became visible. The enzyme activities were determined on the supernatant samples. The endo-1,4-β-glucanase, exo-1,4-β-glucanase, and βglucosidase were assayed using carboxymethyl cellulose (CMC), Avicel, and salicin (β-salicyl alcohol glucoside) as a substrate respectively. Reducing sugars were determined by using 3,5-dinitrosalicylic acid (DNSA).28 One international unit (IU) of enzyme activity was defined as the amount of enzyme releasing 1 μmol of reducing sugar per minute. Thermostability of the secretome was investigated by incubating the enzyme solution in 50 mM sodium acetate buffer (pH 5.0) at different temperatures (40−70 °C) for 30 min. Then the remaining activity of each secretome sample was measured by the DNSA28 method. Lignocellulose biomass hydrolysis potential of A. f umigatus LF9 was analyzed using crude enzymes (secretome). Enzymatic hydrolysis of sawdust by secretome collected from 20, 30, 40, 50, and 60 °C SSF was carried out for 2 h. Sawdust (5 g) was moistened in 0.1 M sodium acetate buffer (pH 5.0), and then 50.0 mL of secretome from 20, 30, 40, 50, and 60 °C SSF was incubated at 40 ± 2 °C with shaking for 2 h. The experimental design contained three flasks for each test. Reducing sugar produced after 60 and 120 min was analyzed by using the DNSA method.28

Data Mining

Database search was conducted using a Mascot (version 2.2.06, Matrix Science, Boston, MA) search engine against an in-house built database. This database was constructed from predicted proteins using concatenated target and decoy sequences. A. f umigatus Af293 protein sequence database downloaded from the genome project Web site (http://www.aspgd.org/, http:// genome.jgi.doe.gov/Aspfu1/Aspfu1.home.html) contained only the accession number, amino acid sequence, and protein identity. A. f umigatus Af293 (Af71 (NCPF 7098) and Af294 (NCPF 7102)) is a clinical isolate whose genome is 29.4megabase and consists of 9,926 predicted genes, and other details have been described by Nierman et al.21 To obtain the protein function, each protein entry in the database was systematically searched using UniProt DB online. The first four hits from the BLAST were used as the protein name. If there was not a good hit, the protein was named as hypothetic protein. In DB search of MS/MS spectra, enzyme limits were set at full tryptic cleavage at both ends; a maximum of two missed cleavages; mass tolerances of 10 ppm for peptide precursors. Mass tolerance of 0.8 Da was set for fragment ions in Mascot searches. Quantification was performed using exponentially modified protein abundance index (emPAI) values reported by Mascot search engine which is based on equations as stated below:22

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RESULTS

Identification of Strain and Characterization

The internal transcribed spacer region (ITS) of rRNA is highly conserved within species and often used in species identification, differentiation, and description. Comparisons of ITS rRNA sequence of the isolated strain (A. f umigatus LF9) with those deposited in GenBank revealed that the strain belongs to the genus Aspergullus sp. in the phylum Ascomycota and its ITS rRNA sequence shares 99% sequence similarity with A. f umigatus isolate 15-F (accession no. GU244530), A. f umigatus isolate 13-F2 (HQ149772), and A. f umigatus isolate SZ8M-18 (JN227000). The phylogenic relationships between strain A. f umigatus LF9 and representative strains of Aspergullus sp. indicated its close relatives as A. f umigatus isolate 15-F

emPAI = 10 Nobserved / Nobservable − 1 C



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Figure 1. Phylogenetic tree based on ITS rRNA sequences, constructed with the neighbor-joining methods. Numbers at nodes indicate bootstrap values (expressed as percentage of 1000 replications); values greater than 50% are shown.

(accession no. GU244530), A. f umigatus isolate A2S3_D7 (JX501382), and A. f umigatus strain AF-IA-N07 (JX729022) with 100 bootstrap value (Figure 1). Lignocellulosic mass could be an excellent substrate for enzyme production by SSF and practically useful for lignocellulosic bioenergy, hence we used sawdust as a substrate. The isolated strain A. f umigatus LF9 grows as a thick mat on sawdust indicating its potential to use lignocellulosic biomass as a substrate. The growth behavior as monitored by total protein secretion at different temperatures from 20 to 70 °C indicated significantly higher growth in temperature range of 30 to 50 °C (Figure S1 in the Supporting Information). With further increase in temperature to 60 °C, fungal growth on sawdust was lower than for other tested temperatures (30−50 °C). Based on these results, this strain could be classified as a thermophilic fungus.

which has been recently documented.25,29 As can be seen in Figure 2, overlap of ligninocellulolytic enzymes at different

Temperature Dependent Expression of Enzymes

Figure 2. Venn diagram summarizing proteins identified in the secretome of strain A. f umigatus LF9 when cultured using sawdust as a substrate at different temperatures.

A. f umigatus LF9 was cultivated in solid-state fermentation with sawdust as lignocellulosic biomass, and production of lignocellulolytic enzymes was monitored at different temperatures like 20, 30, 40, and 50 °C. With FDR ≤1.0%, this study identified 667 ± 30 proteins. The N-terminal region of the gene encoding for the protein facilitated in assessing its secretion pathway and predicting protein from cell lysis. Some proteins were identified with no signal peptide and could be due to cell death/lysis or secreted by nonclassical secretory mechanism,

temperatures indicated 175 common proteins in all tested temperatures while 93, 53, 65, and 116 unique proteins were detected at temperatures 20, 30, 40, and 50 °C respectively. Further some proteins were common between two or three different culture temperatures (Figure 2). Further classification of these proteins according to their biological functions D



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Figure 3. Functional classification of the proteins secreted by A. f umigatus LF9 during sawdust degradation by solid-state fermentation.

Figure 4. Venn diagrams summarizing proteins identified in the secretome of A. f umigatus. The overlap of carbohydratases (a), lignin degrading proteins (b), peptidases and proteases (c), and hypothetical proteins (d) identified across all four different temperatures.

nase (Afu6g11600) was 1.61, 2.79, 2.79, and 1.92. Similarly, GH6 cellobiohydrolase (Afu3g01910) secretion at 20, 30, 40, and 50 °C was registered with emPAI 1.76, 3.00, 3.97, and 2.56 respectively, and GH7 cellobiohydrolase D (Afu6g07070) with 2.35, 2.69, 2.76, and 2.42 respectively. Comparisons of these proteins and other cellulolytic proteins as presented in Table 1 revealed their abundance as a function of temperature. The emPAI values of GH6 cellobiohydrolase (Afu3g01910), GH5 endoglucanase (Afu6g11600), GH7 cellobiohydrolase D (Afu6g07070), GH17 glucanase (Afu3g00270), exo-β-1,3-

indicated significantly higher number of hypothetical proteins (314 proteins) followed by cellulases, hemicellulases, and then proteins involved in lignin degradation (Figure 3). Comparisons of carbohydratases by A. fumigatus LF9 at different temperatures suggested 84 common carbohydratases (Figure 4a). Although 84 common proteins were found, their abundance as estimated by emPAI revealed variable expressions, e.g., the emPAI value of endoglucanase (Afu7g06150) at temperatures 20, 30, 30, and 50 °C was 1.28, 4.80, 4.57, and 3.51 respectively, while the corresponding value of endoglucaE



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Table 1. Cellulases, Amylases, and Lyases Identified during Sawdust Fermentation by A. f umigatus LF9 Using Solid-State Fermentation Technology SSF accession

name

Afu7g06150 Afu3g01910 Afu6g11610 Afu6g11600 Afu6g07070 Afu1g16190 Afu3g00270 Afu2g03980 Afu6g13270 Afu3g03080 Afu2g00920 Afu2g00760 Afu2g00690 Afu6g01800 Afu1g14710 Afu8g01490 Afu2g00710 Afu6g07480 Afu2g12850 Afu8g07120 Afu2g00680 Afu2g17620 Afu3g03870 Afu1g17410 Afu1g05770 Afu6g14540 Afu4g07850 Afu2g00510 Afu2g05340 Afu8g02130 Afu4g08960 Afu6g08700 Afu5g01830 Afu2g03120 Afu2g01170 Afu1g04730 Afu3g03950 Afu1g11460 Afu2g00220 Afu3g14890 Afu5g08030 Afu1g04260 Afu8g06970 Afu1g14450 Afu3g00900 Afu2g09350 Afu7g06650 Afu1g09470 Afu7g06610

endoglucanase, putative (235 aa) cellobiohydrolase, putative (455 aa) 1,4-β-D-glucan-cellobiohydrolyase (533 aa) endoglucanase, putative (398 aa) cellobiohydrolase D (453 aa) glucanase Crf1 (396 aa) glucanase, putative (447 aa) α-1,3-glucanase, putative (497 aa) exo-β-1,3-glucanase, putative (805 aa) endo-1,3(4)-β-glucanase, putative (286 aa) glycosyl hydrolase, putative (397 aa) pectate lyase A (322 aa) glucan 1,4-α-glucosidase, putative (632 aa) endoglucanase, putative (461 aa) β-glucosidase 1 (484 aa) Avicelase III (793 aa) α-amylase, putative (631 aa) endoglucanase C (307 aa) 1,3-β-glucanosyltransferase Gel3 (545 aa) β-1,6-glucanase, putative (489 aa) glycosyl hydrolase, putative (376 aa) cellobiose dehydrogenase (806 aa) endo-1,4-β-glucanase, putative (343 aa) β-glucosidase, putative (770 aa) β-glucosidase, putative (874 aa) endo-1,3(4)-β-glucanase, putative (286 aa) endoglucanase, putative (251 aa) cellulose-binding GDSL lipase/acylhydrolase 1,3-β-glucanosyltransferase, putative (549 aa) 1,3-β-glucanosyltransferase, putative (538 aa) GPI anchored protein, putative (387 aa) β-glucosidase, putative (889 aa) extracellular endoglucanase, putative (576 aa) cell wall glucanase (Utr2), putative (444 aa) 1,3-β-glucanosyltransferase Gel1 (453 aa) endoglucanase, putative (239 aa) endoglucanase, putative (220 aa) 1,3-β-glucanosyltransferase Bgt1 (306 aa) hydrolase (525 aa) pectate lyase (400 aa) cellulase CelA, putative (345 aa) endo-1,3-β-glucanase Engl1 (975 aa) β-glucosidase (584 aa) exo-β-1,3-glucanase Exg0 (948 aa) α-amylase AmyA (562 aa) endo-β-1,6-glucanase, putative (397 aa) α/β hydrolase, putative (298 aa) aminotransferase, class V, putative (387 aa) isochorismatase family hydrolase, putative (238 aa) GPI anchored glucanase, putative (653 aa) GPI anchored protein, putative (399 aa) cell wall glucanase, putative (358 aa) exo-β-1,3-glucanase (Exg1), putative (417 aa) GPI anchored protein, putative (222 aa) exo-β-1,3-glucanase, putative (810 aa) GPI anchored protein, putative (243 aa) cell wall glucanase, putative (451 aa)

Afu2g14360 Afu1g01300 Afu6g03230 Afu1g03600 Afu8g04370 Afu3g07520 Afu6g02800 Afu6g08510

protein mass

GH family

25883 48392 57675 43019 49130 40487 44852 54330 84587 31410 43468 33858 67572 49315 54804 85353 69137 33532 57898 51683 41198 86231 35962 83086 95093 30935 26354 43955 59747 58252 40298 95345 62570 47251 48390 25412 23866 33291 57180 42741 35952 105148 65538 101357 62825 44995 32881 42052 25170

GH12 GH6 GH7 GH5 GH7 GH16 GH17 GH71 a GH16 GH62 a GH15 GH7 GH1 a a GH5 GH2 GH30 GH76 a GH61 GH3 GH3 GH16 GH61 a GH72 GH72 a GH3 GH5 GH16 GH72 GH12 GH61 GH17 a a a GH81 a a a GH2 a a a

1.28 1.76 2.27 1.61 2.35 2.20 1.13 1.01 1.17 0.98 1.04 3.22 4.64 0.50 0.98 0.25 1.51 0.09 0.56 1.08 0.94 0.36 0.33 1.31 0.72 0.44 0.11 0.36 1.36 0.34 0.15 0.21 0.05 0.61 2.62 0.34 b 2.96 1.04 b 0.78 0.79 0.28 0.79 0.42 0.11 0.16 0.19 0.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

66466 43947 37795 45886 22476 87413 24821 47304

GH16 a GH16 GH2 a

0.07 b b 1.23 b 0.94 0.35 0.32

± 0.02

a GH16 F

20 °C 0.11 0.08 0.26 0.08 0.16 0.27 0.00 0.10 0.06 0.08 0.11 0.32 0.39 0.00 0.27 0.05 0.05 0.00 0.10 0.10 0.06 0.04 0.05 0.04 0.07 0.00 0.00 0.16 0.00 0.00 0.00 0.05 0.00 0.00 0.00 0.07

± 0.16 ± 0.05 ± ± ± ± ± ± ± ± ±

0.13 0.05 0.03 0.02 0.08 0.03 0.05 0.04 0.09

± 0.00 ± 0.08 ± 0.07 ± 0.04

30 °C 4.80 3.00 3.38 2.79 2.69 2.75 1.27 2.06 1.55 1.44 1.43 3.09 4.20 1.28 2.55 0.96 1.25 0.52 1.06 1.45 0.94 0.86 0.60 1.97 0.82 0.72 0.48 0.60 1.11 0.46 0.32 0.64 0.26 0.75 1.81 0.39 0.53 1.39 0.76 b 0.49 0.53 0.30 0.44 0.31 0.32 b 0.19 0.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.30 0.29 0.18 0.12 0.10 0.00 0.00 0.20 0.08 0.00 0.07 0.00 0.59 0.06 0.41 0.09 0.04 0.00 0.05 0.07 0.06 0.07 0.00 0.10 0.04 0.00 0.08 0.09 0.10 0.03 0.00 0.11 0.00 0.10 0.16 0.00 0.08 0.10 0.04

± ± ± ± ± ±

0.16 0.07 0.08 0.03 0.05 0.04

0.09 0.35 b 0.67 b 0.39 0.21 0.25

± 0.00 ± 0.04

± 0.04 ± 0.00

± 0.09 ± 0.04 ± 0.06 ± 0.03

40 °C

50 °C

SignalP

4.57 3.97 3.01 2.79 2.76 2.20 1.76 1.76 1.63 1.54 1.53 5.73 4.95 1.50 1.46 1.36 1.34 1.28 1.20 1.14 1.04 0.99 0.86 0.86 0.80 0.76 0.73 0.71 0.64 0.63 0.58 0.58 0.51 0.51 0.44 0.41 0.39 0.38 0.37 0.32 0.31 0.29 0.26 0.25 0.21 0.21 0.20 0.17 0.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.49 0.16 0.36 0.46 0.33 0.22 0.29 0.09 0.20 0.14 0.19 0.77 0.31 0.01 0.21 0.08 0.18 0.25 0.18 0.08 0.08 0.06 0.01 0.00 0.13 0.06 0.12 0.06 0.04 0.00 0.07 0.04 0.06 0.01 0.05 0.03 0.09 0.07 0.06 0.07 0.08 0.04 0.05 0.00 0.01 0.00 0.01 0.07 0.06

3.51 2.56 2.26 1.92 2.42 2.59 1.37 1.80 1.10 1.23 1.48 9.26 5.04 1.92 1.41 0.29 1.28 0.40 0.22 1.24 1.07 0.51 0.44 0.15 1.65 0.83 0.64 0.26 0.58 0.32 0.88 0.55 0.18 0.40 0.73 0.39 0.59 1.14 0.33 0.30 0.44 0.38 0.17 0.45 0.29 0.56 b 0.37 0.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N N Y Y Y N N N

0.16 0.15 0.14 0.13 0.13 0.12 0.12 0.11

± ± ± ± ± ± ± ±

0.02 0.01 0.03 0.00 0.00 0.04 0.00 0.03

0.24 b 0.16 0.06 b 0.55 0.12 0.18

± 0.00

0.00 0.26 0.13 0.09 0.18 0.23 0.07 0.14 0.06 0.00 0.00 1.15 0.32 0.08 0.10 0.02 0.08 0.00 0.00 0.06 0.07 0.02 0.05 0.06 0.04 0.08 0.08 0.04 0.04 0.03 0.18 0.02 0.02 0.04 0.16 0.00 0.00 0.15 0.03 0.00 0.05 0.02 0.02 0.02 0.03 0.13

± 0.12 ± 0.00

± 0.00 ± 0.00 ± 0.05 ± 0.00 ± 0.03

Y Y Y Y Y Y Y Y



Article

Table 1. continued SSF accession

name

Afu3g02280

α,α-trehalose glucohydrolase, putative (1073 aa) α-amylase (Amy1), putative (495 aa) glycosyl hydrolase, putative (621 aa) putative hydrolase (330 aa) glucoamylase (353 aa) transaldolase (325 aa) GPI anchored protein, putative (367 aa) glycosyl transferase, putative (478 aa) pectin lyase B (432 aa) exo-β-1,3-glucanase, putative (1006 aa) glucosyltransferase (594 aa) α-amylase AmyA (569 aa) β-glucosidase 1 (489 aa) Amidohydrolase family, putative (534 aa) β-glucosidase 3 (1034 aa) patatin-like serine hydrolase, putative (590 aa) patatin-like serine hydrolase, putative (626 aa) hydrolase, putative (654 aa) α-glucosidase B (882 aa) pectate lyase, putative (235 aa) α/β hydrolase, putative (293 aa) endo-1,4-β-glucanase, putative (374 aa) β-glucosidase 1 (498 aa) cellobiose dehydrogenase (821 aa) dienelactone hydrolase (458 aa) endoglucanase, putative (478 aa) PAF acetylhydrolase family protein (385 aa) glycosyl hydrolase, putative (457 aa) α/β hydrolase, putative (309 aa) glycosyl transferase, putative (3015 aa) cell wall glucanase, putative (424 aa) α/β hydrolase, putative (298 aa) glycosyl hydrolase family 35, putative (566 aa) β-glucosidase (798 aa) cell wall glucanase, putative (689 aa) catabolite repressor protein (CreC), putative (524 aa) 1,3-β-glucanosyltransferase Gel2 (476 aa) dienelactone hydrolase family protein (246 aa) phytase, putative (528 aa) glucanase, putative (471 aa) β-D-glucoside glucohydrolase (740 aa) pectate lyase, putative (243 aa) endoglucanase, putative (238 aa) cell wall glucanase (Scw11), putative (617 aa) α-1,3-glucanase, putative (475 aa) α-1,3-glucanase, putative (738 aa)

Afu4g10130 Afu2g03270 Afu3g01660 Afu4g10140 Afu5g09230 Afu2g01710 Afu1g17030 Afu7g05030 Afu2g00430 Afu3g07700 Afu2g03230 Afu3g12600 Afu5g01480 Afu6g03570 Afu1g09110 Afu2g07870 Afu2g14520 Afu1g16250 Afu1g01120 Afu1g11400 Afu1g12560 Afu1g16400 Afu2g01180 Afu2g05810 Afu2g09520 Afu2g17720 Afu3g00340 Afu3g01280 Afu3g07860 Afu3g09250 Afu3g14990 Afu5g00670 Afu5g07080 Afu5g08780 Afu6g07860 Afu6g11390 Afu6g12740 Afu7g01240 Afu7g05610 Afu7g06140 Afu7g06400 Afu7g06740 Afu8g05610 Afu8g06030 Afu8g06360 a

protein mass

GH family

20 °C

30 °C

40 °C

50 °C

SignalP

117287

GH65

0.15 ± 0.01

0.16 ± 0.02

0.11 ± 0.01

0.20 ± 0.03

Y

54408 69718 36404 38795 35598 40166 53325 45531 106293 67357 64057 55911 58724 113808 66445 69095 74345 99225 25526 31885 38588 57208 85608 52076 51179 41931 50670 34215 340154 47283 32709 63463 87565 72993 58547

a GH76 GH43 GH15 a a a a a a a GH1 a GH3 a a GH2 GH31 a a GH61 GH1 a a GH5 a GH76 GH87 a GH16 GH31 GH42 GH3 GH17 a

b b 0.26 b 0.24 0.07 b 0.18 b b b 0.05 0.05 0.03 0.04 b 0.04 0.03 b 0.55 0.24 0.05 0.09 b 0.06 0.07 0.12 b b 0.18 0.09 b b 0.07 b

b b 0.55 0.07 b 0.07 b 0.15 0.10 0.04 b 0.05 b 0.04 0.04 b 0.05 b b 0.26 0.24 b b 0.06 0.30 b b b b 0.06 b b 0.03 0.08 b

0.11 0.08 0.08 0.08 0.08 0.07 0.06 0.06 0.05 0.05 0.05 0.05 0.05 0.04 0.04 0.04 0.04 0.03 b b b b b b b b b b b b b b b b b

b 0.04 0.40 b 1.80 0.07 0.05 b b b b 0.05 b b b b 0.15 0.18 0.74 b b b b b b b b 0.09 0.01 b b 0.05

Y Y Y N N Y N N N N Y N N N N N Y Y Y Y Y N Y N Y Y Y N N Y N N Y N N

52084 27211 57525 55337 78731 25951 25427 64439 52619 83338

GH2 a a GH5 GH3 a GH45 GH17 GH71 GH87

0.39 b 0.53 b b b b b 0.06 0.32

± 0.00 ± 0.05 ± 0.00 ± 0.03

± ± ± ±

0.00 0.00 0.00 0.00

± 0.00 ± 0.00 ± ± ± ±

0.00 0.00 0.00 0.03

± 0.00 ± 0.00 ± 0.00

± 0.03 ± 0.00

± 0.02 ± 0.06 ± 0.08

± 0.00 ± 0.00

± 0.06 ± 0.00 ± 0.00 ± 0.03 ± 0.01 ± 0.00 ± 0.00 ± 0.01 ± 0.00 ± 0.02

± 0.05 ± 0.00

± 0.00 ± 0.03

± 0.00

± 0.00 ± 0.00

0.06 ± 0.00 b 0.05 ± 0.00 b b b b b b 0.10 ± 0.02

b b b b b b b b b b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.00 0.02 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00

± 0.00 ± 0.10 ± 0.18 ± 0.00 ± 0.00

± 0.00

± 0.02 ± 0.06 ± 0.25

± 0.00 ± 0.00

± 0.00

0.04 ± 0.00 0.05 ± 0.00 0.27 1.43 0.12 0.11 0.07 0.11 0.12 0.17 b b

± ± ± ± ± ± ± ±

0.06 0.11 0.05 0.00 0.02 0.00 0.00 0.02

Y N Y N Y Y N N Y N

Data not available. bNot identified.

exo-, α-, β-, and 1,3-glucanases were abundant at 20 °C SSF. The majority of endoglucanases were clustered under cluster C4 and significantly upregulated when the SSF temperature was 40 °C, while proteins clustered under clusters C5 and C6 (endoglucanases, GPI-anchored glucanases, and other GH proteins) were significantly abundant when the SSF temperature was 50 °C (Figure 5). GPI-anchor of GPI-anchored glucanases acts like CBM domain, facilitates enzyme binding to substrate, and enhances substrate degradation. Proteins

glucanase (Afu6g13270), GH16 endo-1,3(4)-β-glucanase (Afu3g03080), GH62 glycosyl hydrolase, (Afu2g00920), and GH7 endoglucanase (Afu6g01800) demonstrated their optimum production at 40−50 °C. On the contrary, emPAI values of GH1 β-glucosidase (Afu1g14710), GH3 β-glucosidase (Afu1g05770), and GH15 glucan 1,4-α-glucosidase (Afu2g00690) suggested their abundance at 50 °C. Cluster analysis of the proteins having cellulolytic activity showed eight clusters. Proteins clustered under cluster C1 including endo-, G



Article

Figure 5. Hierarchical cluster analysis of cellulolytic proteins identified in the secretome of A. f umigatus LF9. Cluster analysis was performed by Gene Pattern using Pearson correlation as the column and row distance. Red color indicates high abundance; blue color indicates low abundance; and shades of red and blue indicate intermediate abundances.

dase (Afu6g14620), and GH31 α-galactosidase (Afu6g02560) suggested their higher abundance at 50 °C. Further analysis of expressed hemicellulolytic proteins with cluster analysis demonstrated temperature dependent regulation of xylanases, i.e., xylan hydrolyzing xylanases clustered under H2 and H5 were upregulated when the SSF temperature was 50 °C, proteins grouped under H1 and H6 were highly expressed when the SSF temperature was 40 °C, and protein of clusters

clustered under clusters C5 and C6 including GPI-anchored proteins that play roles in cell wall polysaccharide remodeling, cell morphogenesis, and substrate binding were also upregulated at 40 °C SSF. The protein abundances as measured by emPAI values of hemicellulolytic protein including GH47 α-mannosidase (Afu1g14560), GH10 endo-1,4-β-xylanase (Afu6g13610), GH28 polygalacturonase (Afu1g17220), α-L-arabinofuranosiH



Article

Figure 6. Hierarchical cluster analysis of hemicellulolytic proteins identified in the secretome of A. f umigatus LF9. Cluster analysis was performed by Gene Pattern using Pearson correlation as the column and row distance. Red color indicates high abundance; blue color indicates low abundance; and shades of red and blue indicate intermediate abundances.

H3 and H4 were upregulated at 30 and 20 °C SSF (Figure 6). These data and comparative abundance of other carbohydratases (Table 1 and Table 2) at different temperatures revealed abundance of carbohydratases at 50 °C which strongly supports thermophilic nature of fungus A. f umigatus LF9. Interestingly, cellulolytic proteins like GH45 endoglucanase (Afu7g06740), GH17 glucanase (Afu7g05610), GH35 glycosyl hydrolase (Afu5g00670), catabolite repressor protein (Afu6g07860), GH87 α/β hydrolase (Afu3g01280), GH17 cell wall glucanase (Afu8g05610), and GH3 β-D-glucoside

glucohydrolase (Afu7g06140) and hemicellulolytic proteins such as GH31 α-xylosidase (Afu2g05400), carboxylesterase (Afu6g10800), and rhamnogalacturonase (Afu5g10530) were uniquely secreted only at 50 °C. Pectate lyase (Afu2g00760), the enzyme involved in the maceration and soft rotting of plant tissue that catalyzes the eliminative cleavage of pectate, yielding oligosaccharides with 4-deoxy-α-D-mann-4-enuronosyl groups at their nonreducing ends, was significantly abundant with an emPAI value 9.26 at 50 °C. Again, pectate lyases (Afu7g06400 and Afu1g01120) were uniquely expressed only at 50 °C. I



Article

Table 2. Hemicellulolytic Proteins Identified in the Secretome of A. f umigatus LF9 during Sawdust Fermentation at Different Temperatures by Using Solid-State Fermentation SSF accession

name

Afu1g14560 Afu6g13610 Afu1g17220

α-mannosidase (494 aa) endo-1,4-β-xylanase, putative (398 aa) extracellular polygalacturonase, putative (379 aa) α-L-arabinofuranosidase (507 aa) α-galactosidase (513 aa) endo-1,4-β-mannosidase, putative (467 aa) rhamnogalacturonase B, putative (529 aa) endo-1,4-β-xylanase (XlnA), putative (229 aa) acetyl xylan esterase (Axe1), putative (293 aa) extracellular polygalacturonase, putative (369 aa) extracellular arabinanase, putative (417 aa) endo-1,4-β-xylanase, putative (314 aa) α-1,2-mannosidase family protein (818 aa) possible β-xylosidase, family 43 of glycosyl D-xylose reductase (Xyl1), putative (316 aa) α-1,2-mannosidase, putative subfamily (793 aa) acetyl xylan esterase, putative (372 aa) extracellular exopolygalacturonase, putative (410 aa) β-mannosidase (927 aa) arabinogalactan endo-1,4-β-galactosidase, endo-1,4-β-xylanase (XynG1), putative (222 aa) xylosidase/arabinosidase, putative (373 aa) xylosidase (328 aa) endo α-1,4 polygalactosaminidase, putative (319 aa) extracellular endo-1,4-β-xylanase, putative (325 aa) extracellular endo-1,5-α-L-arabinase, α-1,2-mannosidase, putative (793 aa) α-L-arabinofuranosidase (501 aa) esterase, putative (288 aa) arabinofuranosidase (333 aa) β-xylosidase (772 aa) arabinosidase (483 aa) α-L-rhamnosidase A precursor (677 aa) endo-1,4-β-mannosidase (478 aa) xylosidase: arabinofuranosidase (559 aa) α-galactosidase (892 aa) α-mannosidase (1089 aa) esterase family protein (396 aa) xylitol dehydrogenase (349 aa) alfa-L-rhamnosidase, putative (898 aa) carboxylesterase, putative (531 aa) β-galactosidase (977 aa) extracellular rhamnogalacturonase, putative (522 aa) phosphoglucomutase (556 aa) carboxylesterase, putative (552 aa) endoarabinase, putative (325 aa) α-xylosidase (760 aa) D-arabinono-1,4-lactone oxidase; α-1,2-mannosidase family protein, putative (840 aa)

Afu6g14620 Afu6g02560 Afu8g07030 Afu4g03780 Afu3g00320 Afu3g00420 Afu4g13920 Afu6g00770 Afu3g00470 Afu5g10520 Afu2g00930 Afu1g04820 Afu6g13760 Afu8g06570 Afu8g06890 Afu6g08840 Afu1g06910 Afu6g12210 Afu2g04480 Afu8g04710 Afu3g07890 Afu4g09480 Afu3g14620 Afu7g04720 Afu1g09900 Afu5g09860 Afu2g12770 Afu3g02090 Afu2g00650 Afu1g01660 Afu7g01070 Afu2g13190 Afu4g03580 Afu3g08200 Afu8g06350 Afu7g02550 Afu6g14610 Afu6g10800 Afu5g14090 Afu5g10530 Afu3g11830 Afu3g09230 Afu2g14750 Afu2g05400 Afu1g14950 Afu1g10790

a

protein mass

GH family

20 °C

30 °C

40 °C

50 °C

SignalP

53978 42566 38914

GH47 GH10 GH28

3.86 ± 0.42 2.25 ± 0.38 1.12 ± 0.14

4.59 ± 0.36 4.26 ± 0.00 3.37 ± 0.40

3.82 ± 0.37 3.80 ± 0.27 2.96 ± 0.23

4.42 ± 0.59 4.39 ± 0.33 1.22 ± 0.13

Y Y Y

52972 56917 50831 56986 24536 30055 38542

a GH31 GH2 RGL4 GH11 a GH28

1.49 0.98 0.16 0.57 1.89 0.54 0.71

± ± ± ± ± ± ±

0.11 0.05 0.03 0.11 0.30 0.07 0.11

1.98 1.62 0.48 1.22 1.99 0.92 0.63

± ± ± ± ± ± ±

0.15 0.06 0.07 0.15 0.16 0.00 0.06

2.90 1.35 1.21 1.10 0.96 0.92 0.76

± ± ± ± ± ± ±

0.27 0.20 0.24 0.11 0.16 0.13 0.16

1.40 2.21 0.45 1.26 1.38 0.59 0.75

± ± ± ± ± ± ±

0.12 0.27 0.08 0.05 0.12 0.00 0.06

Y N N Y Y Y Y

45248 33345 89708 57762 35692 87576 39740 43378

GH43 GH11 GH92 GH43 a GH92 a GH28

0.76 0.52 0.44 0.34 0.79 0.92 0.21 b

± ± ± ± ± ± ±

0.09 0.00 0.02 0.00 0.18 0.05 0.08

0.72 0.66 0.44 0.53 0.99 1.14 0.57 0.07

± ± ± ± ± ± ± ±

0.05 0.00 0.02 0.04 0.19 0.09 0.05 0.00

0.75 0.70 0.66 0.54 0.50 0.45 0.45 0.37

± ± ± ± ± ± ± ±

0.00 0.05 0.07 0.10 0.07 0.10 0.06 0.06

0.72 0.61 0.44 0.77 1.65 0.09 0.57 0.45

± ± ± ± ± ± ± ±

0.05 0.07 0.04 0.04 0.19 0.01 0.10 0.05

N Y Y N N Y Y Y

103899 43981 23852

GH2 GH53 GH11

0.22 ± 0.05 0.38 ± 0.00 0.12 ± 0.00

0.37 ± 0.08 0.44 ± 0.04 0.12 ± 0.00

0.35 ± 0.05 0.31 ± 0.02 0.28 ± 0.02

0.44 ± 0.04 0.51 ± 0.09 0.12 ± 0.00

Y N Y

40920 37367 34848

GH43 GH43 a

0.32 ± 0.00 0.08 ± 0.00 0.08 ± 0.00

0.41 ± 0.08 0.11 ± 0.04 0.14 ± 0.04

0.24 ± 0.01 0.23 ± 0.03 0.21 ± 0.04

0.55 ± 0.05 1.18 ± 0.08 b

Y N Y

35656

GH10

0.08 ± 0.00

0.61 ± 0.11

0.18 ± 0.01

b

Y

36194 88118 56705 31749 36634 83742 52930 73678 53249 62310 97414 124797 44153 37170 101515 60220 109600 55512

GH43 GH92 a a GH62 GH3 GH43 a GH2 GH43 a GH38 a a a a GH2 a

0.14 0.12 0.45 b b b 0.43 b b 0.05 0.12 b 0.12 b b b b b

60692 61224 36049 86128 104817 93205

PL3 a GH43 GH31 a GH92

0.57 0.07 0.69 b b 0.18

± 0.04 ± 0.03 ± 0.09

± 0.09

± 0.00 ± 0.00 ± 0.03

± 0.09 ± 0.02 ± 0.06

± 0.01

0.08 0.23 0.37 b 0.08 b 0.56 0.04 b 0.05 0.09 0.02 0.07 b 0.03 b 0.03 b 0.05 0.05 0.20 b b 0.10

± 0.00 ± 0.05 ± 0.07 ± 0.00 ± 0.04 ± 0.00 ± ± ± ±

0.00 0.00 0.00 0.00

± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.04

± 0.03

0.18 0.16 0.15 0.09 0.08 0.07 0.06 0.05 0.05 0.05 0.03 0.02 b b b b b b b b b b b b

± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.02 0.02 0.00 0.00 0.03 0.00 0.02 0.00 0.00 0.00 0.00

0.17 0.17 0.05 2.25 0.08 0.03 0.56 0.24 0.05 b 0.54 0.08 b 0.08 b 0.05 b 0.05 0.37 b 0.64 0.03 0.03 b

± ± ± ± ± ± ± ± ±

0.00 0.02 0.00 0.14 0.00 0.00 0.08 0.02 0.00

± 0.09 ± 0.01 ± 0.00 ± 0.00 ± 0.00 ± 0.03 ± 0.06 ± 0.00 ± 0.00

Y Y N N Y Y N N N N N N N N N N Y Y Y Y Y N N Y

Data not available. bNot identified. J



Article

Similarly, amylases such as α-amylase AmyA (Afu2g03230) and α-amylase Amy1 (Afu4g10130) were expressed only when cultivation temperature was 40 °C. The removal of lignin opens the way for cellulases and hemicellulases. Lignin degradation is an oxidative process where hydrogen peroxide generating enzymes and proteins belonging to the oxidoreductase family play a major role in the initial lignin degradation process.30,31 Thus, differential expression of proteins like laccase, peroxidase, glutathione peroxidase, cytochrome c peroxidase, alcohol oxidase, cytochrome c oxidase, Cu−Zn superoxide dismutase, NADH-dependent oxidoreductase, and several proteins belonging to the oxidoreductase family were profiled when A. fumigatus LF9 was cultivated using sawdust (Table ST1 in the Supporting Information). Cu−Zn superoxide dismutase (Afu5g09240) belonging to ubiquitous family of enzymes, which catalyze the dismutation of superoxide anions to molecular oxygen and hydrogen peroxide, was significantly abundant with emPAI values 7.91 and 8.22 at temperatures 40 and 50 °C. Similarly, isoamyl alcohol oxidase (Afu6g03620) with emPAI 0.99, 0.88, 0.96, and 1.36 was identified at temperatures 20, 30, 40 and 50 °C respectively. Surprisingly, lignin degrading laccase (Afu1g15670) was expressed only when cultivation temperature was 20 °C while peroxidase (Afu4g02780) was identified when A. f umigatus LF9 was cultivated at temperature 30 °C. The proteins belonging to the peroxidase family are hemecontaining enzymes that exploit hydrogen peroxide as the electron acceptor to catalyze a vast number of oxidative reactions. However, cytochrome c peroxidase, a hemecontaining enzyme of the peroxidase family that takes reducing equivalents from cytochrome c and reduces hydrogen peroxide to water, was only detected at 40 °C SSF. Again, enzyme Fecontaining alcohol dehydrogenase (Afu1g06800, Afu2g07610) was detected only at temperature 40 °C while oxidoreductases (Afu6g13830, Afu8g06840, Afu3g13450), cytochrome (Afu2g13110, Afu3g06190, Afu1g01290, Afu1g13220), lactoylglutathione lyase (Afu6g07940), etc. were specific at 50 °C SSF. The results indicate that the fungus employs different enzymes/ mechanisms to degrade lignin at different ambient temperatures. The detailed comparative analysis of temperature dependent expression of the proteins involved in lignin degradation is presented in Figure S2 in the Supporting Information. This study also identified proteases and peptidases, transport proteins, and proteins involved in cell morphogenesis, and their abundances are listed in Table ST3 in the Supporting Information. Hierarchical clustering of proteases and peptidases by A. f umigatus LF9 is presented in Figure S3 in the Supporting Information. Further, this work identified 314 hypothetical proteins that were expressed during SSF of sawdust at temperatures 20−50 °C (Table ST3 in the Supporting Information).

Figure 7. Enzyme activities of the secretome by A. f umigatus LF9. The secretome of A. f umigatus LF9 was collected from solid-state fermentation of sawdust at temperatures 20, 30, 40, and 50 °C.

temperatures demonstrated highest activity of 618.9 ± 14.7 U g−1 at 50 °C while the corresponding activity at 40 °C was 605.3 ± 30.5 U g−1 substrate. Further zymographic analysis of secretome by A. f umigatus LF9 for carbohydatases was performed by zymography and is presented in Figure S4 in the Supporting Information. The CMC degrading enzymes reacted with CMC present in the gel and produced six distinct bands. The major bands were excised, digested, and analyzed by LC−MS/MS. The analysis of band B2 by LC−MS/MS revealed more than 72 carbohydratases (Table B2 in the Supporting Information) with high abundant glucan 1,4-αglucosidase (Afu2g00690), 1,4-β-D-glucan-cellobiohydrolyase (Afu6g11610), exo-β-1,3-glucanase (Afu6g13270), etc. Similarly, band B3 demonstrated 68 cellulolytic proteins (Table B3 in the Supporting Information). Sawdust degradation potential of the crude enzymes (secretome) by A. f umigatus LF9 is presented in Figure 8. When crude enzymes were tested using sawdust as a substrate, sugars produced by secretome of 30−50 °C SSF ranged between 61.53 and 67.50 mg g−1 substrate; while it was 48.42 mg g−1 substrate when secretome of 20 °C SSF was incubated with sawdust for 2 h. Thermostability of

Enzyme Activities, Zymographic Analysis, and Thermostability of Secretome

The enzyme activities of endo-1,4-β-glucanase, exo-1,4-βglucanase, β-glucosidase, and xylanases determined on the supernatant samples are presented in Figure 7. The activity of endo-1,4-β-glucanase at 20 °C was 289.5 ± 17.9 U g−1 substrate, and its titer increased with increase in temperature achieving maximum production (444.2 ± 18.9 U g−1) at 50 °C. A similar trend was noted for exo-1,4-β-glucanase. However, no significant differences in enzyme activities at 30, 40, and 50 °C were noted. Xylanase activities determined at different

Figure 8. Production of sugar from sawdust by A. f umigatus LF9 secretome (crude culture filtrate from different temperature SSF experiments) at different temperatures. K



Article

cellulases and xylanases by A. f umigatus LF9 during SSF of sawdust at 30, 40, and 50 °C is presented in Figure 9.

thermostable enzymes in biorefining process proficiently catalyzes the conversion of renewable bioresources such as agricultural crop residues, agricultural and forest wastes, and cellulosic wastes into chemicals, commodities, and fuels.33,34 Other advantages of thermostable enzymes for bioprocesses include their prolonged stability at room temperature, reduced risk of process contamination, and increased tolerance to organic solvents.33,35,36 Some species of fungi belonging to Aspergillus are pathogenic while some are so beneficial in biomass recycling, and a large number of Aspergillus species are of biomedical and industrial significance. Hence, the genomes of A. clavatus,37 A. flavus,38 A. f umigatus,21 A. nidulans,39 A. niger,40 A. oryzae,41 and A. terreus have been sequenced. A. nidulans acts as a key fungal model system for genetics and cell biology that contributed to our understanding of classical genetics, and A. niger has been exploited for the production of citric acid, enzymes, and the heterologous expression of various proteins; whereas A. oryzae plays a key role in the fermentation process of several traditional Japanese beverages and sauces.39,41−43 In addition, A. oryzae has been exploited in the production of various industrial enzymes and to achieve high levels of heterologous proteins, such as aspartic proteinase and lipase44 and active human proteins (lactoferrin and lysozyme).45,46 Fungi play a significant part in industrial enzyme production, and their importance in the sustainable future on our planet has been reviewed by Lange.44 A. f umigatus is a saprotroph, widely spread in nature, and plays an important role in carbon and nitrogen recycling. Their spores are ubiquitous in the atmosphere and upon inhalation; typically these are eliminated by the immune system in healthy individuals. However, in immunocompromised individuals the fungus is more likely to become pathogenic. The strain of A. f umigatus grown on certain building materials has been reported to produce genotoxic and cytotoxic mycotoxins, such as gliotoxin.47 Comparisons of two strains, A. oryzae 3.042 and A100-8, with a particular interest in proteins involved in soy sauce flavor formations have been documented.48 A. f umigatus is a saprophytic fungus and plays an essential role in recycling environmental carbon and nitrogen.

Figure 9. Thermostability of the secretome by A. f umigatus LF9 from 30 to 50 °C solid-state fermentation. Thermostability was tested at different temperatures from 40 to 70 °C.

Thermostability of enzyme activities was absolutely stable over the temperature range 40−50 °C. When the thermostability was tested at 60 °C, 80% activity was retained which declined to 70−60% when the temperature was increased to 70 °C.

Enzyme Activities at Elevated Temperatures

A. f umigatus produces substantial extracellular cellulose and hemicellulose degrading enzymes on several cellulosic substrates49,50 and hence receiving more attention. A. niger P47C3 and A. f umigatus P40M2 isolated from the Amazon rainforest and grown on different carbon sources such as wheat bran, sugar cane bagasse, soybean bran, and orange bagasse showed 105.8 U g−1 β-glucosidase and 1055.6 U g−1 xylanase activity, but their cellulase activity was significantly low (56.6 Ug1− substrate).49 A. f umigatus Z5 isolated from the compost of a fertilizer factory demonstrated endoglucanase activity of 526.3 U g−1 of dry corn stover,51 while isolate A. f umigatus LF9 of this study showed endoglucanase activity in the range of 398.9− 444.2 U g−1 sawdust. Again xylanase activity by A. f umigatus LF9 ranged between 618.9 ± 14.7 and 605.3 ± 30.5 U g−1 at 50−40 °C. Based on the degree of lignifications and lignin content of biomass, sawdust could be considered as a complex biomass compared to crop residues like corn stover. Softwood consists of 45−50% cellulose, 25−35% hemicellulose, and 25−35% lignin; while the corresponding values in hardwood were 37− 40, 23−29, and 21−23% respectively.12,52,53 The crude

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DISCUSSION This study isolated A. f umigatus LF9 from a composting place and discovered its secreted enzymes while growing under a mimicked natural habitat on sawdust at different temperatures. The protein secretion profile as determined by total proteins at different temperatures revealed the thermophilic nature of the isolated strain. The biomass hydrolysis at enhanced temperature promotes heat induced disorganization of biomass structure that boosts enzyme penetration and further increase in biomass hydrolysis.32 Several researchers attempted to improve the thermotolerant nature of cellulases and hemicellulases by introduction of disulfide bridges, ionic pairs, tightly bound water molecules, protein packing, and oligomerization.32 However, despite the improvement in the specific activity and thermoactivity of these engineered cellulases and hemicellulases, their instability remains a major issue, and hence naturally occurring thermostable enzymes from themophilic strains are in high demand. It is certain that the use of L



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such as A. nidulans encodes 247 GHs, A. f umigatus 263 GHs, and A. oryzae 285 GHs;60 which are significantly higher than the GHs by T. reesei that emphasizes lignocellulose degradation potential of Aspergillus sp. Cluster analysis of the secretory cellulolytic enzymes suggested the majority of cellulolytic proteins (clusters C2−C8) were upregulated when SSF temperature was within 30 and 50 °C. Fascinatingly, endoand exoglucanases, cellobiose dehydrogenase, GPI-anchored glucanases, and GPI-anchored proteins were significantly upregulated when A. f umigatus LF9 was cultured at 40−50 °C; while the highest expression of glucosidases was noted at 30 °C. Additionally, proteins like GH1 β-glucosidase 1 that play a major role in glycan metabolism and cellulose degradation were upregulated at all tested SSF culture condition. The majority of thermostable endoglucanases belong to GH family 12, but thermostable endoglucanases belonging to families 5, 8, and 45 have also been reported.61,62 This study identified endoglucanases belonging to GH2, GH5, GH7, GH12, GH16, GH17, GH61, and GH81, and they could be thermostable since the isolated strain is thermophilic. Exoglucanases or cellobiohydrolases (CBHs) are of significant importance to cellulolytic systems that facilitate the production of mostly cellobiose which can readily be converted to glucose by β-glucosidases. The secretome of A. f umigatus LF9 revealed high abundance of GH6 and GH7 cellobiohydrolases at 30−50 °C. The themostability data of cellulases indicated about 80% activity at 60 °C and 60% activity at 70 °C. Most thermostable CBHs from bacteria Clostridium thermocellum have optimal activity at temperature 60 °C. Thermostable CBHs also occur widely in fungi, such as Thermoascus aurantiacus, Talaromyces emersonii, and Cladosporium spp. Sharma et al.6 also tested A. f umigatus isolated from a compost pile of mixed industrial waste and found to produce a spectrum of cellulases and hemicellulases. β-Glucosidases are important enzymes since they are responsible for the regulation of the entire cellulolytic process. Thus, β-glucosidases not only catalyze conversion of cellobiose into glucose but also reduce cellobiose-mediated repression. This study identified two GH1 and six GH3 β-glucosidases in the thermostable secretome of thermophilic isolate A. f umigatus LF9. The β-glucosidase of A. f umigatus has been shown to have far superior heat stability compared to the previously characterized β-glucosidases of A. niger and A. oryzae.63 Several copies of enzymes like β-1,6-glucanase, α/β hydrolase, α-1,3glucanase, α-amylase, α-glucosidase, glucan 1,4-α-glucosidase, and glycosyl hydrolase with cellulolytic activities were identified in the secretome of isolated A. f umigatus LF9. The secretome of the tested fungus showed several α/β-1,3-glucanases which have multiple functions. Based on the mode of sugar breakdown pattern, they are classified as endo- and exoglucanases and play a role in biomass hydrolysis. These enzymes are presumed to be involved in defense system,64 morphogenetic−morpholytic processes during fungal development and differentiation,65−67 mobilization of glucans during carbon and energy source exhaustion,68 and fungal pathogen− plant interactions.69 The cell wall glucanases that play a role in cell expansion during growth, in cell−cell fusion during mating, and in spore release during sporulation were identified in the secretome of this studied fungal strain. These enzymes hydrolyze both 1,3-β- and 1,6-β-linkages and also function biosynthetically as transglycosylases. Cluster analysis revealed temperature dependent regulation of GPI-anchored glucanases and GPI-anchored proteins. Aspergilli have both GPI-anchored and non-GPI-anchored protein, and some secreted non-GPI

enzymes of A. f umigatus LF9 were found to hydrolyze sawdust, producing sugars 67.50−61.53 mg g−1 sawdust, which was significantly higher than the sugars obtained during alkalipretreated rice straw hydrolysis by different strains of Aspergillus (BCC 125, BCC 199, BCC 309, BCC 4435, and BCC 14405), Penicillium (BCC 4441 and BCC 14374), Trichoderma (BCC 7041), Mamillisphaeria (BCC 8893), Talaromyces (BCC 366), Periconia (BCC 2871), and Guignardia (BCC 12206).54 Mirzaakhmedov et al.55 compared the cellulolytic ability of Panus tigrinus, Pleurotus ostreatus, Fomes fomentarus, and A. terreus using plant wastes (pulp from cotton plants and birch chips) as substrates at 30 °C and showed that the strain belonging to Aspergillus sp. (A. terreus) was the best producer of cellulases. Using grass, corncob, bagasse, and cellulose as a substrate for SSF culture at 35 °C, the production kinetics of endoglucanase and β-glucosidase of A. niger MS82 has been documented.56 Thus, comparisons of endoglucanase, exoglucanase, xylanase, and β-glucosidase activities by isolate A. f umigatus LF9 with other Aspergillus strains suggested enzymes of isolate A. f umigatus LF9 could be useful in lignocellulosic biorefinery provided these enzymes heterologously expressed in a GRAS (generally recognized as safe) organism and then used. Thus, heterologous expression of these enzymes in a GRAS organism would minimize possible pathogenicity of A. f umigatus. Proteomic Characterization of Secretome

Proteomics is an excellent tool in profiling, discovering, and identifying proteins produced in response to a particular environment. Extracellular proteome of A. terreus grown on different carbon sources such as sucrose, glucose, or starch at 30 °C revealed production of hydrolases, glycosylases, and proteases; while A. niger strain N400 (CBS 120.49) cultured with D-maltose, D-xylose, and sorbital at 30 °C demonstrated induced production of hydrolases, oxidoreductases, and transferases.57,58 Culturing A. niger strain N400 with D-maltose and D-xylose resulted in an increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose. However, proteomic analysis of A. f umigatus for lignocellulolytic enzymes is rare. Again, most of the strains including T. reesei, A. niger, and P. chrysosporium9−12 have been studied for their cellulolytic and lignin degrading enzymes but they were cultivated at room temperature (30 °C). Strain A. f umigatus LF9 showed growth at 60 and 70 °C, but it was low when compared to growth at 30−50 °C. This study identified 667 proteins when cultivated using sawdust, and this was significantly higher than the 196, 166, 172, and 182 proteins secreted by A. nidulans when cultivated using sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7, and 14 days at 37 °C.59 Using proteomics techniques, this study identified 78, 75, 78, 78 cellulolytic proteins at temperatures 20, 30, 40, and 50 °C respectively, when A. f umigatus LF9 was cultivated on sawdust by SSF; and this was higher than the 65 cellulases secreted by T. reesei QM6a and Rut C30 during cellulose and lignocellulose degradation.9 Endoglucanases, exoglucanases, and β-glucosidases are the key enzymes in cellulose hydrolysis. This study identified 16 endoglucanases, 10 each exoglucanases and β-glucosidases, 2 cellobiose dehydrogenases, and several GH proteins that emphasize cellulose degradation potential of isolated A. f umigatus LF9 isolate. T. reesei is an efficient polysaccharide degrader, and its genome encodes 200 glycosyl hydrolases (GHs) genes. On the contrary, the genome of Aspergillus sp. M



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temperatures when A. f umigatus LF9 was cultivated with sawdust. In addition to lignocellulolytic proteins, this study identified 41 peptidases and proteases when isolated fungal strain A. f umigatus LF9 was cultivated using sawdust. The genomes of A. oryzae, A. f umigatus, and A. nidulans contain 135, 99, and 90 secreted proteinase genes, respectively21,50,55,80 According to Cassab et al.84 proteins are an integral part of the plant cell wall and they play a major role in morphogenesis and in the formation of β-pleated sheets. Thus, the presence of the proteins in the plant cell wall and their possible cross-links with carbohydrates themselves explain the expression of a vast number of peptidases and proteases in the secretome of A. f umigatus LF9. Extracellular degradation of biomass results in the formation of sugars and other nutrients that are subsequently transported into the cell. Thus, 56 transport proteins were detected; among them were monosaccharide transporter, hexose transporter, sugar transporter, fructose symporter, and glucose transporter indicating the sugar transport pathway was active during sawdust degradation. ABC multidrug transporters that play an important role in normal fungal physiology by protecting from xenobiotics and endogenous metabolites were also abundantly expressed. This study also identified Sec23/Sec24 family protein and Sec7 domain protein indicating sec dependent transport system in A. f umigatus LF9. Yet 18 ABC transport proteins were expressed and identified. This study identified a significantly high number of hypothetical proteins during SSF of sawdust at different temperatures. The abundances of hypothetical proteins were significantly high, e.g., proteins Afu3g01130, Afu3g00960, Afu5g14560, and Afu5g02100 were identified with emPAI 17.93 ± 1.24, 5.78 ± 0.44, 7.86 ± 0.55, and 4.10 ± 0.23 respectively during SSF of sawdust at 50 °C indicating their possible role in lignocellulosic biomass hydrolysis.

enzymes are involved in degradation of plant cell wall material to acquire a carbon source for growth.66 The secretome of A. f umigatus LF9 highlighted the presence of hydrophobins Hyp1 and conidial hydrophobin RodB that plays a major role in mediating contact and communication between the fungus and its environment. Hsp70 chaperonic proteins that play a major role in stabilizing partially folded protein and aid in transmembrane transport of proteins were also identified in this study. Hemicellulose is very complex and heterogeneous, and hence its hydrolysis into simpler constituents like monomers, dimers, or oligomers requires a wide spectrum of enzymes. Numerous mesophilic bacterial and fungal species produce hemicellulases, however, themophilic hemicellulases by Thermobif ida f usca,70,71 Bacillus licheniformis,72 Geobacillus sp.,73 xylanilyticus,74 Thermoactinomyces vulgaris,75 Ureibacillus terrenus,76 and some other species77 have been documented. The secretome of A. f umigatus LF9 revealed expression of 35−41 hemicellulolytic proteins. Similarly, 37 hemicellulolytic proteins were iTRAQ quantified in the secretome of T. reesei QM6a and Rut C30,9 while only 16 hemicellulolytic proteins were detected in P. chrysosporium when this fungus was cultivated in cellulosic or lignin culture medium.13 A. oryzae cultivation in SSF and submerged culture condition with wheat bran as a major carbon source revealed 43 and 37 secretory proteins that had either lignocellulolytic or proteolytic activity.78 The most important of these hemicellulolytic enzymes is endoxylanase, which cleaves β-1,4 linked xylose backbone, and this study identified 6 endo1,4-β-xylanases. Several xylosidases that hydrolyze xylooligomers were detected in the secretome of A. f umigatus LF9 when this fungal strain was cultivated on sawdust. In addition, a variety of debranching enzymes α-arabinofuranosidase, arabinanase, α-, β-galactosidase, α-1,2-, endo-1,4-βmannosidase, acetyl xylan esterase, α-L-rhamnosidase, galactosidases, carboxylesterases, polygalactosaminidases, arabinosidase, arabinases, and polygalacturonase were identified and differentially expressed at different temperatures during sawdust degradation by A. f umigatus LF9. Cluster analysis clearly indicated upregulation of these debranching enzymes when SSF temperature was 50 °C, while esterases were abundant at lower temperatures. Lignin is an amorphous high molecular mass polymer of lignocellulosic biomass composed of phenylpropane subunits that are interconnected by a variety of nonhydrolyzable bonds. Relatively few groups of microorganisms that have potential to cleave Cα−Cβ, β-aryl ether, C1−Cα bonds, and aromatic rings can degrade lignin. Aspergilli are able to transform a wide spectrum of lignin-related aromatic compounds and species like A. f umigatus, A. japonicus, A. niger, and A. terreus have been tested for their ability to metabolize14C-labeled aromatic compounds.79,80 Aspergillus sp. fungal strain F-3 isolated from forest soil has been shown to degrade alkali lignin.81 Peroxidases catalyze lignin degradation while cellobiose dehydrogenases (CDH) also contribute to lignin degradation. This study identified peroxidase (Afu4g02780), glutathione peroxidase (Afu3g12270), cytochrome c peroxidase, (Afu4g09110), and 2 CDH during sawdust degradation. Again, proteins such as laccase, alcohol oxidase, bifunctional catalase-peroxidase, glutathione reductase, and glutathione Stransferase glyoxalase were also detected in the secretome of isolated A. f umigatus LF9. Dioxygenase that catalyzes a key step in the degradation pathway of vanillate,82,83 which is an intermediate in lignin degradation, was expressed at all the

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CONCLUSIONS Thermostable enzymes have potential applications in lignocellulose biomass hydrolysis to its component sugars and significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. Phylogenic characterization of isolated strain revealed its affiliation to Aspergillus while characterization of its growth profile and enzyme activities indicated maximum activities in the range 40−50 °C, demonstrating the thermophilic nature of the isolate A. f umigatus LF9. Proteomics characterization of secretome suggested a large number of cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins. The abundances of lignocellulolytic proteins as estimated by emPAI and further their hierarchical clustering revealed temperature dependent expression. The characterization of its secretome for heat sensitivity demonstrated that the secreted proteins were thermostable. Thus, this study reports the new thermostable GH proteins of A. f umigatus LF9.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S4, Tables ST1−ST3, and Tables B2 and B3. This material is available free of charge via the Internet at http:// pubs.acs.org. N



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AUTHOR INFORMATION

Corresponding Author

*S.K.S.: tel, +65: 6514-1006; fax, +65: 6791-3856; e-mail, [email protected]. S.S.A.: tel, (65) 6316-2852; fax, (65) 67913856; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from Nanyang Technological University (RG 157/06, RG 61/06 and RG 51/10).

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