A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774

A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774

Journal of Microbiological Methods 83 (2010) 74–81 Contents lists available at ScienceDirect Journal of Microbiological Methods j o u r n a l h o m ...

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Journal of Microbiological Methods 83 (2010) 74–81

Contents lists available at ScienceDirect

Journal of Microbiological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774 Yuanshan Lin a,b, Guiguang Chen a, Min Ling a, Zhiqun Liang a,⁎ a b

College of Life Science & Technology, Guangxi University, Nanning 530004, China College of Bioscience & Biotechnology, Hunan Agricultural University, Changsha 410128, China

a r t i c l e

i n f o

Article history: Received 27 March 2010 Received in revised form 23 July 2010 Accepted 25 July 2010 Available online 4 August 2010 Keywords: β-glucosidase Electroelution Native gradient-polyacrylamide gel electrophoresis Trichoderma koningii

a b s t r a c t In this study, we used native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) to purify enzymatic proteins from Trichoderma koningii AS3.2774. With this method, we purified eight enzymatic proteins and classified them to the cellulase system by comparing secretions of T. koningii in inductive medium and in repressive medium. It resulted in 24-fold β-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6 IU mg− 1 protein. The final yield of BG reached 8 μg under purifying procedure of NGGEE. We also identified BG using the enzyme assay with thin-layer chromatography and MALDI-TOFMS. This BG had one subunit with a molecular mass of 69.1 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The hydrolytic activity of the BG had an optimal pH of 5.0, an optimal temperature of 50 °C, an isoelectric point of 5.68 and a Km for p-nitrophenyl-β-D-glucopyranoside of 2.67 mM. Taken together, we show that NGGEE is a reliable method through which μg grade of active proteins can be purified. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cellulose is a major polysaccharide constituent of plant cell walls, which is β-1, 4 linked linear polymer of 8000–12,000 glucose units. Three major enzymes are involved in the degradation from cellulose to glucose: endoglucanase (endo-1, 4-β-D-glucanase, EG, EC 3.2.1.4), cellobiohydrolase(exo-1, 4-β-D-glucanase, CBH, EC 3.2.1.91), and βglucosidase (1, 4-β-D-glucosidase, BG, EC 3.2.1.21). EG acts in a random fashion, cleaving β-linked bonds within the cellulose molecule; CBH removes cellobiose units from the nonreducing ends of the cellulose chain; and BG degrades cellobiose and cellooligosaccharides to glucose. The Trichoderma reesei cellulase system has been the most widely studied among the cellulolytic fungi (Saha Badal and Bothast Rodney, 2009). BG splits the cellobiose into two glucose monomers, removing the inhibition exerted by cellobiose on certain cellulase, such as CBH I of T. reesei (Gong et al., 1977). BGs are also important in the regulation of cellulase genes because they are the key enzymes in the synthesis of sophorose, an efficient inducer of the cellulolytic system of T. reesei (Mandels et al., 1962). Cellulase can be induced by Molasses alcohol stillage (Ling et al., 2009). Recently, BGs have become the focus of many applied studies because they are essential not only in the cellulose breakdown but also in the synthesis of oligomers and other complex molecules (e.g., alkyl-glucosides) by transglycosylation ⁎ Corresponding author. College of Life Science & Technology, Guangxi University, Nanning 530004, China. Tel.: +86 771 3270732; fax: +86 771 3270730. E-mail addresses: [email protected] (Y. Lin), [email protected] (Z. Liang). 0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2010.07.019

(Hanssona and Adlercreutza, 2002). The structural organization of this enzyme is a considerably diversified. The native BG from Pyrococcus furiosus has a molecular mass of 230 kDa and is composed of four subunits, each with a molecular mass of 58 kDa (Kengen et al., 1993). In contrast, the BG from Clostridium stercorarium is a monomer with a molecular mass of 85 kDa (Bronnenmeier and Staudenbauer, 1988). Some studies indicate that some BGs of white-rot fungi also have a high molecular mass. For example, two BGs from Sporotrichum pulverulentum have high molecular masses of 165 kDa and 182 kDa, respectively (Deshpande et al., 1978). Moreover, BGs with similar molecular masses have been purified: the BG from Pisolithus tinctorius, 450 kDa composed of three homo subunits; and the BGs from Volvariella volvacea, 158 kDa and 256 kDa (Cai et al., 1998; Cao and Crawford, 1993). Stachybotrys microbispora has a rich BG system consisting of more than four enzymes. Two of them were purified as dimeric with each monomer of 85 kDa (Amouri and Gargouri, 2006). Due to their wide biological activities, the preparation of BGs with high purity has been of great interest to biologists. However, it is very difficult to obtain pure active proteins by conventional separation methods, such as column chromatography, two-dimensional electrophoresis and thin-layer chromatography (TLC). Combining with transfer membrane blotting, blue native electrophoresis (BN-PAGE) has been successfully used in the preparative separation of active proteins (Offord et al., 1991; Schägger, 1994). So far, no studies have been reported about two-cycle native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) without transfer membrane blotting for isolating and purifying active proteins. Here we

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reported an efficient method for the preparative isolation and purification of BG. 2. Materials and methods 2.1. Microorganisms, culture conditions and preparation of cellulase Trichoderma koningii AS3.2774 was used for cellulase production, which was obtained from the Institute of Microbiology, Chinese Academy of Sciences. Spore suspension of T. koningii (approximately 108 conidia ml− 1) was prepared from one potato dextrose agar slant after being maintained at 28 °C for 4 days. The enzyme production was carried out in 250-ml Erlenmeyer flasks in inductive medium or repressive medium, respectively. Inductive medium contained 40mesh straw powder (inductor) 8.0 g, wheat bran 4.0 g, and tap water 20 ml, with natural pH. Repressive medium contained 40-mesh straw powder 8.0 g, wheat bran 4.0 g, glucose (repressor) 2.0 g and tap water 20 ml, with natural pH. Both of them were sterilized for 25 min at 121 °C. Each medium was inoculated with 2 ml spores, equably suspended and statically incubated at 28 °C for 4.5 days. The mould koji of medium was turned over axenic once a day for ventilation. Cultures of mould koji were harvested at the late exponential phase (4.5 days), and each was mixed with 100 ml citric acid-sodium citrate buffer (50 mM, pH 4.8) and slowly stirred in the flask shaker at room temperature for 1 h. Then, the extract was filtered through two-layer gauze. Filtrates were followed by centrifugation at 12,000 × g at 4 °C for 20 min. The supernatant, crude cellulase was used to purify cellulolytic enzymes and for the activity assay.

Step 3

Step 4

2.2. Zymogram analysis The cellulase of T. koningii AS3.2774 was revealed by the zymogram analysis. For this purpose, ultra-condensates of cellulase excreted from inductive medium and repressive medium were loaded on the same native gradient acrylamide gel (4.0%–8.0% separating gel and 4% stacking gel without SDS and β-mercaptoethanol). When the run finished, the gel was stained with Coomassie brilliant blue R-250 and destained in a solution composed of 10% acetic acid and 20% absolute methanol to identify whether a protein band belonged to the cellulase system or not. The cellulase-related protein strips induced from inductive medium were named P1 to P10, respectively (Fig. 2). The cellulase excreted from inductive medium was also loaded on each gel under different purifying conditions.

Step 5

2.3. Purification of cellulase Protein concentration was determined by the method of Bradford (1976), with bovine serum albumin as the standard. Unless stated otherwise, all the purification procedures were performed at 4 °C. Isolation of cellulase excreted from inductive medium by T. koningii AS3.2774 was performed as follows. Step 1 Ultrafiltration The crude cellulase was ultra centrifugal filtrated 8 fold with Amcion Ultra-4 (Millipore, MW 10000, 4 ml, USA). The condensate was used for both NGGEE and zymogram analysis. Step 2 Native gradient-polyacrylamide gel electrophoresis To purify native BG, NGGEE was performed with a vertical electrophoresis system, which contained 4.0%–8.0% native gradient acrylamide separating gel and 4.0% stacking gel without SDS and β-mercaptoethanol. The glass plate of gel electrophoresis system apparatus (Model DYCZ-24A, 200 × 200 × 1.0 mm, 20 pockets) was purchased from Beijing Liuyi Instrument Factory, China. The electrophoresis conditions described by Schagger were modified as follows (Schagger et al., 1994): the voltage and current of 4% stacking

Step 6

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gel were limited to 80–100 V and 20–30 mA, respectively. For the separation gel, the voltage was limited to 180–200 V, and the current was no more than 50 mA. The electrophoresis buffer was 12.5 mM Tris-glycin 96 mM at pH 8.8. The protein sample was 0.5–5 mg ml− 1, and the sample load was 10 μl pocket− 1. The electrophoresis was run at 4 °C for 10–12 h. When the run finished, the gel was subjected to the next step as quickly as possible. Location of cellulase in gel Location of cellulase bands in the gel is the key of this method. Gel (20 lanes) after electrophoresis was cut into five parts vertically. Part 1 (lanes 1 and 2), part 3 (lanes 10 and 11) and part 5 (lanes 19 and 20) were cut out and subjected to staining with Coomassie brilliant blue R-250 and background destaining at 50 °C for 10–20 min, respectively. Part 2 (lanes 3 to 9) and part 4 (lanes 12 to 18) were stored on the glass plate at 4 °C. To locate protein bands, each part of a gel between stained and unstained was also marked by oblique cut along the vertical dotted line beforehand, because the gels became longer after staining and destaining. Then, all the gels (stained or unstained) were pieced together again, and according to the results of the zymogram analysis, native protein bands unstained in gels were cut out, respectively. Recovering native cellulase bands from gels In this case, all gels (stained or unstained) were put together according to the declining dotted line. Native protein bands in gels (part 2 and part 4) were cut horizontally according to the stained gels (part 1, part 3 and part 5) by comparing with the declining dotted lines as described in the section “Location of cellulase in gel”. Protein strips in part 2 and part 4 named P1 to P10 were cut out and collected, respectively. Electroelution and recovery of native proteins Each native cellulase strip collected from gels of part 2 and part 4 named P1 to P10 was transferred and sealed in a dialysis bag (DM-21, cutoff value of 12–14 kDa, USA) with electrophoresis buffer, respectively. Dialysis bags were dipped into the cathodal sides of the electrophoresis tank and applied to electroelution during native electrophoresis as described in the section “Native gradient-polyacrylamide gel electrophoresis”. In this case, any air bubble in the dialysis bag was removed. Electroelution should be run for 2–3 h during NGGEE till native proteins migrated out from gels to solution in the dialysis bag. After electroelution, native proteins sealed in the dialysis bag were transferred together to a tank with a magnetic stirrer and dialyzed against citric acid-sodium citrate buffer (20 mM, pH 4.8). During the dialysis, the buffer was changed with fresh ones at least third times within 4 h. After the dialysis, the gel strips were discarded, the liquid part was collected, and then the material was condensed by lyophilization. By then, the first purification cycle of cellulase was finished. Second time electrophoresis on the uniform gel After the components of native proteins named P1 to P10 were purified for the first time, the materials of native proteins were applied to electrophoretic isolation for a second time on the uniform gel. For this purpose, the separating gel was appropriately adjusted according to migrations in the first electrophoresis. Gels for P1, P2, P3 and P4 with low migrations were changed to 4%–5.0%, whereas gels for P5, P6, P7, P8 and P10 with high migration were changed to 5%–8.0%. Each native protein isolated in the first electrophoresis was isolated solely in the second electrophoresis. When the procedures were applied to the step “Electroelution and recovery of native proteins” for the second time, the isolation was finished. The purified components of cellulytic enzymes were stored at 4 °C for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), identification and characterization studies.

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2.4. Molecular mass determination and isoelectric focusing SDS-PAGE was carried out at 4 °C on 11% separating gel and 4% stacking gel to determine the purity and the molecular mass of the enzyme with the help of standard mixtures of marker proteins, as described by Laemmli (1970). The gel was stained with Coomassie brilliant blue R-250 and destained in a solution composed of 10% acetic acid and 20% absolute methanol. Isoelectric focusing was performed with 5.0% polyacrylamide gels (0.5 × 10 cm) supplemented with ampholytes (Ampholine 0.4% pH 4–6 and pH 3–10, Sigma) as described by O'Farrell (1975). 2.5. Enzyme assay The total cellulase activity was investigated based on filter paper activity (FPA). According to the Mandels method (Mandels et al., 1976), one international unit (IU) FPA is defined as the amount of enzyme required to produce 1 μmol reducing glucose min− 1. With glucose as the standard, the reducing sugar was determined with the 3,5-dinitrosalicylic acid (DNS) method of Ghose (1987). The BG activity was determined with p-nitrophenyl-β-D-glucopyranoside (p-NPG, Sigma, USA) as substrate, called pNPGase, as follows: 1 ml of 5 mM p-NPG (in 50 mM citric acid-sodium citrate buffer pH 4.8) was incubated with the enzyme solution at 50 °C for 10 min. The reaction was stopped by adding 1 ml sodium carbonate (1.0 M, pH 10); the liberated p-nitrophenol was measured at 400 nm. Then, 1 IU of enzymatic activity was determined as the amount of enzyme required to release 1 μmol of p-nitrophenol min− 1 under the assay conditions (Lin et al., 1999). 2.6. TLC assay To identify the BG function, each protein purified by NGGEE was also incubated with 20 μl of 10 mM cellobiose solution (in 50 mM citric acid-sodium citrate buffer pH 4.8) at 50 °C for 4 h. Hydrolytic products from cellobiose were also analyzed by TLC. TLC was performed with the solvent system butant-1-ol/acetic acid/water (2:1:1, v/v/v) as the developing solvent described by Sugimura (Sugimura et al., 2003). Sugars were visualized by incubating the silica gel plate (Qingdao Haiyang Chemical Co., Ltd, China) at 100 °C for 10 min after being sprayed with the solvent system (aniline 2.0 g soluble in solvent of diphenylamine 2 ml, 85% H3PO4 10 ml and acetone 100 ml). 2.7. MALDI-TOFMS analyses Samples used for MS analysis were prepared from protein bands in-gel digest. Conditions for preparation of the digest according to Olaf Jahn's protocols (Jahn et al., 2006) were modified partially as follows: About 1 mm3 sample of protein band in gel was destained with 50 μl 25 mM ammonium bicarbonate-50% acetonitrile at 50 °C for 30 min, dehydrated with 50% acetonitrile at RT for 15 min, then dried enough in rotory evaporator at 50 °C. Sample in gel was reduced with 50 μl 10 mM dithiothreitol in 50 mM fresh ammonium bicarbonate at 55 °C for 1 h and excess dithiothreitol was removed. For alkylation of the protein sulfhydryl groups, 50 μl 55 mM iodoacetamide in 50 mM fresh ammonium bicarbonate was added to the gel and incubated in the dark at RT for 45 min. After alkylation, sample was washed with ammonium bicarbonate twice, then dehydrated with 50% and 100% acetonitrile successively and dried in rotory evaporator as described previously to ensure efficient up-take of trypsin. After cooling, the gel was rehydrated on ice for 30 min with 10 μl trypsin working solution (0.1 μg μl− 1) prepared fresh from a cooled stock solution (0.1 μg μl− 1 in 15 μl 100 mM hydrochloric acid-135 μl 50 mM fresh ammonium bicarbonate). After rehydration, excess trypsin was removed and 5 μl 25 mM ammonium bicarbonate was added to gel for keeping wetness.

The enzymolysis by trypsin was allowed to proceed at 37 °C over night (16 h). The digest was stopped by acidification with 0.5% trifluoroacetic acid (TFA) and the peptides were extracted for 30 min while shaking. MALDI-TOFMS analyses were performed on an Autoflex II mass spectrometer (Bruker Daltonik GmbH, Bremen, USA) (Suckau et al., 2003) equipped with a nitrogen laser. Analyses were carried out with the matrix solutions: a-cyano-4-hydroxycinnamic acid (HCCA) saturated in acetonitrile/water (1:1, v/v) containing 0.1% TFA. HCCA matrices were used for the analysis of peptides and glycopeptides, respectively. In each case, 0.5 μl of analyte solution and 0.5 μl of matrix solution were deposited and thoroughly mixed on the target. For peptide mass fingerprinting analysis, positively charged ions in the mass-to-charge (m/z) range of 800–4000 were analyzed in the reflector mode with an acceleration of 19 kV and a delayed extraction time of 110 ns, whereas the laser energy ranged from 20% to 30% depending on the peptide and matrix. The peak of keratin was wiped off by peakErazor software. External calibration was achieved by the use of standard mixtures that were purchased from Bruker Daltonik GmbH. The spectra were processed with Autoflexanalysis software (Bruker Daltonics, USA) and Mascot search engine on the website (http://www.matrixscience.com/ cgi/search_form.pl?FORMVER=2&SEARCH=PMF). The search of Mascot peptide mass fingerprint was performed against the NCBI nonredundant database without any limitations by means of taxonomies on the website. Carboxamidomethylation of cysteine was set as fixed and oxidation of methionine was set as variable modification. The monoisotopic mass tolerance was set to 100 ppm, and one missed cleavage was allowed. The error of peptide mass values was less than 0.2 Da. 2.8. Determination of optimal pH and temperature The optimal pH of BG activity was determined by incubating the purified enzyme at 50 °C for 10 min in different buffers: citrate (100 mM, pH 3–6), phosphate (100 mM, pH 6–8). To determine the optimal temperature, the enzyme was incubated in citrate buffer (100 mM, pH 4.8) for 10 min at different temperatures: from 40 °C to 70 °C. To determine the thermostability of BG activity, the purified enzyme was incubated at different temperatures (40 °C, 50 °C, 60 °C and 70 °C) in the absence of substrate. After they were kept for certain time (0–72 h), the residual BG activity was determined as described previously. 2.9. Determination of kinetic parameters and effect of metals and reagents The values of the Michaelis constant (Km) and the maximum velocity (Vmax) were determined for the BG by incubating in 100 mM citrate buffer pH 4.8 at 50 °C with p-NPG at concentrations ranging from 0.5 to 30 mM. The values for Km and Vmax were determined from Lineweaver–Burk plots with standard linear regression techniques. Effects of various metal ions and reagents of 0.1 mM on the BG activity were determined by pre-incubating the enzyme with individual reagents in 20 mM pH 4.8 citrate buffer at 30 °C for 30 min. Then, the activities were measured at 50 °C for 10 min in the presence of metal ions or reagents. The activity assayed in the absence of metal ions or reagents was recorded as 100%. 3. Results and discussion 3.1. Selection of suitable electrophoretic condition During the process of electrophoretic isolation and purification of native proteins, the critical step is to select electrophoretic conditions including voltage, current, concentration of separating gel, concentration of electrophoresis buffer and length of separating gel. As shown in Fig. 1, in terms of the separating efficiency, the gradient gel of 5%–10% (B) was better than the uniform gel of 10% (A), the gradient

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Fig. 1. Electrophoretic condition selection of extra-cellular proteins of T. koningii AS3.2774 in inductive medium. (A) electrophoresis on the uniform separation gel (10.0%), 150 mm in length, 180–200 V, b40 mA, pH 8.8, 25 mM Tris-glycin 192 mM; (B) electrophoresis on the gradient separation gel (5.0%–10.0%), 150 mm in length, 180–200 V, b 40 mA, pH 8.8, 25 mM Tris-glycin 192 mM; (C) electrophoresis on the gradient separation gel (4.0%– 8.0%), 150 mm in length, 180–200 V, b 40 mA, pH 8.8, 25 mM Tris-glycin 192 mM; (D) electrophoresis on the gradient separation gel (4.0%–8.0%), 100 mm in length, 180–200 V, b 30 mA, pH 8.8, 12.5 mM Tris-glycin 96 mM; (E) electrophoresis on the gradient separation gel (4.0%–8.0%), 180 mm in length, 180–200 V, b 30 mA, pH 8.8, 12.5 mM Tris-glycin 96 mM.

gel of 4%–8% (C) was better than the gradient gel of 5%–10% (B); electrophoresis of lower buffer (D and E, 12.5 mM Tris-glycin 96 mM) was better than that of higher buffer (C, 25 mM Tris-glycin 192 mM); and the longer gel (E, 180 mm in length) was better than the shorter gel (D, 100 mm in length). During the electrophoresis, if the gel is run beyond the voltage limit, it would increase the risk of cracking glass plates; if electrophoresis buffer matches with conventional BN-PAGE described by Schägger or SDS-PAGE described by Laemmli, it would raise the risk of cracking gel as well. Therefore, good resolution, separation and migration of protein bands depend on voltage, gel length, gel concentration and ion concentration of the buffer. From the previous results, we found that the best condition for separating the cellulase system of T. koningii AS3.2774 was electrophoresis running for 10–12 h on the separation gel (4%–8%) of 180 mm in length, 180– 200 V, b30 mA, pH 8.8 and 12.5 mM Tris-glycin 96 mM. In particular, the best concentration of the electrophoresis buffer was only a half of that described by Schägger and Offord (Offord et al., 1991; Schägger, 1994). 3.2. Induction and repression of the cellulase system The enzyme system for converting cellulose to glucose involves at least three types of cellulases such as EG (EC 3.2.1.4), CBH (EC 3.2.1.91) and BG (EC 3.2.1.21) (Joo et al., 2009). In the NGGEE, media design for induction and repression of cellulase are very important. The metabolism of T. koningii AS3.2774 was controlled on different media according to the repression of terminal products, such as glucose. Extra-cellular proteins were classified as cellulase or BG. The FPA prepared from inductive medium and repressive medium reached 0.37 IU ml− 1 and 0.08 IU ml− 1, respectively; while BG reached 5.82 IU ml− 1and 1.84 IU ml− 1 respectively. The levels of total cellulase activity (FPA) and BG measured in the extra-cellular fraction strongly depended on the carbon source, including the straw powder, insoluble cellulose, induced total cellulase system as well as BG. When 2% glucose was added to inductive medium, this medium was changed to repressive medium because terminal product inhibition (glucose) and secretions of cellulases were repressed. Thus, it is very easy to determine the difference of extra-cellular proteins in the native gel between two media. Fig. 2 shows the results

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Fig. 2. Native gradient PAGE of extra-cellular cellulase of T. koningii AS3.2774 in inductive medium and repressive medium. Electrophoresis was carried out with a vertical electrophoresis system (actual size of gel 200 × 200 × 1.0 mm) and on 4.0%–8.0% native gradient acrylamide separating gel and 4.0% stacking gel. Lanes 1, 2, and 3, cellulase extracted from repressive medium (inductive medium + glucose 2.0%). Lanes 4, 5, and 6, cellulase extracted from inductive medium (straw powder 8.0 g, wheat bran 4.0 g and tap water 20 ml).

of extra-cellular cellulase of T. koningii AS3.2774 in inductive medium (lanes 4, 5, and 6) and repressive medium (lanes 1, 2, and 3). There were eight protein bands invisible in the native gel named as P1, P2, P3, P4, P5, P6, P7 and P10 induced by straw powder and repressed by glucose distinctly. Meanwhile, glucose had no effect on P8 and P9, so it is possible that P1, P2, P3, P4, P5, P6, P7 and P10 are the components of the cellulase system. However, their functions remained to be further explored after purification Fig. 2. 3.3. BG purification It is very difficult to purify each component of cellulase by a single method. Generally speaking, in order to purify a homogenous enzyme or protein, more than three separation methods should be used, such as (NH4)2SO4 precipitation, gel filtration chromatography, anionexchange chromatography, affinity chromatography, isoelectric focusing and blue native electrophoresis (Schägger, 1994). A BG from Fomitopsis pinicola KMJ812 was purified to be sequentially homogenous by a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography (Joo et al., 2009). A CBH I from T. reesei was also purified by gel filtration, native gel electrophoresis and fast protein liquid chromatography (Offord et al., 1991). In this study, we purified BG as described in the section “Materials and methods”, and our results indicated that the crude cellulase of T. koningii AS3.2774 on inductive medium was ultra centrifugal filtrated 8 fold with Amcion Ultra-4. Active fraction by ultrafiltration increased the specific activity for about two fold and recovered 81% of the BG activity. The active fractions were applied to the first cycle of electrophoresis and electroelution. This step resulted in a 17-fold purification of BG with a recovery rate of 10.6%. For the second cycle of electrophoresis, the separation gels were appropriately adjusted to the uniform gel according to migration in the first electrophoresis. Gels for P1, P2, P3 and P4 with low migrations were changed to 4.0%–5.0%. For example, 5.0% gel for P3, whereas gels for P5, P6, P7, P8 and P10 with high migration were changed to 5.0%– 8.0% in order to give a good resolution. This step resulted in a 24-fold BG purification with a recovery rate of 5.5%. The final yield of BG reached 8 μg under purifying procedure of NGGEE. Fig. 3 shows the purity and molecular mass of the BG from T. koningii AS3.2774 purified by main steps. Lane 1 showed that the

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Fig. 3. Purity and molecular mass determination of purified BG from T. koningii AS3.2774 by SDS-PAGE and isoelectric focusing. Lane pI, isoelectric focusing of purified P3 on 5.0% polyacrylamide gels (0.5× 10 cm) supplemented with Ampholine 0.4%, pH4–6 and pH3– 10; lane 1, P3 separated by the first cycle of “native gradient-polyacrylamide gel electrophoresis and electroelution” loaded on 11.0% SDS-PAGE; lane 2, P3 purified by the second cycle of “5% uniform polyacrylamide gel electrophoresis and electroelution” loaded on 11.0% SDS-PAGE; lane M, standard molecular mass marker proteins such as rabbit phosphoorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43.0 kDa), bovine carbonic anhydrase (31.0 kDa), trypsin inhibitor (20.1 kDa) and hen egg white lysozyme (14.4 kDa).

purity of P3 was not enough by the first electrophoresis isolation and a further separation was required. As shown in lane 2, P3 showed that a single band was pure enough after the second electrophoresis on the native uniform gel of 5.0% with a molecular mass of 69.1 kDa. Thus, the two-cycle NGGEE is a reliable method to separate many more proteins on μg grade at the same time, which is enough for further functional characterization (Fig. 3). 3.4. Functional identification To identify the function of purified proteins, the BG activity of each purified protein was determined by the methods of p-NPG and TLC as described in Materials and methods section. The enzyme assay showed that P3 had a specific BG activity of 994.6 IU mg− 1 protein, as determined by p-NPG. In this case, P3 had a strong ability to release p-nitrophenol and glucose from p-NPG. Fig. 4 shows the products hydrolyzed from cellobiose by each purified protein. In this case, cellobiose was completely hydrolyzed to glucose by P3 and the P3 was confirmed to be the BG of T. koningii AS3.2774. Fig. 5 shows MALDI-TOFMS analysis of P3 and Mascot search results from the NCBI database (the inset in Fig. 5). Protein scores higher than 83 were identified as significant (p b 0.05). Among 50 mass values we searched, 15 were matched with a sequence covered more than 30%. The inset showed that the Mascot search results of P3 obtained 134 scores for gi|201066459, a BG of Trichoderma sp (Mr: 78524; pI: 5.84). So we confirmed again that P3 was the BG from T. koningii AS3.2774 (Figs. 4 and 5). 3.5. Optimal pH and temperature The optimal pH of the BG was 5.0, with 88% and 95% of the maximum activity at pH 4.5 and 5.5, respectively, indicating that the BG had a narrow pH range of 4.5–6.0 (Fig. 6). Effect of pH on this BG appeared to be similar to that of many other BGs from F. pinicola (Joo et al., 2009) and AspergiUus niger strain NIAB280 (Rashid and Siddiqui, 1997). The optimal temperature for the hydrolysis reaction was 50 °C with 78% and 93% of the maximum activity at 45 °C and 55 °C, respectively (Fig. 7). The BG activity declined very sharply when

Fig. 4. Thin-layer chromatography for the functional identification of BG. Lanes 1 to 10, hydrolytic products from cellobiose by purified P1 to P10, respectively. Lane M, marker, G1: glucouse, G2: cellobiose, G3: cellotriose, G4: cellotetraose. TLC was performed with the solvent system butant-1-ol/acetic acid/water (2:1:1, v/v/v) as the developing solvent. Sugars were visualized by incubating the silica gel plate at 100 °C for 10 min after being sprayed with the solvent system (Aniline 2.0 g soluble in the solvent of diphenylamine 2 ml, 85% H3PO4 10 ml and acetone 100 ml).

temperature was above 60 °C. Moreover, we examined the stability of the BG in the standard buffer. The thermal stability of the purified BG exhibited a half-life of about 4.5 h at 60 °C, and the purified enzyme was rapidly inactivated over 70 °C (Figs. 6 and 7). 3.6. Effects of metal ions, pI, compounds and kinetics The BG activity was measured in the presence of metal ions or various other compounds (Table 1), and it was neither stimulated by 2+ Mg2+, Mn2+, Zn2+, Co2+, NH+ (each at 0.1 mM), nor 4 and Fe activated by EDTA at 1 mM. In contrast, Ca2+ ion increased 1.3% of the BG activity, and Cu2+ and Pb2+ significantly inhibited the BG activity (Table 1). Fig. 3 shows that the isoelectric point of the purified BG (pI) was 5.68 (lane pI), which is acidic extracellular BG of Trichoderma. Initial velocities were determined in the standard assay mixture with pH 4.8. Substrate tests showed hyperbolic saturation curves, and the corresponding double-reciprocal plots were linear. The concentration of p-NPG varied from 0 to 50 mM. Fig. 8 shows typical Michaelis–Menten type kinetics for the BG activity with the increase of p-NPG concentration. We obtained the Lineweaver–Burk plot (the inset in Fig. 8) for the conversion of p-NPG under standard assay conditions, and it showed a Km of 2.67 mM. Isolation and purification of active proteins with a high purity has been of great interest to biologists, because only purified proteins can be further studied for their structures and functions. Many conventional protein separation methods have been widely used, such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, electrophoresis and TLC (Simpson, 2004). Due to its high resolution and reliability, electrophoresis has been intensively developed to SDS-PAGE, BN-PAGE, IEF and 2D-PAGE for protein purification. Combining with transfer membrane blotting, blue native electrophoresis (BN-PAGE) has been successfully employed for preparative separation of active proteins (Offord et al., 1991; Schagger, 1995; Schagger et al., 1994; Schagger and von Jagow, 1991). BN-PAGE is a very reliable method for determining the molecular mass of proteins or protein complexes (Schagger et al., 1994). For example, the molecular mass of the ATP synthase determined by BNPAGE is consistent with the value previously reported for this enzyme (Kügler et al., 1997; Walraven and Bakels, 1996). NGGEE was

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Fig. 5. MALDI-TOFMS analyses of P3 T. koningii AS3.2774 and Mascot Search Results from Matrix science database. MALDI-TOFMS of P3 was performed with different matrix solutions: a-cyano-4-hydroxycinnamic acid (HCCA) saturated in acetonitrile/water (1:1, v/v) containing 0.1% trifluoroacetic acid (TFA). The inset showed Mascot search result: P3 reached 134 scores for gi|201066459, glucosidase of Trichoderma sp.

developed and innovated from BN-PAGE, and it is obviously different from others native electrophoresis. Firstly, similar to BN-PAGE, proteins purified by NGGEE are native and active. But this method does not need to transfer membrane to electroblotting, or incubate with Coomassie-blue, so the recovery of native proteins is inexpensive. Proteins ranging from μg to mg can be purified by NGGEE, which

Fig. 6. Effect of pH on the activity of purified BG from T. koningii AS3.2774. The enzyme activity was assayed by the standard assay method by changing the buffer to obtain the desired pH. The buffer used were citrate (pH 3.0 to 6.0), phosphate (pH 6.0 to 7.0). Each value represents the mean of triplicate measurements and the variation from the mean is no more than 10%.

is sufficient for functional determination in the enzyme analysis, Nterminal protein sequencing in the cyclic-Edman degradation, and protein identification by MALDI-TOFMS and PMF. Secondly, the NGGEE procedure is more gently than that of BN-PAGE. The concentration of electrophoresis buffer is 12.5 mM Tris-glycin

Fig. 7. Effect of temperature on the activity of purified BG from T. koningii AS3.2774. The enzyme was assayed at various temperatures by the standard assay method. Each value represents the mean of triplicate measurements and the variation from the mean is no more than 10%.

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Table 1 Effect of various chemicals on the activity of BG purified from T. koningii AS3.2774. Chemicals

Concentration (mM)

Enzyme activity (IU mg− 1 protein)

Relative activity (%)

None EDTA Ca2+ Co2+ Cu2+ Fe2+ Mg2+ Mn2+ Zn2+ NH+ 4 Pb2+

– 1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

994.6 947.8 1007.5 890.2 554.0 877.2 949.6 906.1 903.1 975.8 676.3

100 95.3 101.3 89.5 55.7 88.2 95.48 91.1 90.8 98.1 68.0

The purified enzyme was assayed in the standard assay condition with various compounds. Each value represents the mean of triplicate measurements and the variation from the mean is no more than 10%.

96 mM at pH 8.8, which is only half of that in Schägger system (Schagger et al., 1994; Schagger and von Jagow, 1991). Therefore, this method needs more time, and it often takes over three days to purify proteins. Thirdly, during NGGEE gels stained and destained at high temperature (usually at 50 °C) are necessary. High temperature may accelerate staining and destaining and save time. If gels are stained and destained at low temperature, much more time is required, resulting in diffusions of native protein in unstained gels due to protein immobile and low resolution. Forth, the declining dotted line cut between gels for locations of native proteins before staining is necessary for NGGEE, because staining and destaining make gels (especially for gradient gels) longer than those without treated ones. Thus, the locations of native proteins in the gel are very hard to identify. Finally, NGGEE may provide new insights into the role of novel proteins. Generally speaking, cellulases can be classified into EG, CBH and BG, all of which degrade cellulose to glucose together (Saha Badal and Bothast Rodney, 2009). In our study, protein P1 purified by NGGEE from T. koningii AS3.2774 seemed no activities of EG, CBH and BG. However, this protein could be induced by cellulose and repressed by glucose (Fig. 2), and it seemed to be the cellulase system of T. koningii AS3.2774. The new function of P1 deserves more investigation. Although the cellulase purified by NGGEE in this study is acidic, alkaline proteins may be purified if the buffer of electrophoresis is changed according to the method of Schagger (Schagger et al., 1994; Schagger and von Jagow, 1987).

Fig. 8. Effects of substrate concentration on the activities of BG. The BG activity of the enzyme (IU) was measured in the presence of the indicated concentrations of p-NPG at pH 4.8. The inset shows Lineweaver–Burk plot of initial velocity versus various fixed p-NPG concentrations. Each value represents the mean of triplicate measurements and the variation from the mean is no more than 10%.

4. Conclusions Our results showed that as a separation technique, NGGEE was an effective method for isolation and purification of cellulase components from T. koningii AS3.2774. Selection of electrophoretic conditions and location of native protein bands in the gel are the critical steps of this technique. The best conditions for separating the cellulase system of T. koningii AS3.2774 were electrophoresis running for 10–12 h on the separation gel (4.0%–8.0%), 180 mm in length, 180–200 V, b30 mA, pH 8.8 and 12.5 mM Tris-glycin 96 mM. Through the combination of suitable extraction, the zymogram analysis and TLC analytical method, we purified the cellulase system on μg scale and identified eight enzymatic proteins by a two-cycle NGGEE. NGGEE resulted in a 24-fold BG purification with a recovery rate of 5.5%, and a specific activity of 994.6 IU mg− 1 protein. The yield of BG reached 8 μg under purifying procedure of NGGEE. Moreover, we determined a native protein as BG by MALDI-TOFMS, which had one subunit with a molecular mass of 69.1 kDa. This purified BG had an optimal pH of 5.0, an optimal temperature of 50 °C, an isoelectric point of 5.68 and a Km for p-nitrophenyl-β-D-glucopyranoside of 2.67 mM. To identify the cellulase system, the metabolism control of T. koningii AS3.2774 including both induction and repression is the key to visualize the components of the cellulase system in gel. With the difference of extra-protein extracted from repressive medium and inductive medium, it is very convenient to detect cellulase. Thus, NGGEE is a reliable method through which μg grade of active proteins can be purified. Acknowledgements This study was supported by the National Natural Science Foundation of China (20066001) and the Youth Foundation of Hunan Agricultural University (05QN20). References Amouri, B., Gargouri, A., 2006. Characterization of a novel β-glucosidase from a Stachybotrys strain. Biochem. Eng. J. 32, 191–197. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Bronnenmeier, K., Staudenbauer, W.L., 1988. Purification and properties of an extracellular β-glucosidase from the cellulolytic thermophile Clostridium stercorarium. Appl. Microbiol. Biotechnol. 28, 380–386. Cai, Y.J., Buswell, J.A., Chang, S.T., 1998. β-Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea. Enzyme Microb. Technol. 22, 122–129. Cao, W., Crawford, D.L., 1993. Purification and some properties of β-glucosidase from the ectomycorrhizal fungus Pisolithus tinctorius strain SMF. Can. J. Microbiol. 1, 125–129. Deshpande, V., Eriksson, K.E., Pettersson, B., 1978. Production, purification and partial characterization of 1,4-β-glucosidase enzymes from Sporotrichum pulverulentum. Eur. J. Biochem. 90, 191–198. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Gong, C.S., Ladisch, M.R., Tsao, G.T., 1977. Cellobiase from Trichoderma viride: purification, properties, kinetics, and mechanism. Biotechnol. Bioeng. 19, 959–981. Hanssona, T., Adlercreutza, P., 2002. Enzymatic synthesis of hexyl glycosides from lactose at low water activity and high temperature using hyperthermostable βglycosidases. Biocatal. Biotransform. 20, 167–178. Jahn, O., Hesse, D., Reinelt, M., Kratzin, H.D., 2006. Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry. Anal. Bioanal. Chem. 386, 92–103. Joo, A.R., Jeya, M., Lee, K.M., Sim, W.I., Kim, J.S., Kim, I.W., et al., 2009. Purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola. Appl. Microbiol. Biotechnol. 83, 285–294. Kügler, M., Jänsch, L., Kruft, V., Schmitz, U.K., Braun, H.P., 1997. Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). Photosynth. Res. 53, 35–44. Kengen, S.W., Luesink, E.J., Stams, A.J., Zehnder, A.J., 1993. Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur. J. Biochem. 213, 305–312. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

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