Dynamic synergistic effect on Trichoderma reesei cellulases by novel β-glucosidases from Taiwanese fungi

Bioresource Technology 102 (2011) 6073–6081

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Dynamic synergistic effect on Trichoderma reesei cellulases by novel b-glucosidases from Taiwanese fungi I-Son Ng a,b, Shau-Wei Tsai c, Yu-Ming Ju d, Su-May Yu b,e,⇑, Tuan-hua David Ho b,d,f,⇑ a

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China Biotechnology Center in Southern Taiwan, Academia Sinica, Tainan, Taiwan c Institute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan, Tao-Yuan 33302, Taiwan, ROC d Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC e Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, ROC f Department of Biology, Washington University, St. Louis, MO 63130, USA b

a r t i c l e

i n f o

Article history: Received 3 September 2010 Received in revised form 28 December 2010 Accepted 30 December 2010 Available online 12 January 2011 Keywords: b-Glucosidase Dynamic synergistic effect Trichoderma reesei Fungal cellulases Taiwanese fungi

a b s t r a c t Dynamic synergistic effects in cellulosic bioconversion have been revealed between Trichoderma reesei cellulases and b-glucosidases (BGLs) from six Taiwanese fungi. A high level of synergy (8.9-fold) was observed with the addition of Chaetomella raphigera BGL to T. reesei cellulases. In addition, the C. raphigera BGL possessed the highest activity (Vmax/Km = 46.6 U/mg mM) and lowest glucose inhibition (Ki = 4.6 mM) with the substrate 4-nitrophenyl b-D-glucopyranoside. For the natural cellobiose substrate, however, the previously isolated Aspergillus niger BGL Novo-188 had the highest Vmax/Km (0.72 U/mg mM) and lowest Ki (59.5 mM). The demonstrated dynamic synergistic effects between some BGLs and the T. reesei cellulase system suggest that BGLs not only prevent the inhibition by cellobiose, but also enhance activities of endo- and exo-cellulases in cellulosic bioconversion. Comparisons of kinetic parameters and synergism analyses between BGLs and T. reesei cellulases can be used for further optimization of the cellulosic bioconversion process. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The potential importance of cellulose hydrolysis in the context of conversion of plant biomass to fuels and chemicals is well recognized. Cellulose hydrolysis by cellulases produced by numerous microorganisms has been widely employed for producing sustainable bio-based products and bioenergy to replace depleting fossil fuels (Lynd et al., 2005; Zhang et al., 2006). Currently, the utilization of cellulosic biomass for bioethanol poses significant technical and economic challenges, and its success largely depends on the development of highly efficient and cost-effective biocatalysts for conversion of pretreated biomass to fermentable sugars. One of the most efficient and successful ways of finding new biocatalysts or enzymes is to screen a large number of diverse microorganisms seeking different characteristics with improved versatility. The effective conversion of cellulose to fermentable sugars requires three classes of enzymes: (1) endoglucanases (EC 3.2.1.4) which randomly cut cellulose chains to yield glucose and cello-

⇑ Corresponding authors at: Biotechnology Center in Southern Taiwan, Academia Sinica, Tainan, Taiwan. Tel.: +886 2 2787 1188; fax: +886 2 2782 1605 (T.-h.D. Ho), tel.: +886 2 2788 1695 (S.-M. Yu). E-mail addresses: [email protected] (S.-M. Yu), [email protected], [email protected] (T.-h.D. Ho). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.110

oligosaccharides, (2) exoglucanases (EC 3.2.1.91) which exolytically attack the reducing or non-reducing end of celluloses to yield cellobiose, and (3) b-glucosidases (EC 3.2.1.21) which hydrolyze cellobiose and cello-oligosaccharides to form glucose. Cellulases from the fungal genus Trichoderma have received considerable attention due to their ability to effectively hydrolyze crystalline cellulose. The biomass-degrading fungus Trichoderma reesei contains a cellulase mixture consisting of many catalytically active proteins (Martinez et al., 2008), of which at least two exoglucanases or cellobiohydrolases (i.e., Cel7A [CBH1] and Cel6A [CBH2]), five endoglucanases (EG1 to EG5), several b-glucosidases, and hemicellulases have been identified by two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry analysis (Vinzant et al., 2001). CBH1, CBH2, and EG2 are the three main components of the T. reesei cellulases system, representing 60%, 20%, and 12% of total cellulase proteins, respectively. As a result, the main activity of T. reesei on insoluble cellulosic substrates is to produce cellobiose. Therefore, to completely hydrolyze cellulose to glucose in this hydrolytic system, a supplement of b-glucosidase is required. Ubiquitous DGLs exist in all the living kingdoms, from bacteria to highly evolved mammals. They comprise the main part of the cellulase enzyme system in bacteria and fungi and are responsible for the rate-limiting step in the hydrolysis of short-chain oligosaccharides and cellobiose. Based on amino acid sequence similarities,

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Nomenclature EG endoglucanase CBH cellobiohydrolase BGL b-glucosidase CB cellobiose G1, G2 glucose Vmax, Km, Ki kinetic constants and glucose inhibition constant for BGL Vmt, Kmt apparent kinetic constants for T. reesei with BGL

BGLs differing in their specificities for the aglycone part (aryl-, alkyl-, or amino-) linked to the glycosyl group has been classified into either family 1 or family 3 of glycosyl hydrolase families. Family 1 enzymes include most bacterial, archaeal, plant and animal BGLs, whereas family 3 includes some bacterial and all yeast and fungal enzymes (Bhatia et al., 2002). Synergism occurs when the activity exhibited by mixtures of components (such as cellulases) is larger than the sum of each individually evaluated activity of these components. In the past decades, several groups have reported on synergisms between Trichoderma endo- and exocellulases (Medve et al., 1998; Nidetzky et al., 1994; Valjamae et al., 1999, 2003), however, none of them demonstrated effects of BGLs in the system encompassing CBHs and EGs. In this study, we have examined, for the first time, potential kinetic and dynamic synergism between BGLs and EGs/CBHs. Six fungi with high BGL activities have been isolated from screening of 42 fungi collected in Taiwan, and these BGLs were used for the synergism studies. As shown in Fig. 1, the T. reesei cellulases mainly hydrolyze cellulose to cellobiose (CB); only a minor fraction is hydrolyzed to glucose (G1). By adding extra BGLs, large amounts of glucose (G2) are produced, hence greatly improves the degree of hydrolysis of cellulose to glucose (Gtot). We furthermore determined kinetic parameters of BGLs in the hydrolysis of different substrates in the absence and presence of glucose and identified BGLs by mass spectrometry. 2. Methods 2.1. Preparation of fungi and enzymes All fungi, except Penicillium citrinum YS40-5, which was isolated from rice straw compost (Ng et al., 2010), were collected previously by Prof. Yu-Ming Ju, Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan. T. reesei cellulases (Celluclast 1.5L) and Novozyme 188, a commercial Aspergillus niger bglucosidase, were purchased from Sigma. The minimal requirement (MR) medium consisted of 1 g soy-peptone, 1.4 g (NH4)2SO4, 2.0 g KH2PO4, 0.34 g CaCl22H2O, 0.30 g MgSO47H2O, 5 mg FeSO47H2O, 1.6 mg MnSO4H2O, 1.4 mg ZnSO47H2O and 2.0 mg CoCl26H2O per liter of H2O, was adjusted to pH 6.5 with 0.1 N NaOH. All materials were autoclaved for 15 min at 121 °C. The

Cellulose

T. reesei cellulases

CB + G1 extra BGL

G tot

G2 Fig. 1. Dynamic synergism between extra BGL and Trichoderma reesei cellulases on cellulosic bioconversion.

Vglu, VCB enzymatic rate for glucose and cellobiose Novo-188 Novozyme 188 D2 Chaetomella raphigera DNS dinitrosalicylic acid pNPG 4-nitrophenyl b-D-glucopyranoside

fungal cultures were maintained on PDA medium for 5 days at 30 °C for sporulation. Five discs of PDA with a diameter of 0.8 cm containing approximately 106–107 spores per disc were inoculated into a 250-mL Erlenmeyer flask containing 2 mM cellobiose in 100 mL MR medium at 30 °C under shaking at 125 rpm. The time course of BGL activity was then analyzed daily. BGLs of the fungi, for which high enzymatic activities were measured, were subsequently concentrated 80-fold by using AmiconÒ Ultra centrifugal filter devices (10k NMWL, Millipore, MA, USA). 2.2. BGL assays 4-Nitrophenyl b-D-glucopyranoside (pNPG) from Sigma was used as the substrate for measuring BGL activity. The assay was performed by mixing 100 ll of 1 mM pNPG and 100 ll of enzyme solution in 50 mM sodium phosphate buffer (pH 7.0). After incubation for 15 min at 55 °C, the reaction was quenched by adding 600 ll of 1 M sodium carbonate. Subsequently, the absorbance of 4-nitrophenol in the reaction mixture was measured at 405 nm with a spectrophotometer. One unit (U) of BGL activity was defined as 1 lmol 4-nitrophenol released per minute. Protein concentrations were determined by Bradford assay using the Bio-Rad Protein Assay Kit) with bovine serum albumin as the standard. 2.3. Native PAGE analysis with MUG-zymogram For the detection of in-gel BGL activity, samples were analyzed by native PAGE using 10% and 4% polyacrylamide as separation and stacking gels, respectively. Gels were run in Tris–glycine buffer (pH 8.3) at a constant current of 20 mA per slab for 3 h at 4 °C, washed with distilled water, and overlaid with 0.5 mM 4-methylumbelliferyl b-D-glucopyranoside (MUG, Sigma–Aldrich) in 0.1 M succinate buffer (pH 5.8), followed by incubation for 15 min at 55 °C. The presence of a fluorescent reaction product was then detected by visualization at 365 nm for 5 min. Gels were stained with Coomassie brilliant blue R-250 after being photographed under UV light. The gel bands with enzyme activities were excised for subsequent MS/MS analysis. 2.4. Tandem mass spectrometry and protein identification A nanoLC-MS/MS analysis was performed on an integrated system (QSTAR XL) comprising a LC Packings NanoLC system with an autosampler and a QSTAR XL Q-Tof mass spectrometer (Applied Biosystems, Lincoln, CA) fitted with nano-LC sprayer. Injected samples were first trapped and desalted on a LC-Packings PepMap™ C18 l-Precolumn™ Cartridge (5 lm, 5 mm  30 lm I.D.; Dionex, Sunnyvale, CA). Subsequently, peptides were eluted from the precolumn and separated on an analytical LC-Packings PepMap C18 column (3 lm, 15 cm  75 lm I.D.) connected in-line to the mass spectrometer, at a flow rate of 200 nl/min using a 40-min gradient of 5–60% acetonitrile in 0.1% formic acid.

I-Son Ng et al. / Bioresource Technology 102 (2011) 6073–6081

Online nanoESI-MS survey scan and data dependent acquisition of CID MS/MS were fully automated and synchronized with the nanoLC running under the full software control of AnalystQS. Prior to online analysis, the nano-LC sprayer source parameters were tuned and optimized. Argon was used as the collision gas for CID MS/MS. Calibration was performed with the product ions generated from fragmentation of the doubly charged molecular ion of rennin. For routine protein identification analysis, 1 s survey scans were acquired over the mass range m/z 400–1600 and a maximum of two concurrent MS/MS acquisitions was triggered for 2+, 3+ and 4+ charged precursors detected at an intensity above the predefined threshold. Each MS/MS acquisition was completed and switched back to the survey scan when the precursor intensity fell below a predefined threshold or after a maximum of 6 s acquisition. After data acquisition, the individual MS/MS spectra for each of the precursors within a single LC run were combined and a single Mascot-searchable peak list file was generated. The peak list files were used to query the Swiss-Prot database using the Mascot program with the following parameters: peptide mass tolerance, 150 ppm; MS/MS ion mass tolerance, 0.15 Da; allowing up to one missed cleavage. Only significant hits as defined by Mascot probability analysis were considered. In addition, a minimum total score of 30 comprising at least a peptide match of ion score was arbitrarily set as the threshold for acceptance. 2.5. HPLC analysis Concentrations of cellobiose and glucose concentrations in reaction samples were analyzed by the AllianceÒ HPLC system (Milford, MA), with a 2424 evaporative light scattering (ELS) detector after nebulization of 80% at 60 °C and evaporation by nitrogen at 40 psi. Separation of glucose and cellobiose was carried out by using a Shodex column (Asahipak, NH2P-50 4E, 4.6 mm I.D.  250 mm, Showa Denko, Japan) equilibrated at 40 °C, resulting in a retention time of 6.1 and 8.2 min, respectively. The mobile phase was a mixture of acetonitrile/Mini-Q water (7/3, v/v) at a flow rate of 1 mL/min at a constant concentration (isocratic elution) for 10 min for each sample. Peak areas for cellobiose and glucose showed linear correlation with standard curves within the range of 2–10 mM. The standard deviation of three repeated injections was normally below 3%. Cellulase activities are often determined through measuring total reducing sugars released by the method of Somogyi (1952) or Miller (1959), i.e. the DNS method. However, this method has the inherent problem to underestimating other types of products such as cellobiose and glucose. As the glucose production was more precisely measured by HPLC than by the DNS method, we used the latter in the detection of total reducing sugar and the former for the estimation of synergistic effects. 2.6. Kinetic analysis and glucose inhibition of BGLs For BGLs, enzymatic hydrolysis using pNPG as the substrate was carried out in the VersaMax™ microplate reader (Molecular Devices, CA) controlled at 45 °C in the kinetic mode. Samples were prepared in 96-well microplates with 3 lg/mL of different BGLs and 200 ll of pNPG substrate (ranging from 0.25 to 2.0 mM) in 50 mM sodium acetate buffer (pH 5.0). In order to study the product inhibition effect, analogous experiments were performed in the presence of 1 mM glucose. The enzymatic hydrolysis of cellobiose was also carried out in 1.5 ml microcentrifuge tubes, with incubation for 10 min at 45 °C, to determine initial rates. The activity of BGLs (15 lg/ml) was determined in 200 ll of 50 mM sodium acetate buffer (pH 5.0) with cellobiose concentrations varied from 2.5 to 20 mM. To investigate product inhibitory effects, corresponding experiments were performed with additional 10 mM of

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glucose. The cellobiose and glucose concentration in each reaction were assessed by HPLC. One unit of BGL activity corresponds to 1 lmol 4-nitrophenol and 1 lmol glucose equivalent per minute, respectively. 2.7. Kinetic analysis of synergism Kinetic studies with T. reesei cellulases were carried out in a volume of 0.5 mL containing 50 mM sodium acetate buffer (pH 5.0), 100 lg/ml of T. reesei cellulases (ATCC 26921), 40 lg/ml of different BGLs, and 2.6 mg, 5.2 mg, 7.8 mg, 10.4 mg or 15.6 mg of Whatman No.1 filter paper, respectively. Reactions were carried out for 1 h at 50 °C with shaking at 300 rpm. The cellulose residue was removed by centrifugation at 15,000g for 5 min, and the reducing sugar in the supernatant was determined by the dinitrosalicylic acid (DNS) method. The cellobiose and glucose concentration for each reaction were determined by HPLC. 2.8. Dynamic synergism effect To a 1.5-mL microcentrifuge tube with 0.5 mL of 50 mM sodium acetate buffer (pH 5.0), 7.8 mg of Whatman No.1 filter paper, 160 lg/mL of T. reesei, and BGLs of varied concentrations (from 16 lg/mL to 32 lg/mL for Novo-188 or from 25 lg/mL to 75 lg/ mL for D2) were added, and the mixture was incubated at 50 °C with shaking. At different time intervals, samples were then centrifuged at 15,000g for 10 min to remove the precipitate for HPLC analysis, from which the initial rates Vglu and VCB, the time course of conversion XGt, and hence the dynamic synergism ratios based on Vglu were calculated. 2.9. Model development An irreversible Michaelis–Menten kinetics with glucose as the non-competitive inhibitor was employed for modeling the BGL hydrolysis of pNPG or cellobiose as follows:

V¼

dðSÞ V max ðSÞ=ð1 þ ðIÞ=K I Þ ¼ dt K m þ ðSÞ

ð1Þ

where (I) and (S) denote glucose and pNPG (or cellobiose) concentrations, respectively. The kinetic constants Vmax, Km and KI, corresponding to the maximum initial rate, the Michaelis–Menten constant, and the inhibition constant, respectively, were determined from the variation of initial Vbgl with (S) at different glucose concentrations. In experiments, in which sufficient BGL concentrations were added in order to study the synergistic effect on T. reesei, the cellobiose inhibition was neglected. For these experiments, an irreversible Michaelis–Menten kinetics was employed for modeling the cellulase hydrolysis as follows:

V¼

dðFÞ V mt ðFÞ ¼ dt K mt þ ðFÞ

ð2Þ

where (F) denotes the filter paper concentration with Vmt and Kmt as the kinetic constants. 3. Results and discussion 3.1. Screening and identification of BGLs The contribution of BGLs to cellulose hydrolysis is significant because cellobiose acts as an inhibitor of both endo- and exoglucanases, and therefore must be removed to allow effective saccharification of cellulose. Research and development effort has been focused on cellulosic hydrolysis with cellulases from T. reesei

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supplemented with extra b-glucosidases. In the past decade, the use of BGL, Novozyme 188 (Novo-188), a commercial A. niger BGL, has been developed rapidly (Berlin et al., 2007; Martins et al., 2008; Chauve et al., 2010). Although several other commercial BGLs exist, Novo-188, has been one of the more often used ones in cellulosic ethanol production. Thus, it was chosen as a benchmark enzyme in this study. To further lower the cost for cellulosic hydrolysis, there is still a need to screen for novel BGLs that are better than the currently available commercial enzymes (Karnchanatat et al., 2007; Rosgaard et al., 2006). Taiwan is located in the temperate and humid climate zone with many high mountains and has characteristic changes of the seasons, which support a wide range of biodiversity including miroorganisms. As a result, sophisticated systems for cataloging and utilization of microorganisms have been established in this region (Ju and Hsieh, 2007). Therefore, efforts were devoted to screen Taiwanese fungi that have been previous collected from decomposing wood. BGLs were screened for enzymatic activity using a set of 42 selected Taiwanese fungi producing extracellular BGLs was shown in Fig. 2. The highest enzymatic activity of most fungi in the cultivated time course occurred at 144–168 h, except for Chaetomella raphigera (D2), Chaetomella sp. 2 (D3) both was at 72 h. This result demonstrated that Chaetomella species grew faster than other fungi. The best six fungi including C. raphigera (D2), Chaetomella sp. 2 (D3), Eutypa sp. (E9), Graphostroma platystoma (E10), Hypoxylon hypomiltum (E13), and Penicillium citrinum YS40-5 (PC), displayed high enzymatic activity at the range from 2 U/mL to 2.5 U/mL among selected fungi, were chosen for further investigation. As these fungi are all new species, their biochemical properties have so far not been reported in the literature. In order to further study the best six selected BGLs from crude mixtures, total protein samples were separated by 10% native PAGE followed by MUG-zymogram analysis (Fig. 3A). Detection of BGL activities with MUG, a substrate containing a fluorescent group for sensitive detection and with a small size (338.3 Da) for rapid mass transfer into the gel matrix by diffusion, has been used to study the properties of this type of enzyme (Kim et al., 2007; McKeon, 1988; Saloheimo et al., 2002). After observation of BGL

(A) MUG-Zymogram Mw 130 95 72 55 M

D2

D3

PC

E9

E10

E13 BSA

Novo-188

Mw 130 95 72 55

(B) Coomassie Blue Stain Fig. 3. Native PAGE analysis with (A) MUG-zymogram and (B) Coomassie blue R250 staining. Lane 1: Marker in kDa, lane 2: D2 (Chaetomella raphigera), lane 3: D3 (Chaetomella sp. 2), lane 4: PC (Penicillium citrinum YS40-5), lane 5: E9 (Eutypa sp.), lane 6: E10 (Graphostroma platystoma), lane 7: E13 (Hypoxylon hypomiltum), lane 8: BSA, lane 9: Novo-188.

activities under UV light, the gel was stained with Coomassie Blue R250 (Fig. 3B) to indirectly estimate molecular weight of the enzymes in their native form. The major fluorescent band of each selected BGL (indicated with arrows in Fig. 3A) were subjected to

3.0

β-glucosidase (U/mL)

2.5

2.0

1.5

1.0

.5

A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 E1 E2 E3 E4 E5 E6 E7 E9 E10 E11 E13 E15 E16 PC

0.0

Fig. 2. BGL activity screening among Taiwanese fungi. Enzyme activity was determined with 1 mM pNPG substrate for 15 min at 50 °C, 50 mM sodium phosphate buffer (pH 7.0). A1: X. atrodivaricata, A2: X. acuminatilongissima, A3: X. griseosepiacea, A4: X. Cirrata, A5: X. Branneovinosa, A6: X. intraflava, B1: X. nigrips, B2: Jumillera cinerea, B3: Daldinia eschscholzii, B4: Rosellinia merrillii, B5: X. schweinitzii, B6: Annulohypoxylon leptascum, C1: Amphisphaeria andropognis, C2: X. deserticola, C3: Rosellinia lamprostoma, C4: Biscogniauxia waitpela, D1: Myxosporium psidii, D2: Chaetomella raphigera, D3: Chaetomella sp. 2, D4: Stilbocrea macrostroma, D5: Pasreutypella sulcata, D6: Roussoella donacicola, D7: Valsaria rubricosa, D8: Coprinus insignis, D9: Valsaria fulvopruinata, D10: Conocybe fragilis, D11: Marasmiellus candidus, D12: Psathyrella velutiina, E1: Ajrekarella sp., E2: Albonectria rigidiuscula, E3: Alternaria sp., E4: Curvularia sp., E5: Leptosphaerulina sp., E6: Leptospora sp., E7: Penicilliopsis clavariaeformis, E9: Eutypa sp., E10: Graphostroma platystoma, E11: Hyphomycete sp., E13: Hypoxylon hypomiltum, E15: X. arbuscula var. plenofissura, E16: X. cubensis. PC: Penicillium citrinum YS40-5. This screening process had been carried out twice, and the average values and standard errors of these duplicated experiments are plotted.

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I-Son Ng et al. / Bioresource Technology 102 (2011) 6073–6081 Table 1 Mascot protein identifications of b-glucosidases from fungi. Protein source

Peptide sequence

N-188

Aspergillus niger b-glucosidase (ABB29285) TMHELYLWPFADAIR 1862.1 NGVFTATDNWAIDQIEALAK 2176.3 DLANWNVETQDWEITSYPK 2308.4 Aspergillus fumigatus b-galactosidase precursor (CAF32131) GPLNEGGLYAER 1274.55 LPVPSLWLDVFQK 1540.84 Pyrenophora tritic b-galactosidase precursor (XP_001931072) LTGNLGGEDYKDISR 1202.29 Humicola grisea var. thermoidea cellulase (BAA09785) YAGVCDPDGCDFNSYR 1896.66 Aspergillus clavatus NRRL 1b-glucosidase (XP_001273447) ANVASVMCSYNK 1342.54 FANPVTAFPVAINAGADWDK 2102.98 Penicillium simplicissimum a-galactosidase (CAA08915) AHFALWAIMK 1202.58 VDAEAFAEWGIDYLK 1725.74 Penicillium brasilianum b-glucosidase (ABP88968) AQDFVSQLTILEK 1490.82 IMAAYFK 842.44 Thermoascus aurantiacus thermostable b-glucosidase (AAZ95587) GIQDAGVIACAK 1201.60 Melanocarpus albomyces cellulose 1,4-b-cellobiosidase (CAD56667) CDANGCDYNPYR 1504.52 Penicillium brasilianum b-glucosidase (ABP88968) GITIQLGPVAGPLGR 1447.83 AQDFVSQLTILEK 1490.81

D2

D3 PC

E9

E10 E13

Observed mass, Da

in-gel trypsin digestion followed by LC–ESI-MS/MS analysis. Detailed peptide sequencing information is summarized in Table 1. The best match from Mascot protein identification agreed with BGLs for Novo-188, PC, E9 and E13, b-galactosidase for D2, cellulase for D3, and cellobiosidase for E10. These enzymes were identified as either BGLs or glycosylhydrolases. However, the results did not predict precisely the hydrolase as BGLs owing to the inadequate database of these novel fungi. Tandem mass spectrometry data have provided information not only about protein identity but also about its potential function (Chir et al., 2002; Shevchenko et al., 1997). It is also much faster and simple than the traditional processes such as protein precipitation and chromatography followed by amino acid sequence determination because with the MS-based analysis no protein purification is needed, and it involves fewer manipulation steps. The results obtained from the MS analysis allowed us to first clone the partial cDNA sequence by using degenerated primers based on determined peptide sequence, followed by cloning the full-length cDNA by 50 -PCR in later experiments. 3.2. Kinetic analysis and glucose inhibition of BGLs To obtain more insight into the different biochemical properties of all BGLs isolated in this work, kinetic parameters were determined at 45 °C from the hydrolysis of pNPG and cellobiose (Fig. 4). Comparing the synthetic substrate pNPG with natural substrate cellobiose, it was observed that BGLs from the selected fungi were more active (based on Vmax/Km) with higher affinity (i.e. lower Km) on pNPG than on cellobiose (Table 2). Moreover, these BGLs had nearly the same Km values (i.e. 0.42–0.91 mM), which is significantly lower than the Km value of Novo-188 (1.77 mM). The best three BGLs, i.e. D2, D3 and PC, also showed less glucose inhibition when compared with Novo-188. The hydrolytic efficiency of Novo-188 (i.e. Vmax/Km = 0.72 U/ mg mM) for cellobiose was slightly higher than those of other BGLs (Table 2). The greater substrate affinity of Novo-188 than that of other BGLs (i.e. smaller Km) resulted in lower glucose inhibition (i.e. larger Ki), which was also valid when pNPG was used as the

Calculated mass, Da

Mascot score

1861.9 2176.1 2308.0

139 139 139

1274.56 1540.86

107 107

1202.62

72

1896.69

48

1342.60 2103.04

72 72

1202.62 1725.82

93 93

1490.80 842.43

99 99

1202.62

82

1503.55

55

1447.85 1490.79

70 70

substrate. Based on the substrate specificity, BGLs have been classified as: (1) aryl BGLs acting on aryl-glucosides, (2) true b-glucosidase hydrolyzing cellobiose to release glucose, and (3) broad substrate specificity enzymes acting on a wide spectrum of substrates (Bhatia et al., 2002). The enzymes we studied in this work appeared to belong to group 3 as they hydrolyzed both artificial and natural substrates to glucose. It has been reported that both a- and b-glucosidases are inhibited by the accumulation of their enzymatic reaction product, glucose (Wang et al., 2004; Xiao et al., 2004). It is intriguing to note that glucose inhibition of BGLs, followed a typical non-competitive inhibition kinetics. As shown in Fig 4, based on the Michaelis–Menten equation, the Lineweaver–Burk double reciprocal plot of BGL activities in the presence or absence of glucose had the same Km, but different Vmax. The same non-competitive inhibition existed with either the synthetic substrate, pNPG (Fig. 4A) or the natural substrate, cellobiose (Fig. 4B). From the Ki values presented in Table 2, it becomes apparent that different BGLs display different sensitivities to glucose inhibition. With pNPG as the substrate, Novo-188 was more sensitive to inhibition than other BGLs, i.e., D2, D3, and PC. In contrast, Novo-188 was surprisingly the least sensitive to glucose inhibition when the natural substrate, cellobiose, was used. Whereas D2 was only slightly more sensitive than N-188, D3 and PC were significantly affected by glucose. Since the glucose inhibition follows a non-competitive kinetics, this observation suggests that the product inhibition of BGLs is likely mediated by a second site on the enzyme molecule away from the active site. However, the mechanisms underlying the glucose inhibition require further investigations. 3.3. Synergistic effect of BGLs and T. reesei cellulases Synergistic effect was carried out with the T. reesei enzyme system, in the presence or absence of supplemental BGLs, with incubation for 1 h at 50 °C. Fig. 5A and B shows the determined total reducing sugar concentration or the sum of cellobiose and glucose concentrations, respectively. The extra BGLs not only eliminated the cellobiose inhibition, but also accelerated the total hydrolytic

I-Son Ng et al. / Bioresource Technology 102 (2011) 6073–6081

-1

0

1

2

3

4

5 -4

-2

0

2

4

6 0.2

D2

Novo-188

0.5

0.1

0.0

0.0

-0.5 0.4

-0.1 0.6

PC

D3

0.2

0.3

0.0

0.0

-0.3

-0.2 -4

-2

0

1/S

(B) 0.0

6 -2

-1

0

1/S

0.2

0.4

1 (mM-1)

2

3

1/S (mM-1) 0.6

0.0

0.2

0.4

0.6 1.2

Novo-188

D2

0.8

0.8

0.4

0.4

0.0

0.0

5.0

5.0

PC

D3

4.0

4.0

3.0

3.0

2.0

2.0

1.0

1.0

0.0

0.0 0.0

0.2

0.4

1/S

(mM-1)

0.6

0.0

1/V (mg/U)

1/V (mg/U)

4

1/S (mM-1) 1.2

1/V (mg/U)

2 (mM-1)

0.2

0.4

1/S

(mM-1)

1/V (mg/U)

1/V (mg/U)

1.0

1/V (mg/U)

1/S (mM-1)

1/S (mM-1)

1/V (mg/U)

(A)

1/V (mg/U)

6078

0.6

Fig. 4. The Michaelis–Menten double reciprocal plot showing the kinetics of BGLs with various substrate concentrations and reaction rate in the hydrolysis of (A) pNPG and (B) cellobiose by different b-glucosidases at 45 °C. Solid symbols represent the reaction rate in the presence of the reaction product, glucose (1 mM in pNPG and 10 mM in cellobiose). Open symbols represent the reaction rate in the absence of glucose. (ds) Novo-188, (jh) D2, (N4) D3, (}) PC and (—) best-fit line with the experimental data.

Table 2 Kinetic parameters of b-glucosidases at 45 °C. Enzymes

Novo-188 D2 D3 PC E9 E10 E13 T. reesei

pNPG

Cellobiose

Vmax (U/mg)

Km (mM)

Vmax/Km (U/mg mM)

Ki (mM)

Vmax (U/mg)

Km (mM)

Vmax/Km (U/mg mM)

Ki (mM)

13.28 20.96 16.75 6.37 6.49 2.98 4.73 4.50

1.77 0.45 0.86 0.75 0.91 0.42 0.63 1.54

7.50 46.58 19.48 8.49 7.15 7.05 7.45 2.91

1.59 4.59 3.45 3.93 nd nd nd nd

12.09 17.98 13.12 6.42 1.31 0.78 1.18 1.03

16.82 36.98 51.20 26.21 5.66 12.05 6.82 24.96

0.72 0.49 0.26 0.24 0.23 0.06 0.17 0.04

59.50 46.33 12.92 5.47 nd nd nd nd

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I-Son Ng et al. / Bioresource Technology 102 (2011) 6073–6081

(A)

14

33.5 %

33.8 % 22 %

Reducing sugar (mM)

12

15 %

13 %

11 % 6%

10 8 6 4 2

8 18 ov o-

T+ E1 3

T+ N

T+ E9 T+ E1 0

3

T+ PC

2

T+ D

T+ D

T

0

(B) Cellobiose and Glucose (mM)

18

422 %

411 %

16 14

resulting in incomplete hydrolysis of cellobiose, which in turn generates product inhibition (Holtzapple et al., 1990). In our results, supplementation with extra BGLs is not only required for more complete hydrolysis of cellulose, but also stimulates the overall cellulolytic reaction in a synergistic manner. Furthermore, this work revealed that BGLs from various fungal sources appear to generate different synergistic kinetics when supplemented with the T. reesei enzyme system. The calculated apparent kinetic parameters (see Table 3) showed that Vmt was highly increased up to 10-fold (i.e. 27.7– 289 for Novo-188 and 27.7–260.7 for D2) with the addition of extra BGLs. However, there was not much change in Kmt thus the Vmt/Kmt was raised from 5.9 to 21.4. This observation suggested the ratedetermining step in hydrolysis was the acylation or deacylation step (higher Vmt) but not the binding of the substrate to enzyme, i.e. substrate affinity (similar Kmt). The BGLs from Novo-188 and D2 performed well among all the selected enzymes, yielding about 10-fold enhancements of the apparent maximum velocity, however, showed reduced substrate affinity when compared to the values obtained in the absence of BGL. Based on the Vmt/Kmt values, the ratios of synergism were 3.2 for Novo-188 and 3.6 for D2, indicating that BGL from D2 is comparable to Novo-188 in this particular quality.

12 239 %

10

3.4. Dynamic synergistic effect

214 % 193 %

8

155 % 134 %

6 100 %

4 2

T+

N

ov

o-

18

8

3

0

E1 T+

E1 T+

E9 T+

3

PC T+

2

D T+

D T+

T

0

Fig. 5. Synergistic effect of BGLs with T. reesei cellulases at incubation for 1 h at 50 °C in 50 mM sodium acetate buffer (pH 5.0). Enzyme concentrations were 100 lg/ml of T. reesei and 40 lg/ml of b-glucosidases. (A) The total reducing sugar was determined by the DNS method. (B) Cellobiose and glucose concentration were measured by HPLC, (j) cellobiose, (h) glucose. This experiment had been performed three times, and the average values and standard errors are plotted.

rate of filter paper as substrate. The production of reducing sugars showed an increase of 33.5% for D2, 22% for D3, 15% for PC, 13% for E9, 6% for E10, 11% for E13, and 33.8% for Novo-188. Moreover, the glucose released due to the synergistic effect by BGLs was significantly higher than without BGLs, i.e. 4.22-fold enhancement for D2 and 4.11-fold increase for Novo-188. The overall cellulase activity is most often determined through measuring total reducing sugars released by the method of Somogyi (1952) or Miller (1959), i.e. the DNS method. However, this method has the inherent problem to underestimating other types of products such as cellobiose and glucose. As the glucose production was more precisely measured by HPLC than by the DNS method, we used the latter in the detection of total reducing sugar and the former for the estimation of synergistic effects. Several studies have addressed the possibility that synergisms occur between EGs and CBHs, the so-called endo–exo synergism, but the results are contradictory (Medve et al., 1998; Nidetzky et al., 1994; Valjamae et al., 1999; Woodward, 1991; Woodward et al., 1988). The main reason for the confusion is probably that different experimental conditions, such as enzyme source and composition, nature of the substrate were used for different studies. Despite the contradictory results in the literature, the fact is in the T. reesei enzyme system contains rather low BGL activities,

To investigate the dynamic synergism between EGs/CBHs and BGLs, we added various amounts of BGL (or called CBU, Cellobiase unit) to T. reesei cellulases at 0.25 FPU (filter paper unit). The results presented in Table 4 demonstrate that the addition of Novo-188 or D2 BGLs boosted the glucose conversion level from 10.86% to 50.60% and 48.71%, respectively (Table 4). A significant increase of glucose production rate (from 1.76 mM/h to 15.67 mM/h) and hence the dynamic synergistic ratio (8.9-fold enhancement) was observed by the presence of D2 BGL in T. reesei cellulases. The dynamic synergistic effect is probably because the extra BGLs can reduce substrate impediment due to steric hindrance, thus stimulate hydrolysis of cellobiose and eliminate product inhibition, leading to an enhanced total cellulolytic activity in T. reesei enzyme system. Although the cellobiose production rate was 4.2 mM/h for T. reesei enzymes, no cellobiose was detected when 0.2 CBU of BGLs was added (Table 4). Further enhancement on initial rate of glucose production (Vglu) in the hydrolysis of filter paper was achieved by an additional increase of the BGL concentration. However, the mechanisms underlying these improvements are not yet clear. Fig. 6 illustrates the time course of the dynamic synergism between BGLs and the T. reesei cellulases. The highest yield at the shortest reaction time of 6 h, i.e. 67% hydrolysis, for the D2 BGL was obtained in comparison with the results reported in the literature (Berlin et al., 2007; Szijarto et al., 2008). It has been well recognized that cellulase activities are inhibited by their reaction product, cellobiose, thus it is expected that Table 3 Apparent kinetic parameters of synergistic cellulases at 50 °C analyzed by HPLC. Enzyme added to T. reesei cellulases

Vmt (lmol/ h mg)

Kmt (mM)

Vmt/Kmt (lmol/ h mg mM)

Syn ratio

None Novo-188 D2 D3 PC E9 E10 E13

27.7 ± 0.59 289.0 ± 6.92 260.7 ± 6.60 157.4 ± 3.60 97.5 99.2 42.8 76.6

4.66 ± 0.13 15.19 ± 3.75 12.19 ± 3.20 11.92 ± 2.20 9.67 12.48 5.26 6.19

5.95 ± 0.10 19.03 ± 0.46 21.39 ± 0.62 13.20 ± 0.21 10.08 7.95 8.13 12.37

1.00 3.20 3.59 2.22 1.69 1.34 1.37 2.08

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Table 4 Effect of b-glucosidase concentrations on dynamic synergism with T. reesei cellulases (with 0.25 FPUa) at 50 °C. Enzyme added to T. reesei cellulases

Vglu (mM/h)

VCBb (mM/h)

Dynamicc synergistic ratio

XGtd (%)

None Novo-188-0.2 CBUe Novo-188-0.4 CBU Novo-188-0.6 CBU D2-0.2 CBU D2-0.4 CBU D2-0.6 CBU

1.76 8.86 10.12 12.92 10.05 12.57 15.67

4.20 0 0 0 0 0 0

1.00 5.03 5.75 7.34 5.71 7.14 8.90

10.86 50.60 54.23 59.97 48.71 61.41 67.66

Although an enhanced total cellulolytic bioconversion can be easily observed when a well established EG system, such as that from T. reesei, is supplemented with extra BGLs, one has to recognize that this constitutes additional cost to the overall process. One of the major constraints in industrial cellulosic ethanol production is the cost for enzymes, thus identification of inexpensive and effective source of BGL, such as demonstrated in this work is of vital significance in the future research and development of this budding industry. 4. Conclusions

a

FPU (filter paper unit) is equivalent of hydrolysis of filter paper to produce 1 lmol glucose per minute. b Cellobiose production rate with various enzymes. c Dynamic synergism ratio based on the glucose production rate (Vglu). d Glucose produced in 6 h in the hydrolytic reaction. e CBU (cellobiase unit) is virtually equivalent to b-glucosidase activity.

Degree of hydrolysis (%)

80

60

40

Six Taiwanese fungi with high BGL activity for the cellulosic bioconversion have been isolated. Enzyme identifications were revealed by native PAGE MUG-zymogram followed by MS/MS. Comparisons of kinetic parameters indicate that properties of these BGLs differ from those of the commercial enzyme, Novo-188. The Vmax/Km value for the C. raphigera D2 BGL was about the same as Nono-188, 0.49 vs. 0.72 U/mg mM, respectively. However, D2 appeared to be much more active than Novo-188 toward the synthetic substrate, pNPG, with Vmax/Km values at 46.58 vs. 7.50 U/ mg mM for Novo-188. Total cellulolytic activities were greatly increased by supplementing the T. reesei cellulases with either C. raphigera D2 BGL (8.9-fold) or Novo-188 (7.3-fold). The synergism between EG/CBH and BGL can be described by a dynamic synergism model, in which BGLs not only hydrolyze cellobiose readily but also significantly enhance cellulose hydrolysis by T. reesei cellulases. Acknowledgements This research was supported by a grant (NSC96-3114-P-001004-Y) from the National Science Council, Taiwan (ROC).

20

References 0 0

1

2

3

4

5

6

Time (h) Fig. 6. Time course of dynamic synergism for BGLs with T. reesei cellulases (160 lg/ mL) at 50 °C. (4) Vglu and (}) VCB of T. reesei only, Vglu of T. reesei with Novo-188: (d) 16 lg/mL, (.) 32 lg/mL and (j) 48 lg/mL, Vglu of T. reesei with D2 (Chaetomella raphigera): (s) 25 lg/mL (5) 50 lg/mL and (h) 75 lg/mL.

the removal of cellobiose by the addition of BGL would enhance hydrolysis of cellulose. However, the actual kinetic features of this process are much more complicated. Although BGL hydrolyzes cellobiose to yield glucose, this enzyme itself is feedback inhibited by glucose. As demonstrated in this work, different BGLs are inhibited by glucose to a different extent, with Novo188 and D2 of C. raphigera being less sensitive to glucose than other BGLs tested. This is most likely the reason these two BGLs displayed much better synergism working together with the T. reesei cellulases. Furthermore, it has been shown that glucose is also an inhibitor to cellulases, albeit weaker than cellobiose (Xiao et al., 2004). Also, since the BGL action is reversible (Fig. 1), it is conceivable that the stronger feedback inhibitor cellobiose can be resynthesized if glucose is not further degraded. Therefore, a gradual shift in inhibition kinetics of cellulase takes place when cellobiose is further hydrolyzed to glucose, and the product feedback inhibition remains in effect until glucose is used up by the fermentation process. It is apparent from this work and previous reports in the literature that a significant synergism exits between BGL and EGs.

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