Proteomic Analysis of pH and Strains Dependent Protein Secretion

ARTICLE pubs.acs.org/jpr

Proteomic Analysis of pH and Strains Dependent Protein Secretion of Trichoderma reesei Sunil S Adav,† Anita Ravindran,† Lim Tze Chao,† Lynette Tan,‡ Sunil Singh,‡ and Siu Kwan Sze†,* † ‡

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 Temasek Engineering School, Temasek Polytechnic, 21 Tampines Avenue 1, Singapore 529757

bS Supporting Information ABSTRACT: Bioenergy, particularly biofuel, from lignocellulosic biomass has been considered as one of the most promising renewable and sustainable energies. The industrial productivity and efficiency of microbial lignocellulolytic enzymes for cellulosic biofuel applications are significantly affected by pH of culture condition. This study established and compared hydrolytic protein expression profiles of Trichoderma reesei QM6a, QM9414, RUT C30 and QM9414MG5 strains at different pH in cellulosic culture media. Liquid chromatographytandem mass spectrometry (LCMS/MS) analysis of secretome of T. reesei cultured from pH 3.09.0 revealed significantly higher hydrolytic protein expressions at acidic pH. The BrayCurtis similarity indices, clustering, and Shannon diversity index elucidated differences in protein secretion at different pHs in individuals and among the strains. This study demonstrated a comparative lignocellulolytic enzyme secretion profile of T. reesei and its mutants at different pHs and provides pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis. KEYWORDS: Trichoderma reesei, cellulase, cellulose degradation, secretome, bioenergy and biorefinery

’ INTRODUCTION Lignocellulosic biomass is a renewable and the most abundant common biopolymer synthesized by plants using solar energy. Its enzymatic hydrolysis to monomeric sugars has great biotechnological application in biofuel and biomaterials. Economic bottleneck in the production of cellulosic biofuel lies in economically viable production of lignocellulolytic enzymes for lignocellulosic biomass hydrolysis which could accelerate development in renewable biofuel for mitigating global warming, fuel shortage and environmental pollution. To enhance enzyme production and improve hydrolytic potential, efforts in genetic manipulation of metabolic pathways, preparation of different recombinants and mutant strains, strain optimization and characterization of their enzymes at genetic and biochemical levels have been reported.14 In spite of these efforts, the required break through has not yet been achieved. Advancement in the development of potential enzyme cocktail that could hydrolyze lignocellulosic biomass into monomeric sugars with lignin as a byproduct is needed to make lignocellulosic biofuel commercially viable. Several bacterial and fungal strains including strains of Trichoderma species have the potential to produce cellulases and hemicellulases.5,6 Trichoderma spp. is a potential degrader of cellulosic biomass and often detected at plant material decomposing sites, rhizosphere of plants7,8 and also isolated from termite guts.9 Trichoderma species are ubiquitous soil-borne ascomycetes filamentous fungi of which T. reesei is extensively studied and developed as an industrial producer of cellulases and xylanases r 2011 American Chemical Society

with applications in food, feed, textile, pulp and paper industries.1013 Due to its biotechnological applications, several researchers are interested in isolating novel cellulases by T. reesei that have higher activity and can tolerate higher pH14,15 while others tried to genetically remodify the enzymes to improve their pH optima and activity.1618 The pH is an important parameter in the production of enzymes by T. reesei and earlier reports showed high pH (7.0) is essential for xylanase production while low pH favors cellulase production.1921 Of the endoglucanase, endoglucanase II is most abundantly produced by T. reesei which has highest activity at pH 4.64.8 and were further investigated by saturation mutagenesis to demonstrate the influence of amino acid at position 342 on the activity and pH optima.18 Several researchers tried to increase the catalytic efficiency by single substitution of amino acid or modifying proteins.16,17,22 T. reesei is known as the most efficient cellulose-degrading organism and its cellulase system is under thorough investigation to establish it in lignocellulosic bioenergy. The genome sequencing of three Trichoderma strains, that is, T. reesei, T. atroviride and T. virens, itself explains massive research efforts in establishing Trichoderma sp. in lignocellulosic biofuel.23 Of these three strains, T. reesei is a potent cellulase producer23 whose several mutants have been generated by random mutation using chemical mutagens and UV light to enhance cellulolytic enzyme production and cellulose Received: May 6, 2011 Published: August 31, 2011 4579

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Journal of Proteome Research hydrolysis. The available literature suggests that T. reesei secretes at least four different endoglucanases (EGI-EGIV), two cellobiohydrolases (CBHI and CBHII) and two beta-glucosidases.24 Recently, Herpoel-Gimbert et al.25 compared secretome of T. reesei RUT-C30 and CL847; however, comparative proteomics secretome analysis of T. reesei and its random mutants that might highlight novel and low abundant important enzymes is limited. Along with recent developments in genome sequencing technology, proteomics becomes an excellent tool in exploring, discovering and identifying expressed proteins in response to environmental stress, carbon source, temperature and pH.26 Particularly, the shotgun LCMS/MS proteomics method allows unbiased identification of thousands of proteins in a complex sample in a single experiment.27 Using label-free quantitation methods with shotgun proteomics approach enables comparative proteomics quantification of multiple samples.28,29 This strategy can be employed to investigate global differential protein expression of any samples suitable for shotgun proteomics analysis. The parameters such as pH, carbon source, temperature etc. affects microbial enzyme production. The pH is a major factor that alters metabolic pathways, affects industrial enzyme production and enzyme synthesis efficiency. Moreover, several hyper cellulase mutants of T. reesei were generated by UV light and chemical mutagens, but these mutagens were not specific to cellulase and hemicellulase gene mutation. Comparing their hydrolytic enzymes production at different pHs will enables optimal production of interested enzymes using appropriate strain in suitable cell culture condition. Therefore, the objective of this study was to comprehensively profile the extracellular lignocellulolytic protein of T. reesei from pH 3.0 to 9.0 using LCMS/MS and to semiquantitatively compare the protein expression levels by different strains at different pH using label-free exponentially modified protein abundance index (emPAI).29 Further, this study reports pH sensitive and resistance hydrolytic protein targets useful for industrial lignocellulose hydrolysis.

’ EXPERIMENTAL PROCEDURES Microorganism Cultivation Conditions and Secretome Extraction

This study used T. reesei QM6a (ATCC 13631), QM9414 (ATCC 26921), RUT C30 (ATCC 56765), and QM9414MG5 (ATCC 46481) strains. These strains were procured from American Type Culture Collection (ATCC) and maintained following supplier protocol. For enhancing cellulolytic potential, wild strain T. reesei QM6a was mutated by linear accelerator to obtain mutant QM 9123 which was further transformed to QM 9414 by high-voltage electrons. While four different series such as L series, VTTD series CL series and MG series were obtained by mutating T. reesei QM 9414. The Rut C30 was obtained by series of mutation using mutagens N-methyl-N0 -nitro-N-nitrosoguanidine and UV light. Figure S1 (Supporting Information) presents detailed strain improvement ancestry. These fungi were grown in potato dextrose broths; cell biomass of each strain was collected by centrifugation, washed with sterilized Milli-Q water and inoculated in flask containing medium with composition 3.1 g L1 (NH4)2SO4; 1.5 g L1 carboxymethyl cellulose; 1.5 g L1 NaCl; 1.2 g L1 KH2PO4; 1.0 g L1 K2HPO4 and micronutrients 0.300 g/L MgSO4.7H2O; 0.005 g L1 FeSO4.7H2O; 0.075 g L1 MnSO4.H2O; 0.03 g L1 CaCl2.2H2O 0.002 g L1 COCl2;

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0.1 g L1 thiamin. Further, cell mass was collected by centrifugation and used for inoculation of test flask that contained above medium and cellulose fibrous as a major carbon source (Cat No. C6663, Sigma, St. Louis, MO). The medium pH was adjusted by adding HCl or NaOH solution and maintained at 3.09.0 by increments of 1.0. In the preliminary experiments, growth behaviors of the four strains in cellulosic medium at different pH were monitored by analyzing total protein content to determine the optimal harvesting period. The experiments were performed in triplicate and the mean of emPAI values with standard deviation were reported. The test flasks were incubated at 30 °C, 80 rpm for 120 h and secretome was collected by centrifugation at 7000 g at 4 °C (Beckman Coulter, Brea, CA). The final pH was measured that showed no significant change in pH. The secretome was further filtered through a 0.2 μm filter and concentrated by freeze-drying. The proteins were dissolved in 10 mM acetate buffer pH 7.0, dialyzed and precipitated using ice cold acetone. The protein content was determined by the Bradford method. Protein Digestion, Peptide Extraction, and Mass Spectrometric Analysis

The protein digestion, peptide extraction and spectroscopic analysis were performed as described previously.3032 In brief, equal amounts of protein (20 μg) from each sample were separated by SDS-PAGE and stained with Coomassie brilliant blue, and each sample lane was sliced separately into five fractions, washed with 50% acetonitrile containing 25 mM ammonium bicarbonate buffer (Sigma, St. Louis, MO) and subjected to destaining. The destained gel pieces were reduced with DTT (10 mM) and then alkylated using iodoacetamide (55 mM). After dehydration with 100% acetonitrile, gel pieces were subjected to sequencing grade modified trypsin (Promega, Madison, WI) digestion at 37 °C overnight. The peptides were extracted using 50% ACN/5% acetic acid and analyzed by mass spectrometer. The LCMS/MS analysis was performed in duplicate using LTQFT Ultra mass spectrometer (Thermo-Finnigan, Bremen, Germany), coupled with an online HPLC system (Shimadzu, Kyoto, Japan). Mass Spectrometric and Data Analysis

For each analysis, 100 μL of the samples reconstituted in 0.1% formic acid was injected from an autosampler (Shimadzu, Japan) and concentrated in a Zorbax peptide trap (Agilent, Palo Alto, CA, USA). The peptide separation was performed in a capillary column (200 μm ID x 10 cm) packed with C18 AQ (5 μm, 100 Å pore, Michrom BioResources, Auburn, CA). Mobile phase A (0.1% formic acid in H2O) and mobile phase B (0.1% formic acid in acetonitrile) were used to establish the 90 min gradient that comprised of 3 min 05% B; 52 min 525% B; followed by 12 min 2560% B; maintained at 80% B for 8 min and finally re-equilibrated at 5% B for 8 min. The HPLC was operated at a constant flow rate of 30 μL/min and a splitter was used to create a flow rate of approximately 500 nL min1. The samples were injected into LTQFT through an ADVANCE CaptiveSpray Source (Michrom BioResources, Auburn, CA) with an electrospray potential of 1.5 kV. The gas flow was set at 2, ion transfer tube temperature at 180 °C and collision gas pressure at 0.85 mTorr. The LTQFT was set to perform data acquisition in the positive ion mode. A full MS scan (3501600 m/z range) was acquired in the FT-ICR cell at a resolution of 100 000 and a maximum ion accumulation time of 1000 ms. The linear ion trap was used to collect peptide ions and to measure peptide ion

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Journal of Proteome Research fragments generated by collision-induced dissociation (CID). The 10 most intense ions above a 500 counts threshold were selected for fragmentation in CID. All MS and MS/MS data were searched using Mascot (version 2.2.06, Matrix Science, Boston, MA) search engines against an inhouse build database. The in-house database was a concatenated target and decoy sequences constructed using predicted proteins (TreeseiV2_FilteredModelsv2.0.proteins.fasta) from genome sequences that were downloaded from http://genomeportal. jgi-psf.org. The T. reesei protein sequence database downloaded from the genome project Web site contained only the accession number and amino acid sequence. To obtain the protein function by homology, the amino acid sequence of each protein entry in the database was automatically BLASTp to the NCBInr protein database download from NCBI Web site on 29 November 2009. 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 search, 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. Furthermore, quantification was performed using emPAI values reported by Mascot search engine which is based on equations as stated below.29 PAI ¼ Nobsd =Nobsdl emPAI ¼ 10PAI 1 Where Nobsd and Nobsbl are the number of observed and observable tryptic peptides per protein respectively. The BrayCurtis similarity indices were determined based on the detection or not detection of the protein in the secretome using software Primer (Plymouth Routines In Multivariate Ecological Research). The structural diversity of the secreted proteins were determined using the Shannon index, calculated by H = ∑(Pi log Pi), where Pi is the importance probability of protein in the secretome. Cellulose Hydrolysis and Enzyme Assay

The residual cellulose was collected after centrifugation at 10 000 g, digested with 67% sulphuric acid at 37 °C for 30 min and released sugars were determined using 3,5-dinitrosalicylic acid (DNSA) method33 with glucose as the standard. The enzyme activities were determined on the samples collected at regular intervals from the test culture flask. The endo-1,4-betaglucanase, exo-1,4-beta-glucanase, beta-glucosidase were assayed using carboxymethyl cellulose (CMC), Avicel and salicin as a substrate, respectively. The reducing sugars were determined using 3,5-dinitrosalicylic acid.33 One international unit (IU) of enzyme activity was defined as the amount of enzyme releasing 1 μmol of reducing sugar per minute. The activity of the cellulase and glycoside hydrolases was determined by zymogram analysis on polyacrylamide gel electrophores using 10% separating gel containing 1% CMC. The gels were run in Mini Protean (Bio-Rad) gel running apparatus at 70 V for 120 min. The gel staining was performed by removing SDS using Triton X-100. The renaturation of enzyme was carried out by overnight incubation of gels at 4 °C in 2.5% Triton X-100. After overnight incubation, the gels were incubated at 42 °C for 60 min in acetate buffer pH 5.5. The gels were then stained with 1% Congo red stain for 30 min and destained in 1 M NaCl for 15 min.

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’ RESULT Features of T. reesei Proteins at Different pH

The abundance of protein in the conditioned medium is measured by the emPAI value. The secretion amount of GH7 cellobiohydrolase I/exoglucanase 1, GH54 arabinofuranosidase B and glyoxal oxidase at different pH in T. reesei wild and mutants were presented in Figure 1, while other cellulose and hemicellulose hydrolyzing proteins by T. reesei QM6a, QM9414, RUT C30 and QM9414MG5 at different pH were depicted in figures G1G25 and H1H20 (Supporting Information). The cellulases and hemicellulases secreted by T. reesei and its mutants at different pH were listed in Table 3. While, cellulase, hemicellulase, lignin degrading proteins, peptidases and proteinases, chitinases, phosphatases, lipases, transport and hypothetical proteins with siganlP and GH family under different pH conditions were listed in Table TS1 to TS7 (Supporting Information). On the basis of the number of lignocellulolytic proteins identified, major protein expressions of T. reesei QM6a, QM9414, RUT C30 and QM9414MG5 were in the pH range of 5.07.0, 4.08.0, 4.07.0 and 4.08.0, respectively (Figure S2, Supporting Information). The comparisons of the specific identical proteins identified at each pH in the secretome of wild and mutant strains of T. reesei and their similarities are listed in Table 1. At pH 5.0, BrayCurtis similarity indices between T. reesei QM6a and QM9114, RUT C30 and QM9414MG5 were in the range of 56.7662.80%, while the corresponding values at pH 9.0 and pH 3.0 were 37.4330.65% and 49.2250.00%, respectively (Table 1). The comparison of secretory protein profile of T. reesei QM6a at different initial pH suggested higher BrayCurtis similarity indices in pH range 5.0 to 7.0 (77.7555.34%) while BrayCurtis similarity indices for the secretory proteins by mutants T. reesei QM9114, RUT C30 and QM9414MG5 vary with pH (Table 2). The clustering pattern (Figure S3, Supporting Information) and BrayCurtis similarity indices indicated that the extracellular protein secretion as a dynamic nature. The effects of pH on diversity of secretory proteins by T. reesei QM6a, QM9114, RUT C30 and QM9414MG5 were further determined using the Shannon diversity index (H) (Table S1, Supporting Information). The H index of T. reesei QM6a at pH 3.0 was 5.34 that decreased to 4.934.30 when pH was 8.09.0. The initial H index of T. reesei QM9414 was 4.77 which increased with increase in pH, achieved optimum at pH 8.0 and then decreased with more alkaline pH. T. reesei RUT C30 and T. reesei QM 9414 MG5 showed higher H index at pH 5.0 and 8.0, respectively. Expression of Cellulolytic Proteins at Different pHs by T. Reesei and Their Mutants

As can be seen from the profile of emPAI values (Figure G1G25, Supporting Information, Table 3) by T. reesei QM6a, QM9414, RUT C30 and QM9414MG5 at different pHs, hydrophobin-2 (jgi|Trire2|119989) and hydrophobin-1 (jgi|Trire2| 123967) were expressed at all tested pH in T. reesei wild and mutants and variably regulated, while other cellulose hydrolyzing protein expressions were pH and strain dependent. On the basis of the emPAI values (Table 3), GH15 glucoamylase F was significantly higher in T. reesei QM6a followed by QM9414, at acidic pH 3.05.0. The expression of protein GH7 cellobiohydrolase I increased with pH and achieved maximum at pH 6.0, 4.0, 5.08.0 and 5.06.0 and then decreased in T. reesei QM9414, RUT C30, QM9414MG5 and QM6a, respectively (Figure 1). The expression of GH17 glucan 1,3-beta-glucosidase was higher in QM9414 followed by RUT C30 and QM6a and achieved 4581


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Figure 1. Profiles of emPAI values of proteins in the secretome of T. reesei wild and mutants at different pH.

highest emPAI values at pH 4.0, pH 4.0 and pH 6.0 respectively (Figure G5, Supporting Information). The emPAI values of alpha/beta hydrolase suggested higher production at acidic pH 4.06.0 with better expressions in T. reesei QM9414, RUT C30 and QM6a (Figure G7, Supporting Information) while GH5 endoglucanase EG1 was significantly higher in QM9514MG5 (Figure G4, Supporting Information). Thus, majority of cellulases were expressed abundantly at acidic pH 4.06.0. Interestingly, GH5 endoglucanase III was peaked at pH 8.0 in T. reesei QM9414MG5 indicating its activity at above neutral pH (Figure G8, Supporting Information). Thus, the expressions of cellulolytic enzymes at different pHs suggested several pH resistance enzyme targets useful for lignocellulosic bioenergy. The detailed analysis of the cellulases and glycoside hydrolases including total number of identified proteins, common proteins in all and among the strains are presented in Figure 2. Of the total identified proteins in the secretome of T. reesei QM9414, RUT C30, QM9414MG5 and QM6a at pH range 4.0 to 8.0, 3649, 2346, 2240 and 2644 proteins were having cellulolytic function. Lowest number of proteins was detected at pH 9.0. In addition to numerous cellulose hydrolyzing proteins, this study identified novel CBM1 swollenin, GH16 cell wall glucanase, GH16 cell wall glucanosyltransferase, beta-glucosidases etc. The novel swollenin had high sequence similarity to the plant expansins protein and ability to hydrolyze crystalline cellulose at acidic pH. Although swollenin was produced by all the tested

strains, it was significantly higher in T. reesei QM9414MG5 at pH 6.08.0 and RUT C30 at pH 4.06.0 followed by QM9414. Profile of Hemicellulases, Lignin Depolymerizing and Other Proteins

Hemicelluloses hydrolyzing proteins such as xylanse, endo1,4beta-xylanase, arabinofuranosidase, exorhamnogalacturonase, betamannase, alpha-,beta-mannosidase, acetylxylan esterase, ferulic acid esterase, alpha-beta-xylosidase, arabinosidase, endo-beta1,6galactanase, mannan-binding lectin and mannan endo1,6-alphamannosidase were identified in the secretome (Table 3). The GH11 endo1,4-beta-xylanase I was comparatively higher in T. reesi QM9414 (Figure H1, Supporting Information) while GH10 endoxylanase II was expressed at all pH above 3.0 in T. reesei QM9414, RUT C30 and QM9414MG5 (Figure H10, Supporting Information). This study noted the higher expressions of GH54 arabinofuranosidase B in the secretome of T. reesei QM9414 followed by RUT C30 and QM6a, with optimum expressions at pH 4.06.0 in T. reesei wild and other tested mutants (Figure 1). The GH11 xylanase III was only expressed in T. reesei QM9414 and QM9414MG5 (Figure H20, Supporting Information). The detailed analysis with identical proteins among T. reesei wild and mutants (Figure S4, Supporting Information) and overlap proteins suggested only 3 to 5 common proteins in all tested strains with hemicellulolytic function. 4582



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Table 1. BrayCurtis Similarity Indices for the Secretory Proteins by T. reesei Wild and Its Mutants at Different pH during Cellulose Hydrolysis T. Reesei QM6a

T. Reesei

T. Reesei

QM9414 RUT C-30

Table 2. BrayCurtis Similarity Indices for the Secretory Proteins by T. reesei Wild and Its Mutants at Different pH during Cellulose Hydrolysis

T. Reesei QM

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

47.15

100.00

9414 MG5 T. reesei QM6a

pH 3 T. reesei QM6a

pH 3

100.00

100.00

pH 4

68.21

T. reesei QM9414

49.22

pH 5

65.48

77.05

T. reesei RUT C-30 T. reesei QM 9414 MG5

50.00 49.85

pH 6 pH 7

61.46 55.34

69.92 60.42

77.75 64.43

71.10

pH 8

42.75

47.01

48.26

51.41

58.93

pH 9

29.68

31.37

32.43

36.64

36.82

pH 3

100.00

pH 4

59.64

pH 5

59.77

71.33

pH 6

63.08

71.57

75.09

pH 7 pH 8

43.60 34.76

51.17 40.83

56.91 44.08

60.87 48.46

68.51

pH 9

27.62

24.90

27.23

29.06

36.48

34.50

100.00

pH 3

100.00

pH 4

68.20

pH 5

65.66

82.35

pH 6

61.49

76.05

80.89

pH 7

57.36

64.14

65.62

69.39

pH 8 pH 9

41.70 20.00

44.62 17.67

43.90 15.70

50.00 17.35

62.73 25.14

35.86

100.00

pH 3

100.00

pH 4

65.52

pH 5

58.77

71.92

100.00

pH 6

51.97

66.67

70.22

T. reesei QM9414

52.17

pH 7

50.39

62.60

63.11

62.69

T. reesei RUT C-30

43.05

36.36

T. reesei QM 9414 MG5

47.81

57.79

pH 8 pH 9

43.16 35.94

47.35 42.11

48.00 48.94

49.56 42.42

47.06

100.00

37.21 51.87

43.43

100.00

pH 4 T. reesei QM6a

100.00

T. reesei QM9414

T. reesei QM9414

60.36

T. reesei RUT C-30

55.49

53.42


48.46

51.30

43.32

100.00

pH 5 T. reesei QM6a

100.00

T. reesei QM9414

62.80

T. reesei RUT C-30 T. reesei QM 9414 MG5

56.76 57.25

50.15 65.81

43.82

100.00

T. reesei RUT C-30

pH 6 T. reesei QM6a

100.00

T. reesei QM9414

68.48

T. reesei RUT C-30

54.90

55.95


50.93

55.80

40.26

100.00

pH 7 T. reesei QM6a

100.00

T. reesei QM9414 T. reesei RUT C-30

65.73 51.83

51.28


42.14

46.67

T. reesei QM 9414 MG5 39.38

100.00

pH 8 T. reesei QM6a

37.50

100.00

52.48 38.96

pH 9 T. reesei QM6a T. reesei QM9414

100.00 31.33

T. reesei RUT C-30

30.65

23.94


37.43

33.86

29.93

100.00

The protein identification data suggested that the proteins such as GH54 alpha-N-arabinofuranosidase, GH11 endo1,4-beta-xylanase1, acetylxylan esterase and 3-phytase were not expressed in T. reesei RUT C30. Similarly, GH47 alpha1, 2-mannosidase was not expressed in T. reesei QM9414 MG5. The reactive radical generating lignin depolymerizing proteins like laccase, glyoxal oxidase, peroxin 11C, peroxidase/catalase, bifunctional catalase-peroxidase, glutathione transferase, cytochrome oxidase, cytochrome c peroxidase etc. were identified in this study (Table TS1TS7, Supporting Information). The glyoxal oxidase, a H2O2 generating, copper metalloenzyme that play important role in the lignin degradation catalyzed by the fungal lignin (Lip) and manganese peroxidases (MnP) was

identified in wild and all mutant strains at all tested pH. The laccase was detected in all strain with variable quantities except T. reesei QM9414MG5. In addition to cellulases, hemicellulases and lignin degrading proteins, numerous peptidases and proteases were also identified in this study (Table TS1TS7, Supporting Information). Among the peptidases, amidase family protein and hexosaminidase were abundantly produced in wild and mutant T. reesei. Of the detected lignin depolymerizing proteins and petidases, common proteins detected in T. reesei wild and mutants were presented in Figure S5 and S6 (Supporting Information). The other identified protein includes chitinases, phosphatases, lipases, transport proteins and hypothetical proteins. Zymogram, Enzyme Assay and Cellulose Hydrolysis

This study validated LCMS identified proteins by colorimetric enzyme assays and zymography (Table 4 and Figure 3). The zymographic analysis of the secretome by T. reesei wild and mutants showed prominent bands at all pH except pH 9.0. The bands from zymograms were excised; trypsin digested and 4583


QM 6a

3.458 ( 0.7 0.560 ( 0.2 0.601 ( 0.06

2.610 ( 0.00 4.083 ( 1.60 5.151 ( 1.00

1.290 ( 0.00 4.597 ( 1.01 1.072 ( 0.30 0.284 ( 0.04 0.070 ( 0.04

Hydrophobin-2

Hydrophobin-1

4584

Cell wall glucanase

Cel3b

Beta-glucosidase-like protein

Secreted hydrolase Cip1

1.623 ( 0.32

0.158 ( 0.04

0.100 ( 0.00

0.257 ( 0.05

0.302 ( 0.08

0.130 ( 0.00

Endo-beta1,4-glucanase

Beta1,3-glucanase precursor

0.253 ( 0.23

Cellulose hydrolase, putative

0.040 ( 0.02

0.050 ( 0.00

0.210 ( 0.04 0.080 ( 0.04

QM 6a (emPAI)

QM9414 (emPAI)

RUT C-30

0.062 ( 0.03

0.100 ( 0.05 0.100 ( 0.00 0.164 ( 0.06 1.166 ( 0.11 0.081 ( 0.02 1.143 ( 0.40

0.116 ( 0.04 0.150 ( 0.00 0.070 ( 0.04

0.779 ( 0.12 0.130 ( 0.07

0.157 ( 0.08 0.509 ( 0.12 2.292 ( 0.51

0.448 ( 0.05 0.571 ( 0.40

0.200 ( 0.00 0.391 ( 0.05

0.670 ( 0.08 0.756 ( 0.09 0.110 ( 0.06

0.210 ( 0.04 0.127 ( 0.04 0.166 ( 0.09

0.602 ( 0.09 0.448 ( 0.08

0.807 ( 0.22 0.420 ( 0.06 0.617 ( 0.27 0.300 ( 0.00 0.605 ( 0.32

0.105 ( 0.02 0.322 ( 0.02 1.105 ( 0.43

0.250 ( 0.04 0.224 ( 0.04 0.194 ( 0.07

0.090 ( 0.00 0.090 ( 0.00 0.148 ( 0.05

0.260 ( 0.00 0.244 ( 0.04 0.140 ( 0.08

1.273 ( 0.11 2.469 ( 0.16 1.203 ( 0.21

0.811 ( 0.11 0.201 ( 0.04 0.862 ( 0.29

0.350 ( 0.00

1.051 ( 0.09 2.508 ( 0.33 1.897 ( 0.23

0.564 ( 0.06 0.348 ( 0.21

5.783 ( 2.90 2.608 ( 0.55 1.089 ( 0.97

5.151 ( 1.00 5.860 ( 0.00 5.151 ( 1.07

0.373 ( 0.09 0.936 ( 0.49

1.659 ( 0.31 3.648 ( 1.06 0.738 ( 0.20

0.524 ( 0.05 1.048 ( 0.20 1.163 ( 0.38 0.895 ( 0.07 2.310 ( 0.40 0.563 ( 0.25

0.330 ( 0.00 0.845 ( 0.05 1.923 ( 0.38

4.148 ( 0.13 3.831 ( 0.58 0.248 ( 0.03

(emPAI)

pH 4

0.070 ( 0.00

0.384 ( 0.09

0.766 ( 0.00 0.077 ( 0.00 0.492 ( 0.25 0.258 ( 0.06 0.085 ( 0.04 0.470 ( 0.07

0.230 ( 0.00 0.168 ( 0.08

0.807 ( 0.22 0.090 ( 0.05

0.061 ( 0.02 0.370 ( 0.11

Glycosyl hydrolase

xanthan lyase

glucosidase

Glucan endo1,3-alpha-

putative

Endo1,3(4)-beta-glucanase,

Glucan 1,3-beta-glucosidase Glucan 1,3-beta-glucosidase

0.130 ( 0.00 0.095 ( 0.03 0.150 ( 0.04

Endo0,6-beta-D-glucanase

Endoglucanase Cel74a

0.030 ( 0.01 0.090 ( 0.00

0.313 ( 0.02 0.075 ( 0.02 0.100 ( 0.05

0.090 ( 0.00 0.087 ( 0.00 0.201 ( 0.12

Aalpha-glucosidase

Endoglucanase-4

0.649 ( 0.09

0.345 ( 0.04 0.056 ( 0.00 0.407 ( 0.16

0.913 ( 0.09 0.173 ( 0.01 0.598 ( 0.20

Beta1,3-glucanosyltransferase

Alpha/beta hydrolase

0.063 ( 0.02 0.030 ( 0.01

0.487 ( 0.06 0.260 ( 0.05 0.298 ( 0.20

0.377 ( 0.05 0.090 ( 0.04 0.211 ( 0.05

Glucan 1,3-beta-glucosidase

Alpha,alpha-trehalose glucohydrolase

Endoglucanase III

1.320 ( 0.00 1.577 ( 0.24

2.037 ( 0.69 2.279 ( 1.03 0.738 ( 0.20

0.070 ( 0.04 0.389 ( 0.07 0.314 ( 0.00

Extracellular small protein 1

0.731 ( 0.06 0.040 ( 0.00

0.210 ( 0.00 1.201 ( 0.21 0.830 ( 0.26 0.614 ( 0.06 0.661 ( 0.13 0.579 ( 0.19

Exoglucanase 2 Endoglucanase

Endoglucanase EG1

0.228 ( 0.03 2.108 ( 0.10

(emPAI)

QM9414 MG5

3.260 ( 0.30 2.268 ( 0.49 0.210 ( 0.00

(emPAI)

RUT C-30

0.260 ( 0.00 0.741 ( 0.06 0.947 ( 0.46

(emPAI)

QM9414

Glucoamylase F

(emPAI)

Cellobiohydrolase I

protein name

pH 3

hemicellulases

0.150 ( 0.06

0.578 ( 0.05

0.273 ( 0.02

0.197 ( 0.05

0.186 ( 0.01

0.083 ( 0.00

0.050 ( 0.02

0.366 ( 0.02

0.526 ( 0.08

1.392 ( 0.24

1.522 ( 0.47

3.805 ( 0.68

2.042 ( 0.57

0.472 ( 0.04

0.629 ( 0.05 0.169 ( 0.02

1.952 ( 0.35

0.288 ( 0.11

(emPAI)

QM9414 MG5

QM 6a (emPAI)

QM9414 (emPAI)

RUT C-30

0.076 ( 0.05

0.197 ( 0.00

1.639 ( 0.51

0.303 ( 0.02 0.210 ( 0.00 0.164 ( 0.06

0.210 ( 0.01 0.090 ( 0.04 0.070 ( 0.04

0.640 ( 0.00 0.280 ( 0.16

0.581 ( 0.05 0.346 ( 0.02 2.212 ( 0.33

0.622 ( 0.05 0.448 ( 0.05 0.390 ( 0.22

0.484 ( 0.09 0.440 ( 0.09

1.029 ( 0.11 0.616 ( 0.04 0.110 ( 0.06

0.220 ( 0.01 0.080 ( 0.04 0.127 ( 0.04

0.546 ( 0.04 0.230 ( 0.00

1.004 ( 0.09 0.160 ( 0.09 0.476 ( 0.38 0.280 ( 0.01 0.958 ( 0.57

0.485 ( 0.05 0.429 ( 0.03 1.242 ( 0.50

0.186 ( 0.01 0.060 ( 0.03 0.060 ( 0.03

0.138 ( 0.04 0.090 ( 0.00 0.148 ( 0.05

0.549 ( 0.16 0.122 ( 0.04 0.120 ( 0.07

2.063 ( 0.04 1.607 ( 0.34 1.161 ( 0.50

0.366 ( 0.02 0.250 ( 0.00 0.646 ( 0.15

0.025 ( 0.01

1.549 ( 0.18 1.821 ( 0.32 1.787 ( 0.26

0.578 ( 0.05 0.640 ( 0.00 0.270 ( 0.26

1.514 ( 0.20 1.734 ( 0.41 3.276 ( 1.71

4.287 ( 0.27 3.004 ( 0.79 3.817 ( 2.41

0.460 ( 0.16 0.400 ( 0.10 0.622 ( 0.35

1.985 ( 0.11 1.860 ( 0.00 0.968 ( 0.46

0.983 ( 0.02 0.967 ( 0.14 0.666 ( 0.41 0.619 ( 0.05 1.536 ( 0.30 0.585 ( 0.16

0.765 ( 0.10 0.958 ( 0.06 1.414 ( 0.15

3.552 ( 0.61 2.260 ( 0.00 0.284 ( 0.06

(emPAI)

pH 5

0.506 ( 0.11

0.725 ( 0.06

0.110 ( 0.06

0.460 ( 0.00

0.263 ( 0.02

0.090 ( 0.05

0.125 ( 0.02

0.180 ( 0.00

0.452 ( 0.04

0.373 ( 0.08

0.080 ( 0.04

1.517 ( 0.17

2.289 ( 0.55

2.610 ( 0.00

1.419 ( 0.18

0.520 ( 0.00

0.524 ( 0.05 0.132 ( 0.05

2.416 ( 0.21

0.330 ( 0.00

(emPAI)

QM9414 MG5

Table 3. Effect of pH on the Extracellular Secreted Cellulases, Glycoside Hydrolases and Hemicellulases by T. reesei and Its Mutants When Cultivated Using Cellulosea

Journal of Proteome Research ARTICLE


QM 6a (emPAI)

RUT C-30

0.100 ( 0.04 0.065 ( 0.04

Swollenin

Trehalase precursor

1,3-beta-glucanosyltransferase

4585

0.100 ( 0.05

0.328 ( 0.13

Transaldolase, putative Cellulose or protein binding

Putative glycosyl hydrolase

Glycosyl hydrolase, putative

Beta-Ig-H3/Fasciclin

Glycosyl hydrolase,

Alpha amylase catalytic region

Metallo-beta-lactamase Endoglucanase, putative

Dienelactone hydrolase

Fumarylacetoacetate hydrolase

HAD-superfamily hydrolase

Alpha-galactosidase 3

0.295 ( 0.15

0.050 ( 0.00

0.070 ( 0.04

Glycosyl hydrolase

Cell wall glucanase, putative

domain

Glycosyl hydrolase

0.365 ( 0.04

Exobeta 1,3-glucanase

protein

Cellulose signaling related

gel2

Endoglucanase-7 Glycoside hydrolase

Glucanase B

0.080 ( 0.00

0.060 ( 0.00

Maltose permease

0.137 ( 0.03

Mutanase

Alpha-amylase

0.196 ( 0.07

0.639 ( 0.09 0.150 ( 0.00

Extracellular beta glucosidase

Beta1,3-glucanosyltransferase Aldose 1-epimerase

Glucan endo1,3-alpha-glucosidase

0.889 ( 0.56

(emPAI)

QM9414

0.480 ( 0.09

(emPAI)

pH 3

Cell wall glucanosyltransferase

protein name

Table 3. Continued

0.335 ( 0.06

0.416 ( 0.13

0.060 ( 0.03

0.060 ( 0.03

(emPAI)

QM9414 MG5 (emPAI)

QM9414 (emPAI)

RUT C-30

pH 4

0.095 ( 0.03

0.286 ( 0.11

0.110 ( 0.06

0.640 ( 0.37

0.190 ( 0.11

0.137 ( 0.03

0.100 ( 0.00 0.040 ( 0.02

0.069 ( 0.02 0.040 ( 0.02 0.078 ( 0.04

0.060 ( 0.03 0.226 ( 0.21

0.390 ( 0.00 0.180 ( 0.10 0.298 ( 0.20 0.854 ( 0.07 0.070 ( 0.04 0.070 ( 0.04

0.110 ( 0.06

0.116 ( 0.04

0.380 ( 0.00 0.110 ( 0.00 1.000 ( 0.47

(emPAI)

QM 6a

hemicellulases

0.375 ( 0.14

0.053 ( 0.00

(emPAI)

QM9414 MG5

QM 6a (emPAI)

RUT C-30

1.003 ( 0.72

(emPAI)

QM9414

0.070 ( 0.04

0.100 ( 0.05

0.423 ( 0.15

0.110 ( 0.06

0.280 ( 0.00

0.040 ( 0.02

0.190 ( 0.11

0.157 ( 0.03

0.060 ( 0.00

0.078 ( 0.04

0.200 ( 0.01 0.050 ( 0.00

0.083 ( 0.00

0.134 ( 0.03 0.060 ( 0.00 0.252 ( 0.12

0.936 ( 0.04 0.060 ( 0.00 0.291 ( 0.33 0.654 ( 0.11 0.070 ( 0.04

0.102 ( 0.02

0.070 ( 0.07 1.000 ( 0.00

0.533 ( 0.07

(emPAI)

pH 5

0.131 ( 0.02

(emPAI)

QM9414 MG5



QM 6a

0.030 ( 0.00

Glycoside hydrolase family 3

4586

Acetyl esterase

0.080 ( 0.00 0.222 ( 0.06 0.456 ( 0.06

0.140 ( 0.00

0.545 ( 0.05 0.133 ( 0.01 0.317 ( 0.09

Alpha-galactosidase

Endoxylanase II

0.986 ( 0.11

0.201 ( 0.05 0.150 ( 0.08

0.083 ( 0.04 0.142 ( 0.06 0.157 ( 0.04

Beta-xylosidase

Xylosidase/arabinosidase

0.220 ( 0.00

0.210 ( 0.01

0.155 ( 0.02

Beta-mannosidase mndA

Ferulic acid esterase Acetylxylan esterase

0.330 ( 0.00

0.110 ( 0.00

0.080 ( 0.04 0.418 ( 0.05

Beta-mannase

Acetylxylan esterase

1,2-a-D-mannosidase

0.250 ( 0.04

0.130 ( 0.00 0.513 ( 0.10

0.488 ( 0.05 0.322 ( 0.11 0.419 ( 0.13

Alpha-N-arabinofuranosidase

Exorhamnogalacturonase B

0.290 ( 0.00

0.227 ( 0.08 0.557 ( 0.10 0.675 ( 0.05 1.456 ( 0.13 1.686 ( 0.35

Ubiquitin C-terminal hydrolase

Endo1,4-beta-xylanase 1 Arabinofuranosidase B

Glycosyl hydrolase

0.080 ( 0.00

Beta-glucuronidase

0.100 ( 0.04

(emPAI)

QM9414 MG5

Glycosyltransferase

Endoglucanase

Exo-beta1,3-glucanase, putative Endo 1,4-beta-glucanase Cel5b

Glycosyltransferase

0.760 ( 0.00

Putative glycosyl transferase

Peptidoglycan-binding

Probable beta-glucosidase 1

0.040 ( 0.00

Carbohydrate-binding protein,


0.254 ( 0.10 0.111 ( 0.04

0.220 ( 0.00

Beta-glucosidase, putative Endo0,3(4)-beta-glucanase

0.164 ( 0.12

Cellulose hydrolase, 0.130 ( 0.00

(emPAI)

RUT C-30

(emPAI)

QM9414

0.060 ( 0.035

(emPAI)

pH 3

Endoglucanase V

protein name

Table 3. Continued

(emPAI)

RUT C-30

pH 4

0.120 ( 0.06

0.080 ( 0.00 0.137 ( 0.05 0.180 ( 0.11

0.410 ( 0.10 0.233 ( 0.09

0.444 ( 0.05 0.444 ( 0.05 0.216 ( 0.17

0.330 ( 0.00 0.303 ( 0.16

0.210 ( 0.12 0.300 ( 0.07

0.130 ( 0.00 0.142 ( 0.04

0.785 ( 0.10 0.110 ( 0.00 0.360 ( 0.20

0.190 ( 0.00 0.381 ( 0.04 0.060 ( 0.03

0.132 ( 0.05 0.684 ( 0.07 0.307 ( 0.30

0.685 ( 0.06 0.714 ( 0.16 1.181 ( 0.68

0.130 ( 0.00 1.359 ( 0.37

0.290 ( 0.00 0.660 ( 0.00 1.812 ( 0.09 4.200 ( 0.36 3.172 ( 0.63

0.378 ( 0.05

0.126 ( 0.01

0.147 ( 0.03

1.864 ( 0.18

0.156 ( 0.06

0.090 ( 0.05

0.121 ( 0.05

0.123 ( 0.00

0.263 ( 0.02 0.705 ( 0.10

0.070 ( 0.04 0.040 ( 0.02

0.123 ( 0.00

(emPAI)

QM9414 MG5

0.080 ( 0.04 0.110 ( 0.06

0.030 ( 0.01

0.130 ( 0.07

0.164 ( 0.12

(emPAI)

QM9414

Hemicellulases

0.080 ( 0.00

0.020 ( 0.00

(emPAI)

QM 6a

hemicellulases

QM 6a (emPAI)

RUT C-30

0.020 ( 0.12

0.050 ( 0.02 0.350 ( 0.20

0.030 ( 0.01

0.130 ( 0.07

0.200 ( 0.11

(emPAI)

QM9414

0.073 ( 0.00 0.080 ( 0.00 0.080 ( 0.00

0.233 ( 0.09 0.140 ( 0.00

0.400 ( 0.01 0.189 ( 0.04 0.140 ( 0.08

0.394 ( 0.05 0.303 ( 0.16

0.093 ( 0.00 0.010 ( 0.05 0.200 ( 0.01 0.110 ( 0.00

0.112 ( 0.02 0.142 ( 0.02

0.876 ( 0.02 0.180 ( 0.06 0.110 ( 0.06

0.546 ( 0.04 0.976 ( 0.11 0.307 ( 0.21

1.012 ( 0.16 0.474 ( 0.11 0.307 ( 0.30

0.524 ( 0.05 0.315 ( 0.04 1.497 ( 0.77

0.478 ( 0.11 1.228 ( 0.13

0.599 ( 0.05 0.660 ( 0.00 1.845 ( 0.21 3.999 ( 0.30 3.602 ( 0.84

0.030 ( 0.01

0.020 ( 0.00

(emPAI)

pH 5

0.754 ( 0.14

0.140 ( 0.00

0.111 ( 0.04

1.664 ( 0.11

0.220 ( 0.00

0.267 ( 0.07

0.474 ( 0.11

0.130 ( 0.00

0.290 ( 0.00 1.084 ( 0.13

0.130 ( 0.00

(emPAI)

QM9414 MG5



QM 6a

QM 6a

4587

0.060 ( 0.03

Phytase

0.625 ( 0.05 0.040 ( 0.02 0.520 ( 0.00 1.751 ( 0.09 1.638 ( 0.10 0.174 ( 0.05

0.731 ( 0.06 0.923 ( 0.13 0.830 ( 0.26

0.490 ( 0.00 1.080 ( 0.09 0.368 ( 0.09

2.283 ( 0.38 1.860 ( 0.00 0.738 ( 0.20 0.347 ( 0.05 0.498 ( 0.11 0.272 ( 0.13

0.494 ( 0.05 0.985 ( 0.07 0.348 ( 0.21

1.750 ( 0.26 1.452 ( 0.14 1.607 ( 0.34

Exoglucanase 2

Endoglucanase

Extracellular small protein 1 Endoglucanase EG1

Endoglucanase III

Glucan 1,3-beta-glucosidase

0.222 ( 0.06 2.228 ( 0.10

(emPAI)

3.469 ( 0.12 2.419 ( 0.27 0.254 ( 0.10

(emPAI)

0.780 ( 0.00 1.366 ( 0.27 1.456 ( 0.31

(emPAI)

QM9414MG5

0.116 ( 0.04

Glucoamylase F

(emPAI)

RUT C-30

pH 6

Cellobiohydrolase I

protein name

EstA precursor

QM9414


QM 6a

Mannan endo1,6-alpha-

mannosidase

0.116 ( 0.04

Mannan-binding lectin

precursor

0.150 ( 0.08

0.060 ( 0.000

Acetyl xylan esterase Mannose-binding lectin

Ferulic acid esterase

0.067 ( 0.04

Xylanase III

0.142 ( 0.04

Alpha1,2-mannosidase

Beta-mannosidase



(emPAI)

QM9414

(emPAI)

RUT C-30

pH 7

0.150 ( 0.08 0.100 ( 0.04

0.080 ( 0.04

1.299 ( 0.13 0.816 ( 0.21 0.915 ( 0.19

0.394 ( 0.05 0.803 ( 0.15

1.320 ( 0.00 1.659 ( 0.31 0.520 ( 0.00 0.183 ( 0.04 0.347 ( 0.05 0.070 ( 0.04

0.413 ( 0.02 0.270 ( 0.00 0.130 ( 0.07

0.660 ( 0.00 1.008 ( 0.12 0.354 ( 0.14

0.614 ( 0.10 1.000 ( 0.00 0.619 ( 0.05

3.064 ( 0.28 1.096 ( 0.05 0.050 ( 0.02

(emPAI)

QM 6a

hemicellulases

0.070 ( 0.00

0.060 ( 0.00

0.100 ( 0.00

0.200 ( 0.00

0.284 ( 0.06 0.100 ( 0.00

0.847 ( 0.17 0.100 ( 0.04

0.180 ( 0.10

0.150 ( 0.00 0.058 ( 0.03

1.302 ( 0.39 0.175 ( 0.09 0.400 ( 0.23

3-phytase A precursor

Galactomannoprotein, Endo-beta1,6-galactanase

0.095 ( 0.02 0.193 ( 0.04 0.242 ( 0.05 1.264 ( 0.18 0.554 ( 0.28

(emPAI)

RUT C-30

(emPAI)

QM9414

(emPAI)

(emPAI)

QM9414 MG5

0.058 ( 0.01

(emPAI)

RUT C-30

0.085 ( 0.04 0.192 ( 0.11

(emPAI)

QM9414

pH 4


(emPAI)

pH 3

hemicellulases

3-carboxymuconate cyclase

protein name

Table 3. Continued

0.209 ( 0.02

1.673 ( 0.19

0.472 ( 0.04 1.259 ( 0.05

1.708 ( 0.24

2.401 ( 0.30

0.119 ( 0.01

(emPAI)

QM9414MG5

0.166 ( 0.01

0.043 ( 0.00

(emPAI)

QM9414 MG5

QM 6a (emPAI)

QM9414

(emPAI)

RUT C-30

(emPAI)

QM9414

(emPAI)

RUT C-30

pH 8

0.320 ( 0.18 0.130 ( 0.07

0.777 ( 0.11 0.565 ( 0.09 0.735 ( 0.26

0.494 ( 0.05

0.520 ( 0.00 0.738 ( 0.20 0.520 ( 0.00 0.070 ( 0.00

0.111 ( 0.02 0.396 ( 0.02 0.040 ( 0.02

0.692 ( 0.12

0.190 ( 0.00 0.680 ( 0.00 0.295 ( 0.11

1.062 ( 0.05 0.702 ( 0.09 0.050 ( 0.02

(emPAI)

QM 6a

0.060 ( 0.00

0.080 ( 0.04 0.160 ( 0.09

0.180 ( 0.10

0.355 ( 0.04 0.100 ( 0.00

0.166 ( 0.01 0.116 ( 0.01

0.299 ( 0.03 0.147 ( 0.07

0.218 ( 0.03 0.593 ( 0.07 0.554 ( 0.28

0.095 ( 0.02

(emPAI)

pH 5

0.463 ( 0.10

1.831 ( 0.11

0.520 ( 0.00 1.577 ( 0.24

1.816 ( 0.28

2.352 ( 0.19

0.150 ( 0.00

(emPAI)

QM9414 MG5

0.131 ( 0.02

(emPAI)

QM9414 MG5



0.180 ( 0.06

0.448 ( 0.08 0.360 ( 0.00

Endo1,3(4)-beta-glucanase,

4588

0.210 ( 0.00 0.330 ( 0.00 0.284 ( 0.06

0.070 ( 0.00

Secreted hydrolase Cip1

Beta-glucosidase-like protein

0.221 ( 0.04 0.321 ( 0.04

0.040 ( 0.00

0.280 ( 0.00 0.050 ( 0.02

0.153 ( 0.04 0.685 ( 0.06

1.327 ( 0.10 0.425 ( 0.29

Trehalase precursor

Mutanase

Alpha-amylase

Glucanase B

0.040 ( 0.00

0.270 ( 0.00

0.530 ( 0.00 0.070 ( 0.00

Aldose 1-epimerase

Swollenin

0.040 ( 0.02

0.145 ( 0.02 0.442 ( 0.04 0.120 ( 0.06 0.379 ( 0.21

Extracellular beta glucosidase Beta1,3-glucanosyltransferase

0.427 ( 0.09 0.240 ( 0.00 1.262 ( 0.80


Cell wall glucanosyltransferase

0.100 ( 0.00

0.230 ( 0.00 0.070 ( 0.04

Cell wall glucanase 1.204 ( 0.34

0.040 ( 0.00 1.380 ( 0.00

0.309 ( 0.03 0.270 ( 0.00 2.156 ( 0.59 0.130 ( 0.07 1.100 ( 0.00 0.130 ( 0.07

Beta1,3-glucanase precursor Endo-beta1,4-glucanase

Cel3b

0.836 ( 0.13

0.801 ( 0.11 0.307 ( 0.05

0.480 ( 0.00 0.789 ( 0.19 0.420 ( 0.20

Glycosyl hydrolase

0.815 ( 0.14 0.241 ( 0.09 0.085 ( 0.03

Cellulose hydrolase,

0.080 ( 0.04

0.160 ( 0.00


xanthan lyase

0.270 ( 0.11

0.460 ( 0.00 0.080 ( 0.00 0.953 ( 0.24 0.860 ( 0.00 0.750 ( 0.09 2.770 ( 0.55

Glucan 1,3-beta-glucosidase Glucan 1,3-beta-glucosidase

putative

0.444 ( 0.05

0.600 ( 0.03

0.100 ( 0.04

0.090 ( 0.00 0.090 ( 0.00 0.090 ( 0.00

0.232 ( 0.02 0.356 ( 0.02 0.789 ( 0.23

0.090 ( 0.00

0.425 ( 0.08 0.053 ( 0.02 0.100 ( 0.05

Aalpha-glucosidase

Endoglucanase-4

Eendoglucanase Cel74a

0.180 ( 0.00

Endo0,6-beta-D-glucanase

0.400 ( 0.00

0.342 ( 0.08 0.095 ( 0.03 0.428 ( 0.17

0.109 ( 0.01

1.470 ( 0.11 1.077 ( 0.26 0.956 ( 0.28

Alpha/beta hydrolase

Beta1,3-glucanosyltransferase

Alpha,alpha-trehalose glucohydrolase

3.458 ( 0.79 0.560 ( 0.24

(emPAI)

QM9414MG5

1.072 ( 0.31 1.290 ( 0.00 1.290 ( 0.00

(emPAI)

RUT C-30

3.458 ( 0.79 5.860 ( 0.00 5.860 ( 0.00

(emPAI)

QM9414

Hydrophobin-2

(emPAI)

QM 6a

pH 6

Hydrophobin-1

protein name

Table 3. Continued

(emPAI)

QM9414 (emPAI)

RUT C-30

pH 7

0.206 ( 0.04

0.125 ( 0.06

0.767 ( 0.37

5.198 ( 0.49 1.590 ( 0.00

0.685 ( 0.06 0.394 ( 0.09

0.490 ( 0.07 0.137 ( 0.03

0.145 ( 0.02 0.040 ( 0.00 0.090 ( 0.05

0.244 ( 0.04 0.130 ( 0.07

0.530 ( 0.00 0.070 ( 0.00

0.090 ( 0.05 0.285 ( 0.10 1.552 ( 0.14 1.286 ( 0.13 0.341 ( 0.16

0.155 ( 0.09 0.110 ( 0.07

1.930 ( 0.00 1.824 ( 0.17 1.813 ( 0.46

0.311 ( 0.04

0.210 ( 0.00 0.412 ( 0.07

0.070 ( 0.00

0.270 ( 0.05 0.161 ( 0.02 0.684 ( 0.13 0.280 ( 0.00 1.700 ( 0.00

0.448 ( 0.05 0.390 ( 0.00

0.612 ( 0.12 0.120 ( 0.00

0.882 ( 0.05 0.050 ( 0.00

0.210 ( 0.04 0.080 ( 0.00

0.707 ( 0.19 0.230 ( 0.00

0.911 ( 0.08 0.531 ( 0.06 2.165 ( 0.28 1.153 ( 0.11 1.478 ( 0.44

0.200 ( 0.04 0.307 ( 0.05 0.264 ( 0.10

0.090 ( 0.00 0.090 ( 0.00 0.090 ( 0.05

0.206 ( 0.02 0.187 ( 0.06 0.070 ( 0.04

0.649 ( 0.05

0.040 ( 0.02 0.084 ( 0.02

0.374 ( 0.17

0.376 ( 0.08

0.116 ( 0.01 1.501 ( 0.16

1.472 ( 0.07

0.146 ( 0.01

0.843 ( 0.03

0.307 ( 0.05

0.665 ( 0.06

2.258 ( 0.29

(emPAI)

QM9414MG5

1.273 ( 0.11 0.803 ( 0.15 0.293 ( 0.25

0.224 ( 0.04

1.072 ( 0.31 0.740 ( 0.00 1.290 ( 0.00

3.980 ( 0.00 3.458 ( 0.79 5.151 ( 1.08

(emPAI)

QM 6a

hemicellulases

(emPAI)

QM9414 (emPAI)

RUT C-30

0.090 ( 0.06

0.169 ( 0.06

0.183 ( 0.04

0.103 ( 0.09

2.110 ( 0.38 1.092 ( 0.18 0.080 ( 0.04

0.100 ( 0.04 0.179 ( 0.04

0.090 ( 0.05

0.220 ( 0.00 0.160 ( 0.00

0.040 ( 0.00

0.070 ( 0.00 0.070 ( 0.04

0.143 ( 0.05 0.109 ( 0.06 0.769 ( 0.05 0.263 ( 0.15

1.360 ( 0.00 1.275 ( 0.13 1.717 ( 0.32

0.284 ( 0.06

0.080 ( 0.00 0.137 ( 0.05 0.252 ( 0.02 1.280 ( 0.16

0.080 ( 0.00 0.063 ( 0.02

0.050 ( 0.02

0.827 ( 0.21

0.685 ( 0.05

0.501 ( 0.06 1.012 ( 0.06

1.824 ( 0.17

0.712 ( 0.09

0.324 ( 0.05 1.926 ( 0.20

1.445 ( 0.09

0.242 ( 0.14 0.230 ( 0.00

0.938 ( 0.12

0.258 ( 0.06

0.160 ( 0.00 1.423 ( 0.21

0.090 ( 0.05

0.201 ( 0.04

0.030 ( 0.01

1.072 ( 0.31

4.528 ( 0.08

(emPAI)

MG5

QM9414

0.460 ( 0.00 1.026 ( 0.18 1.207 ( 0.32 1.026 ( 0.18

0.145 ( 0.02

0.090 ( 0.00 0.090 ( 0.00

0.112 ( 0.02 0.112 ( 0.02

0.290 ( 0.00 0.660 ( 0.00 0.247 ( 0.06

1.290 ( 0.00 1.290 ( 0.00 1.290 ( 0.00

3.980 ( 0.00 3.458 ( 0.79 5.860 ( 0.00

(emPAI)

QM 6a

pH 8



Glycosyl hydrolase, putative

4589

Probable beta-glucosidase 1

Peptidoglycan-binding

Putative glycosyl transferase

Glycosyltransferase

0.020 ( 0.01

0.020 ( 0.00

Carbohydrate-binding protein,


0.080 ( 0.04 0.139 ( 0.06

Beta-glucosidase, putative Endo1,3(4)-beta-glucanase



Cellulose hydrolase,

HAD-superfamily hydrolase

Endoglucanase V

Fumarylacetoacetate hydrolase

0.050 ( 0.00

0.070 ( 0.04

Glycosyl hydrolase Cell wall glucanase, putative

Endoglucanase, putative

0.110 ( 0.06

Lysozyme

0.640 ( 0.37

0.280 ( 0.16

0.040 ( 0.02

Metallo-beta-lactamase

Alpha amylase catalytic region

Glycosyl hydrolase,

domain Putative glycosyl hydrolase

0.090 ( 0.05 1.000 ( 0.00

Transaldolase, putative

0.180 ( 0.10

0.140 ( 0.08

0.100 ( 0.05

0.070 ( 0.04

Cellulose or protein binding

Exobeta 1,3-glucanase

Glycosyl hydrolase

Cellulose signaling related protein

gel2

Glycoside hydrolase 1,3-beta-glucanosyltransferase

(emPAI)

RUT C-30

0.060 ( 0.03

(emPAI)

QM9414

Endoglucanase-7

(emPAI)

QM 6a

pH 6

Maltose permease

protein name

Table 3. Continued

0.130 ( 0.07

(emPAI)

QM9414MG5

(emPAI)

QM9414

0.020 ( 0.00

0.040 ( 0.02

0.382 ( 0.15 0.050 ( 0.02

0.040 ( 0.02

0.015 ( 0.01

0.280 ( 0.16

0.040 ( 0.02 0.040 ( 0.02 0.160 ( 0.09

0.060 ( 0.03

0.322 ( 0.13 0.090 ( 0.05

0.070 ( 0.00

0.258 ( 0.06 0.300 ( 0.17

0.095 ( 0.03 0.060 ( 0.03

0.320 ( 0.00

(emPAI)

RUT C-30

pH 7

0.350 ( 0.00 0.300 ( 0.07

0.150 ( 0.08 0.271 ( 0.04 0.060 ( 0.03

1.073 ( 0.26

(emPAI)

QM 6a

hemicellulases

0.123 ( 0.00

0.171 ( 0.05

(emPAI)

QM9414MG5

(emPAI)

QM9414

(emPAI)

RUT C-30

0.040 ( 0.02

0.120 ( 0.06

0.130 ( 0.07

0.532 ( 0.11 0.070 ( 0.00

0.416 ( 0.11

0.326 ( 0.08 0.412 ( 0.17

0.120 ( 0.06 0.200 ( 0.00

0.060 ( 0.03

0.190 ( 0.11 0.148 ( 0.05

2.562 ( 0.18

0.180 ( 0.00

0.220 ( 0.12 0.350 ( 0.00

0.740 ( 0.00

0.271 ( 0.04 0.070 ( 0.04 0.130 ( 0.00

0.790 ( 0.00

(emPAI)

QM 6a

pH 8

0.130 ( 0.00

0.212 ( 0.08

0.140 ( 0.08

0.080 ( 0.00

0.090 ( 0.00

0.327 ( 0.05

0.740 ( 0.00

0.271 ( 0.04 0.060 ( 0.00

0.358 ( 0.09

(emPAI)

MG5

QM9414



0.090 ( 0.05

Beta-glucuronidase

Glycosyl hydrolase

4590

0.267 ( 0.07

0.220 ( 0.00 0.651 ( 0.07 0.140 ( 0.00 0.140 ( 0.00 0.080 ( 0.00

0.670 ( 0.00 0.180 ( 0.06 0.510 ( 0.29

0.155 ( 0.02 0.071 ( 0.02

0.100 ( 0.05 0.100 ( 0.00

0.110 ( 0.06 0.330 ( 0.00 0.354 ( 0.11

0.325 ( 0.04 0.140 ( 0.00 0.070 ( 0.04

0.140 ( 0.00 0.410 ( 0.10 0.140 ( 0.00

0.080 ( 0.00 0.080 ( 0.00 0.080 ( 0.04


Beta-mannosidase mndA



Alpha-galactosidase

Endoxylanase II

Acetyl esterase

0.100 ( 0.00 0.392 ( 0.07


Xylanase III


0.040 ( 0.00


0.080 ( 0.04


0.180 ( 0.10 0.060 ( 0.03 0.060 ( 0.03

0.050 ( 0.00

0.131 ( 0.02 0.150 ( 0.00

0.180 ( 0.00 0.730 ( 0.08 0.486 ( 0.20

3-phytase A precursor

Galactomannoprotein, Endo-beta1,6-galactanase

3-carboxymuconate cyclase


1.089 ( 0.11 1.240 ( 0.35 0.395 ( 0.21

Xylosidase/arabinosidase Beta-xylosidase

0.391 ( 0.12

0.967 ( 0.08 0.454 ( 0.19 0.264 ( 0.23

Beta-mannase

1,2-a-D-mannosidase

0.130 ( 0.00

0.331 ( 0.04 1.463 ( 0.38 0.234 ( 0.04 0.210 ( 0.00 1.129 ( 0.49

Alpha-N-arabinofuranosidase Exorhamnogalacturonase B

0.290 ( 0.00 0.782 ( 0.06

0.290 ( 0.00 0.660 ( 0.00

1.865 ( 0.09 3.348 ( 0.30 2.688 ( 0.72

Endo1,4-beta-xylanase 1

(emPAI)

QM9414MG5

Arabinofuranosidase B

Ubiquitin C-terminal hydrolase

Endoglucanase Glycosyltransferase

(emPAI)

RUT C-30

(emPAI)

QM9414

Endo 1,4-beta-glucanase Cel5b

(emPAI)

QM 6a

pH 6

Exo-beta1,3-glucanase, putative

protein name

Table 3. Continued

2.030 ( 0.14

0.145 ( 0.02

0.100 ( 0.00 0.268 ( 0.06

0.188 ( 0.03 0.131 ( 0.02

0.841 ( 0.08

0.283 ( 0.02

0.094 ( 0.06

0.073 ( 0.00

0.180 ( 0.10 0.242 ( 0.05 0.090 ( 0.05

0.471 ( 0.22

0.063 ( 0.00

0.209 ( 0.06

0.323 ( 0.02

0.096 ( 0.01

1.060 ( 0.11

0.180 ( 0.00

0.140 ( 0.00 0.480 ( 0.00 0.233 ( 0.09

0.070 ( 0.00 0.070 ( 0.04

0.116 ( 0.04 0.116 ( 0.04 0.040 ( 0.02

0.175 ( 0.06

0.170 ( 0.00

0.670 ( 0.00 0.180 ( 0.06 0.230 ( 0.13

2.281 ( 0.47 0.619 ( 0.20 0.274 ( 0.04

0.725 ( 0.13 0.595 ( 0.12

0.208 ( 0.03

0.358 ( 0.10

0.100 ( 0.04 0.200 ( 0.00 0.060 ( 0.00 0.130 ( 0.00 0.306 ( 0.09

(emPAI)

QM9414MG5

0.526 ( 0.08

(emPAI)

RUT C-30

pH 7

1.169 ( 0.12 1.491 ( 0.23 1.435 ( 0.37

(emPAI)

QM9414

Hemicellulases

0.153 ( 0.06

(emPAI)

QM 6a

hemicellulases

(emPAI)

QM9414

(emPAI)

RUT C-30

0.155 ( 0.10

1.050 ( 0.09

0.080 ( 0.04

0.410 ( 0.10 0.140 ( 0.00

1.943 ( 0.24

0.213 ( 0.12

0.192 ( 0.11

0.080 ( 0.00

1.086 ( 0.15

0.232 ( 0.02

0.180 ( 0.06

0.205 ( 0.05 0.150 ( 0.08

0.275 ( 0.08

0.220 ( 0.00

1.307 ( 0.10

0.173 ( 0.04

0.967 ( 0.13

0.290 ( 0.00

(emPAI)

MG5

QM9414

0.612 ( 0.09 0.110 ( 0.00

0.430 ( 0.09 0.662 ( 0.09 0.090 ( 0.06

0.130 ( 0.07 0.080 ( 0.00

0.244 ( 0.04 1.084 ( 0.13 0.942 ( 0.32

(emPAI)

QM 6a

pH 8



, Not identified; n = 3, ( SD.

EstA precursor

a

identified using MALDI-TOF (Applied Biosystems; MDS-Sciex, Foster City, CA). The bands in the zymogram analysis showed abundance of GH7 cellobiohydrolase and GH15 glycoside hydrolase (Table 5).The peptide sequences of these bands identified by MALDI-TOF were listed in Table S2 (Supporting Information). These proteins were also abundant in the secretome analysis by LCMS analysis with high emPAI values (Table 3). In addition, enzyme activities, cellulose hydrolysis potential of T. reesei wild and its mutants at pH range 3.0 to 9.0 were determined and presented in Table 4. The LCMS/ MS identified proteins, their emPAI values, enzymes assays, cellulose hydrolysis, zymogram and the zymogram-band identification data has been correlated and discussed in Discussion section.

Phytase


0.060 ( 0.03

0.116 ( 0.04 mannosidase

Mannan endo1,6-alpha-

Mannose-binding lectin precursor Mannan-binding lectin

0.140 ( 0.00

0.180 ( 0.09

0.690 ( 0.00

ARTICLE

Acetyl xylan esterase

Beta-mannosidase

MG5

(emPAI) (emPAI) (emPAI) (emPAI) (emPAI) (emPAI) (emPAI) (emPAI) (emPAI) (emPAI)

RUT C-30 RUT C-30

(emPAI)

(emPAI)

RUT C-30 QM9414 QM 6a

protein name

Table 3. Continued

QM 6a

QM9414

pH 6

QM9414MG5

QM 6a

hemicellulases

QM9414

pH 7

QM9414MG5

pH 8

QM9414


’ DISCUSSION Lignocellulosic bioenergy is most promising renewable, sustainable energy and has been considered as an alternative to the current petroleum or fossil fuel based energy. However, major bottleneck preventing its commercialization is enzyme production cost and lack of efficient lignocellulose hydrolyzing enzyme cocktail. Numerous microorganisms are capable of producing lignocellulose hydrolyzing enzymes; however, fungi are considered most dynamic lignocellulolytic enzyme producers against the most abundantly available natural polymer. Species of Trichoderma play major role in enzymatic depolymerization of lignocellulosic biomass since they produce substantial amount of endoglucanase, exoglucanase,5,6,23 hemicellulases23,34,35 but low levels of beta-glucosidases. This mesophilic fungus is one of the most efficient xylanase and cellulase producers while according to Cherry and Fidantsef,36 industrial strains of T. reesei can achieve protein production levels of up to 100 g/L. The cultivation of T. reesei and its mutants in cellulosic medium resulted into production of 30.169.9 g/L at pH range of 3.0 7.0. However, besides carbon and nitrogen source other parameters such as temperature, pH etc. affects the protein production. This study evaluated protein expressions by T. reesei wild and its mutants at pH 3.09.0 in cellulosic medium and compared their quantitative expressions using emPAI values. Several researchers investigated optimum pH of cellulase and xylanase activities using traditional colorimetric technique. However, colorimetric techniques can not precisely differentiate individual enzymes in a complex secretome secreted by microbial strains. The traditional colorimetric technique suffer from several limitations such as low detection sensitivity, inability of detecting various isoforms, reagent cross reactivity etc.; however, proteomics technology is free of colorimetric reagent, cross reactivity, detects low abundant proteins and isoforms. Again the comparative secretome analysis of wild and mutants of T. reesei is rarely documented. This study, first time reports the comparative analysis of the secretome of T. reesei wild and their three mutants at different pH using proteomics technology. The cellulose hydrolyzing enzymes such as endoglucanases which catalyzes random cleavage of internal bonds of the cellulose chain, exoglucanases (cellobiohydrolases) that attacks the chain end and beta-glucosidase that converts the end product of exoglucanases into glucose were identified at different pH in all test strains with variable expressions. In addition to the proteins that hydrolyses beta-1,4- or -1,6-linkage, proteins with alpha-1,4-linkage hydrolyzing potential were also expressed during fermentation of cellulose. 4591



ARTICLE

Figure 2. Overlap diagram of the cellulases and glycoside hydrolases of T. reesei and its mutants at different pHs. Inside the circles are exclusive total numbers of protein identified in the secretome of respective strains. At the center are common proteins identified in the secretome of all T. reesei strains. (TR1, T. Reesei QM9414; TR2, T. Reesei RUT C-30; TR3, T. Reesei QM9414MG5; TR4, T. Reesei QM6a).

Cluster analysis based on the identification of specific proteins at different pH demonstrated variability of clustering profile within clusters of each strain and percentage similarity suggesting dynamic profile of the secreted enzymes at different pH. The clustering pattern of enzymes at pH 3.07.0 in T. reesei QM6a and RUT C30 showed 6177% similarity while the corresponding values for T. reesei QM9491 and QM9414 were 5870%. The clustering of secreted proteins by T. reesei QM 9414 was totally different from other strains showing three groups with separate cluster of proteins at pH 7.0 and 8.0, while protein profile at pH 3.06.0 clustered together. The correlation of clustering pattern, BrayCurtis similarity indices and emPAI values of the identified lignocellulolytic proteins indicated differential protein secretion response of the strain to pH and also strong flexibility of the strain’s tolerance to stress caused by pH. Although, 1,4-beta-Dglucosidic linkages hydrolyzing GH7 exoglucanase, GH15 glycoside hydrolase/glucoamylase F, cell adhesive hydrophobin were expressed at all the test pH in T. reesei wild and mutants, but their emPAI values varied significantly with pH emphasizing expression of proteins as a pH dependent. Again, GH30 endo-1,6-betaD-glucanase, GH16 endo-1,4-beta-glucanase, GH71 glucan endo1,3-alpha-glucosidase were not expressed in RUT C30, while GH71 glucan endo1,3-alpha-glucosidase, GH71 glucan endo1,3-alpha-glucosidase agn1 were not expressed in T. reesei QM9414MG5 suggesting strain dependent expressions of these

proteins. Restated, expression of the specific hydrolytic proteins were pH and strain dependent and we provide data on protein expressions with wide range of pH in four strain that could be useful for industrial production of specific enzymes. Further, significantly higher emPAI values of cellulose hydrolyzing proteins at acidic pH 3.05.0 and their cellulose degradation potential at corresponding pH suggested that these enzymes secreted in the bulk medium was active and tolerate acidic pH and hence have potent use in conversion of industrial cellulosic waste into bioenergy. According to Angsana et al.,37 pH affects total net protein charge, breaking of ionic bonds that subsequently alters tertiary structure of the enzyme making them unable to catalyze chemical reaction. However, efficient cellulose hydrolysis, higher enzyme activity, emPAI values, zymography and further identification of the specific cellulolytic enzymes at lower acidic pH suggested production of active enzymes at acidic pH (pH 3.04.0) emphasizing T. reesei’s potential to combat acidic pH. Singh et al.38 demonstrated that the change in temperature was less important than the change in pH since pH changes the ionic components of substrate as well as net charge on enzymes. Identifying the exact change in the tertiary structure of individual secreted proteins in the complex secretome remains unknown and challenging. At certain pH, key enzymes such as proteases may alter other cellulolytic enzymes and the mechanisms corresponding to its structural alteration and biological function could not be detailed. 4592



ARTICLE

Table 4. Specific Activities of Cellulytic Enzymes and Cellulose Degradation by T. reesei Wild and Its Mutant Strains at Different pHs specific activity (mg/min/g protein) strain name

T. reesei QM9414

T. reesei RUT C-30

T. reesei QM9414MG5

T. reesei QM6a

pH

total protein (mg/mL)

cellulose degradation (%)

endoglucanase

exoglucanase

beta glucosidase

3

0.301 ( 0.077

59.8 ( 1.7

3.38

0.308

0.23

4

0.358 ( 0.026

59.8 ( 1.8

5.22

0.367

0.25

5 6

0.404 ( 0.026 0.401 ( 0.040

61.1 ( 2.8 64.9 ( 3.4

2.98 1.38

0.764 0.551

0.34 0.24

7

0.699 ( 0.006

59.3 ( 2.2

1.31

0.294

0.19

8

0.456 ( 0.049

49.9 ( 4.0

0.46

0.140

0.23

9

0.404 ( 0.095

7.3 ( 1.3

0.11

0.076

0.22

3

0.485 ( 0.018

46.0 ( 5.2

1.87

0.303

0.57

4

0.426 ( 0.015

46.6 ( 1.8

2.35

0.262

0.64

5 6

0.407 ( 0.008 0.411 ( 0.003

60.1 ( 2.0 60.3 ( 1.8

2.76 2.69

0.272 0.248

0.63 0.74

7

0.468 ( 0.010

44.1 ( 1.6

2.06

0.160

0.17

8

0.376 ( 0.009

39.2 ( 4.0

1.20

0.204

0.21

9

0.298 ( 0.008

11.8 ( 2.1

0.03

0.065

0.05

3

0.444 ( 0.021

46.8 ( 1.9

1.21

0.87

0.80

4

0.588 ( 0.016

56.3 ( 5.1

0.99

0.59

0.48

5 6

0.628 ( 0.030 0.605 ( 0.012

56.8 ( 3.7 58.0 ( 3.4

1.06 1.09

0.69 0.47

0.55 0.47

7

0.619 ( 0.005

56.7 ( 1.3

1.12

0.50

0.41

8

0.722 ( 0.051

45.5 ( 2.0

1.27

0.36

0.49

9

0.568 ( 0.019

10.7 ( 1.3

0.71

0.04

0.08

3

0.438 ( 0.011

69.3 ( 2.5

0.36

0.336

0.476

4

0.430 ( 0.012

68.4 ( 3.4

0.35

0.260

0.490

5 6

0.401 ( 0.004 0.411 ( 0.003

61.9 ( 1.9 61.5 ( 3.4

0.60 0.99

0.276 0.248

0.412 0.634

7

0.464 ( 0.014

56.5 ( 3.6

1.56

0.161

0.173

8

0.376 ( 0.009

55.1 ( 3.6

0.37

0.204

0.205

9

0.298 ( 0.008

14.5 ( 2.4

0.09

0.047

0.045

Of 36, 46, 24 and 42 cellulolytic proteins identified in T. reesei QM9414, RUT C30, QM9414MG5 and QM6a at pH 5.0, respectively, only 16 unique proteins were common in all test strains, while the corresponding values at pH 9.0 were 10, 7, 18 and 9 with only 1 protein in common. At alkaline pH, low protein expressions, lower emPAI values, low cellulose hydrolysis, enzyme activities and undetectable enzyme band in zymogram confirm that T. reesei and its mutants are intolerant to alkaline culture condition. Further, analysis of total protein as an indicator of cell biomass suggested low growth at alkaline pH 8.09.0, confirming pH as an important factor that determines the growth and morphology of T. reesei. The total protein content, significantly reduced cellulose degradation efficiency and low enzyme activity (Table 4) by T. reesi QM6a and Rut C30 when growth at pH 9.0 could correlate lower cell growth. According to Wohlfahrt,39 carboxyl carboxylate pairs of cellulases are involved in catalysis covering the active site tunnel of the catalytic domain and their pH dependent conformational changes at active site determined their activity and stability. Thus, although total protein content that profiles the cell growth pattern was not significantly reduced in T. reesei

QM9414 and QM9414MG5, conformational changes could inactivate the enzyme at alkaline pH. According to Sohail et al.,40 enzyme production by Aspergillus niger MS82 decreased drastically at and above pH 5.0. Watanabe et al.41 reported an optimum pH of 4.5 for beta-glucosidase from A. niger. The production of cellulases by several other microbial strains was higher when pH was below neutrality while its reduced production has been noted with increase in pH above neutrality.4244 The emPAI values of cellobiohydrolase I and exoglucanase 2 produced by T. reesei RUT C30 suggested that they were optimally expressed at pH 4.0 while proteins such as endoglucanase Cel74a, beta1,3-glucanase, betaglucosidase etc. were secreted higher at pH 5.0. Tangnu et al.45 reported high cellulase production by T. reesei RUT C30 when the pH of the culture medium was controlled around 5.0, while Chahal et al.46 noted pH 6.0 as a favorable for cellulase production when lignocellulose was used as a cellulose source. On the basis of emPAI values, GH7 cellobiohydrolase I that cleaves cellobiosyl residues from the reducing ends of cellulose chains and is considered as a key enzymes in cellulose hydrolysis4749 and its expressions were in the order T. reesei QM9414MG5 > RUT C-30 > QM9414 > QM6a. 4593


Journal of Proteome Research Because of physical barriers generated by the lignin that surrounds cellulose and hemicellulose polymers, enzymatic hydrolysis of cellulose and hemicellulose has been hindered.

Figure 3. Zymogram of cellulases produced by T. reesei and its mutant strains. SDS zymogram was performed using carboxymethyl cellulose as a substrate, stained with cango red and destained by 1 M NaCl. The cellulase activity can be seen as a bright white band in zymogram analysis. The bands were excised and further identified using MALDI-TOF.

ARTICLE

Therefore, lignocellulosic biomass acid pretreatment to disrupt the lignin bonds50 and enhance susceptibility to enzymatic hydrolysis is under consideration. The acid-tolerant hydrolytic proteins play a major role in enhancing lignocellulose hydrolysis and controlling process cost. This study identified several acidtolerant cellulolytic proteins, useful for fermenting acid-pretreated lignocellulosic biomass. The hemicellulose hydrolyzing GH11 endo1,4-beta-xylanase, GH11 xylanase III, GH54 arabinofuranosidase, GH5 beta-mannase, GH43 xylosidase/arabinosidase, GH 39 beta-xylosidase, GH5 endo-beta1,6-galactanase, phytase etc. were detected in the secretome at different pHs. The hemicellulose degrading proteins production by T. reesei and its mutants were not significantly affected in pH range 3.07.0; for example, T. reesei QM9414, RUT C30, QM9414MG5 and QM6a produced 15, 12, 11, and 15 hemicellulolytic proteins at pH 4.0 while corresponding numbers were 14, 11, 10, and 14 at pH 7.0. Xiong et al.34 found a maximum activity at pH 6.5 for the xylanase produced by Thermomyces lanuginosus while Thukral et al.51 reported higher rate of xylanase decay at pH 2.0 and 4.0 when compared to its decay at between pH 4.5 and 9.0. On the contrary, our data showed high abundance of hemicellulose hydrolyzing proteins at acidic pH. According to Bailey et al.,19 T. ressei RUT C30 favors xylanase production at pH 7.0 while optimum cellulases production were recorded at pH 4.0 when cellulose and xylan was used as a major carbon source. Of the xylanases IIV produced by wild T. reesei, xylanases I, II and III have optimum pH range of 4.04.5, 4.06.0 and 6.06.5, respectively.5254 Xiong et al.34 reported optimal xylanase I production at pH 4.0 and xylanase II at pH 6.0 on lactose medium. Although, few lignin degrading proteins were expressed during cellulose utilization, their regulation suggested that cellulose alone could not induce their expressions. This study reports optimum pH at which emPAI values were high for each individual protein under category of cellulases, hemicellulases, lignin degrading proteins and peptidases secreted by strains. The shift in pH values above and below its optimum affects enzyme production and was attributed to the low enzyme production and cell growth.55 Similarly, lower enzyme activity at alkaline pH and higher at acidic pH could be attributed to cell growth and enzyme activities.

Table 5. SDS Zymogram Bands (Figure 3) Identification Analysis using MALDI-TOF MSa pH

protein name

protein MW

protein score

3.0

Glycoside Hydrolase, glucoamylase

67787.6

113

Cellobiohydrolase, Exoglucanase

55445.1

354

jgi|Trire2|123989|


67787.6

142

jgi|Trire2|1885|

4.0

accession number jgi|Trire2|1885|


55445.1

483

jgi|Trire2|123989|

5.0


67787.6

153

jgi|Trire2|1885|

6.0

Cellobiohydrolase, Exoglucanase Glycoside Hydrolase, glucoamylase

55445.1 67787.6

348 176

jgi|Trire2|123989| jgi|Trire2|1885|


55445.1

332


67787.6

76

7.0 8.0 9.0

jgi|Trire2|123989| jgi|Trire2|1885|


55445.1

73

jgi|Trire2|123989|


67787.6

68

jgi|Trire2|1885|


55445.1

81

jgi|Trire2|123989|

Not detected

a

The protein name and accession numbers are listed below while detailed information including peptide sequences were presented in Supplementary Table S2 (Supporting Information). These two proteins with variable protein score were identified at pH 3.08.0 in all test strains. 4594



’ CONCLUSION The impact of environmental factors and culture conditions on the productivity of hydrolytic enzymes by T. reesei and its mutants is poorly investigated so far. Therefore, it is a major concern to improve the understanding of microbial hydrolytic protein production and cellular physiology during cellulose utilization. This study profiled hydrolytic enzyme production by filamentous fungus T. reesei QM6a and its mutants QM9414, RUT C30, QM9414MG5 at pH 3.09.0. The identification and quantitation of different cellulases, hemicellulases, lignin degrading proteins, peptidases, Chitinase, and transport proteins suggested that the enzyme production was significantly affected at alkaline pH. The LCMS data demonstrated that the lower pH favors higher protein expressions, cellulose hydrolysis and also enzyme activities. This study identified pH-tolerant protein targets useful for enzymatic hydrolysis of the acid-pretreated lignocellulosic biomass. Despite the fact that Erlenmeyer flask cultures are not fully equivalent to bioreactors, the data presented here serve as a starting point for potentially industrial production of particular hydrolytic enzyme. ’ ASSOCIATED CONTENT

bS

Supporting Information Figures S1S5, Figures H1H20, Figures G1G25, Tables S1, S2 and TS1TS7. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Siu Kwan SZE, PhD, School of Biological Sciences, Division of Chemical Biology & BioTechnology, Nanyang Technological University, 60 Nanyang drive, Singapore 637551. Tel: (+65) 6514-1006. Fax: (+65)791-3856. E-mail: [email protected].

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Proteomic Analysis of pH and Strains Dependent Protein Secretion

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