Implications of cellobiohydrolase glycosylation for use in biomass conversion
© Jeoh et al; licensee BioMed Central Ltd. 2008
Received: 04 January 2008
Accepted: 01 May 2008
Published: 01 May 2008
The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.
It is estimated that more than a billion tons of lignocellulosic plant biomass could be utilized each year to produce liquid biofuels in North America alone . A major focus of research in the biofuels industry has been to address issues of enzyme production costs and performance. Developing low-cost enzymes remains a priority for both the Department of Energy's Office of the Biomass Program and for private industry. To achieve these goals, scientific advances in enzyme technologies, including enzyme production and performance, are required. Tactics proposed to solve the biomass conversion cost goals are diverse and include in planta enzyme expression, consolidated bioprocessing, enzymes engineered by rational design, enzymes improved by directed evolution, the development of hyper-producing fungal hosts, and many more. In each case, understanding those factors that impact the heterologous and/or hyper-expression of enzymes is critical for success.
Heterologous protein expression by fungi and yeast is complex and can be impacted by many post-translational events that take place during the secretion process. Fungal production and secretion of industrial enzymes can be highly productive and is the source for many industrial enzyme products. Yet, the fungal secretory pathway and the effects of post-translational events on protein function are poorly understood. Glycosylation is a common post-translational event that may have an effect on the properties of industrial enzymes. Glycosylation of glycosyl hydrolases, including cellulases, varies with the expression host and culture conditions. There is also evidence that different fungi used by industry, such as the Trichoderma and Aspergillus, glycosylate proteins differently. Therefore, because of the likelihood of critical impact on glycosyl hydrolase function and production, it is important to understand more about the chemical nature of the glycans decorating fungal enzymes, as well as their effect on cellulosic biomass conversion.
Glycosyl hydrolases represent a group of enzymes that hydrolyze the glycosidic bonds in carbohydrates . Cellulases, a subgroup of glycosyl hydrolases, are specific for cleaving the β(1→4) bonds of cellulose, making them key enzymes for the economic production of cellulosic ethanol from ligncellulosic materials. To achieve complete hydrolysis of structured cellulose in biomass requires a synergistic mix of enzymes that include exoglucanase, endoglucanase, and cellobiase activities. The cost of these enzymes has been identified as a key economic barrier to the deployment of liquid fuels from lignocellulosics . Long term strategies for bioethanol cost reduction include engineering noncellulolytic ethanologens to produce cellulases or engineering cellulase producers to become ethanologenic. Nearer term strategies currently rely on the over-expression of cellulase enzymes in heterologous production hosts. For both examples, consideration must be given to the co- and post-translational variations of heterologous proteins which may impact structure and function . Protein glycosylation has been demonstrated to impact the function, structural framework, and stability of proteins . A major challenge, therefore, for improvements in commercial cellulase production is to understand how protein glycosylation is impacted by expression from different hosts and specifically how glycosylation affects the activity of cellobiohydrolases. For example, glycosylation must be considered for the expression and production of cellobiohydrolases in planta, or for the development of advanced biocatalysts for consolidated bioprocessing. The significance of this problem is illustrated by considering the expression of T. reesei Cel7A in yeast. Expression of Cel7A in Pichia pastoris, for example, resulted in over-glycosylation of the enzyme with compromised activity [6, 7].
Recent reports describe the successful co-production of an endoglucanase and β-D-glucosidase in Saccharomyces cerevisiae, as well as the production of ethanol from the hydrolysis of phosphoric acid swollen cellulose . However, the expression of T. reesei Cel7A in S. cerevisiae has been less successful and generally results in the heterologous enzyme preparation having high and polydisperse molecular weights (MW) due to hyperglycosylation . The apparent MW of the heterologous protein prior to secretion (intracellular) or after enzymatic deglycosylation of the purified enzyme was more monodisperse and closer to the native .
T. reesei produces a suite of cellulolytic enzymes with distinctly different activities, including endocellulases, exocellulases, and β-glucosidases which synergistically depolymerize cellulose [10, 11]. Glycosyl hydrolases are classified into families based on amino acid sequence and folding similarities . Family 7 consists of both endoglucanases and exoglucanases and is exclusively derived from eukaryotes. Cellobiohydrolases within the family are recognized as being the most important single enzyme component for the cellulose conversion industry. Of these, Cel7A from the industrial fungus T. reesei, is the most studied. Cel7A is produced abundantly by Trichoderma, typically making up to 60% of the total secreted protein . This enzyme hydrolyzes cellobiose units from the reducing-end of a cellulose chain and is considered to be a processive enzyme . As such, considerable attention has been paid to the study of T. reesei Cel7A [14–16] with the ultimate goal of improving its performance .
Most enzymes secreted by T. reesei, including Cel7A, are glycoproteins with both O- and N-linked glycosylation sites [18–23]. Cel7A consists of a catalytic domain (CD) and a cellulose binding module (CBM) joined by a highly O-glycosylated linker peptide . The catalytic domain has four N-linked motifs, three of which are glycosylated . The type and extent of glycan on the CD is influenced by both the strain  and culture conditions . Fermentation pH is a dominant determinant for the final configuration of the glycans by influencing activities of co-secreted glycanases, such as endoglycosidase H (Endo H) , α-mannosidases [18, 24, 25], and α-glucosidases that modify the glycan structures in the medium .
Various expression systems, including E. coli, Spodoptera frugiperda (insect cells), Pichia pastoris, Saccharomyces cerevisiae, and Aspergillus niger var. awamori have been used for the heterologous expression of the cel7a gene . Successful expression of functional protein has been limited due to stringent co- and post-translational requirements, including formation of disulfide bridges and native-like glycosylation. Of these hosts, A. niger var. awamori and insect cells produce functional enzyme but show greater extents of glycosylation compared to native enzyme . The molecular mass of the recombinant Cel7A (rCel7A) was higher than the native protein and the reported data show slight differences in the activities of the two enzymes on pretreated yellow poplar . A. niger var. awamori expressed enzyme had equivalent thermal stability to T. reesei Cel7A when tested by differential scanning calorimetry (DSC) .
In this study, we examine the effects of heterologous expression of Cel7A from T. reesei on glycosylation and activity. Understanding the heterologous expression of cellobiohydrolases and cellulases in general is important in the future development of fuels from cellulosic biomass.
Materials and methods
Bacterial Cellulose (BC)
BC was produced in static cultures of Gluconacetobacter xylinus sbsp. sucrofermentans (ATCC 700178) in Hestrin Schramm medium  with 1% (v/v) ethanol . Inocula were prepared by growing frozen G. xylinus culture in 50 mL of the same medium (HS + 1% ethanol) at 26°C for 3 d under static conditions. At the end of the three days, the culture flask was shaken vigorously to dislodge the cells from the pellicle. The cells were pelleted by centrifugation and used to inoculate 75 mL media in 750 mL rectangular tissue culture flasks. Production cultures were incubated at 26°C for 5–7 d without agitation. At the end of the production period, the cells were re-pelleted and used in fresh media for growing subsequent batches of BC. The BC pellicles were washed as described by Helbert and co-workers  with the following modifications: 1) following neutralization from the alkali wash, the cellulose pellicles were incubated in a 0.3% bleach solution (in 4 mM sodium acetate buffer) for 2 h at 70°C, and 2) the pellicles were rinsed three times with distilled water to remove the bleach solution and subsequently resuspended in 5 mM sodium acetate buffer with 0.04% sodium azide and homogenized in a food processor. A final concentration of 1.9 mg/mL (standard deviation of 0.12 mg/mL) was determined from triplicate oven dry weights of 3 mL suspensions. The stock BC suspension was stored at 4°C.
Phosphoric acid swollen cellulose (PASC)
PASC was prepared from Sigmacell 50 (Sigma-Aldrich Co., St. Louis, MO). A cellulose slurry of 30 g Sigmacell in water was slowly added to 2.5 L of vigorously stirred, concentrated phosphoric acid (≥ 85 wt. % in water) at 4°C. The suspension was stirred at 4°C for 1 h before precipitating in 10–15 L of water. The precipitated cellulose was washed extensively with water until the final pH equilibrated at pH 5. The PASC was autoclaved and stored at room temperature. Final PASC concentration was 8.39 (± 0.03) mg/mL, as determined by triplicate oven-dried measurements of 3 mL suspensions.
Enzyme Expression and Purification
Trichoderma reesei Cel7A
Cel7A enzyme was purified from Spezyme CP (Genencor International). The cellulase preparation was dialyzed extensively into 10 mM Bis-Tris buffer, pH 6.0 and loaded onto a HiPrep 16/10 DEAE Sepharose FF column anion exchange column. After extensive washing with buffer, bound proteins were eluted using a linear, 0 to 1.0 M NaCl gradient in 10 mM Bis-Tris buffer, pH 6.0. The collected fractions were analyzed by SDS-PAGE, using previously purified T. reesei Cel7A as a standard to identify fractions containing Cel7A. These fractions were pooled, desalted and loaded onto a 6 mL ResourceQ anion exchange column (GE Healthcare Bio-Sciences). The unbound fraction was washed out and the bound fraction was eluted with a linear gradient of 0 to 1.0 M NaCl in 10 mM Bis-Tris buffer, pH 6.0. The presence of Cel7A in the eluted fractions was determined by assaying for activity on p-nitrophenyl β-D-lactobioside (pNPL). Fractions that were active for pNPL activity were pooled, spiked with 1 mM gluconolactone and loaded onto a column packed with p-aminophenyl β-D-cellobioside (pAPC) affinity matrix . The bound fraction was eluted with 10 mM cellobiose in 10 mM Bis-Tris buffer, pH 6.0. The eluted fractions that tested positive for pNPL activity were pooled, concentrated and run through a Superose 12 size exclusion column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) with 20 mM sodium acetate buffer, 100 mM NaCl at pH 5.0 as the running buffer. A single band corresponding to the MW of the T. reesei Cel7A standard on SDS-PAGE verified the purity of the eluted Cel7A.
Cloning and Heterologous Expression/Protein purification
rCel7A was produced in A. niger var. awamori and purified as described previously by Adney et al. .
Cel7A Catalytic Domains (CD)
Catalytic domains (CD) of Cel7A and rCel7A were obtained by proteolytic cleavage of the linker peptide with papain . A ratio of 30:1 (w/w) of papain from Carica papaya, immobilized on Eupergit®C (76221 Sigma-Aldrich Co., St. Louis, MO) to Cel7A was used in 5 mM sodium acetate buffer, pH 5.0, and incubated overnight at room temperature. The digested samples were separated by size exclusion chromatography on a Superdex 200 16/60 column (Amersham Biosciences, Piscataway, NJ) in 20 mM sodium acetate, 100 mM sodium chloride, pH 5.0 at 0.5 mL/min. Fractions containing the CDs were pooled and concentrated in a 10,000 MWCO Amicon Ultra-4 centrifugal filter unit (Millipore Corp., Billerica, MA).
N-glycosylation analysis by Mass Spectrometry
Mass spectrometry analyses were performed with a Waters Q-Tof micro quadrupole mass spectrometer (Waters/Micromass, Milford, MA) with an electrospray ionization (ESI) source. HPLC separations were performed using a Waters 2795 HPLC (Waters, Milford, MA). Both HPLC and mass spectrometer are controlled by the Masslynx service pack 4.0 software (Waters/Micromass, Milford, MA). An injection volume of 15 μL of the Cel7A papain digests were separated at 28°C on a C8 column (4.60 mm × 15 cm × 5.0 μm Agilent Zorbax Eclipse XDB-C8) at a flow rate of 100 μL/min. The optimal gradient determined for separation was 90% solvent A (deionized water with 0.2% formic acid) and 10% solvent B (acetonitrile with 0.2% formic acid), linearly changed to 15% A and 85% B after 30 min and 5% A and 95% B after 75 min. Conditions were returned to original conditions of 90% A and 10% B for an additional 10 min to recondition the column before the next injection. The autosampler chamber was held at 4°C throughout the analysis. The separated sample was flowed directly into the mass spectrometer and ionized by positive mode electrospray. The mass spectrometer was tuned with GFP (Waters, Milford, MA) and calibrated with sodium rubidium iodide (Waters, Milford, MA) to cover the mass range examined. The MS data was recorded as survey scans in the range from 400 to 3500 atomic mass units (amu) in positive ES mode. The inlet conditions were set at a capillary voltage of 3000 V, sample cone voltage of 40 V, source temperature of 100°C and a desolvation temperature of 250°C. A desolvation gas flow rate of 650 L/hr flow and a cone gas flow rate of 30 L/hr were used. The collision energy was set at 10 V, which is optimal for steering the electrons to the time-of-flight (TOF) analyzer. The MCP detector and probe positioning was adjusted for optimal sample sensitivity. Run time was set to correspond with the HPLC program that allowed adequate time to separate, collect, and analyze the samples.
Labeling cellulases with fluorescent tags
The Cel7A enzymes and the CDs were labeled with Alexa Fluor 488 fluorophore (Invitrogen) using the manufacturer recommended labeling protocol. Degrees of labeling (DOL) of 1 to 10 moles dye/mole cellulase were achieved. Fluorescence-labeled cellulases were diluted with unlabeled cellulases to final DOL in the range of 0.5 to 1 for the cellulose hydrolysis assays. The activities of the labeled enzymes on BC and PASC were checked against corresponding unlabeled enzymes, which verified that labeling did not alter the original activity .
Cellulose hydrolysis time course experiments
Reactions containing 1.0 μM Cel7A (intact, CDs, or deglycosylated) and 1.0 mg/mL BC or PASC in 0.25 mL reaction volumes were conducted at 38°C. Triplicate reactions were assayed over the course of 120 h. Each reaction was setup by preparing the appropriate dilution of cellulase in 5 mM sodium acetate buffer (pH 5.0) in 0.5 mL microcentrifuge tubes. The substrate (BC or PASC) was pre-incubated separately at 38°C for a minimum of 30 min. The reactions were initiated by addition of the pre-incubated substrate and incubated at 38°C with end-over-end rotation. At the designated times, the reactions were terminated by separating the liquid and solid phase by filtration in a manifold filtration system equipped with a 96-well 1.0 μM glass fiber filter frit (Innovative Microplate, Chicopee, MA). Reducing sugar concentration was determined by the disodium-2,2'-bicinchoninate (BCA) method  using cellobiose for the standard curve. If fluorescence-labeled enzymes were used, the concentration of Cel7A bound to the cellulose fibers was determined by fluorometry as described previously . The solid phase retained in the filter was re-suspended in 250 μL distilled water. Fluorescence intensity of 150 μL of the re-suspended solids was measured in a Tecan GENios fluorometer (Tecan Systems Inc., San Jose, CA) at fluorescence excitation/emission wavelengths of 485 and 535 nm, respectively. Cellulase concentrations on the solids fractions were determined with a set of purified Cel7A standards of known concentrations that was measured concurrently.
The rCel7A has a higher apparent molecular weight than Trichoderma expressed Cel7A when analyzed by SDS-PAGE (Figure 1). The rCel7A band also appears broader and more diffuse which is typical of protein with polydispersity of molecular weight due to glycosylation (Figure 1). The amidase N-glycosidase F (PNGaseF) specifically cleaves between N-linked glycans and the Asn residue of the glycoprotein backbone. Efficacy of PNGaseF requires a minimum N-glycan size of GlnNAc2Man3. PNGaseF treatment of Cel7A did not cause a visible shift in the protein band on SDS-PAGE, indicating a general lack of N-linked glycans larger than GlnNAc2Man3 on the native Cel7A. However, PNGaseF treatment resulted in a significant shift of the rCel7A band to a lower apparent molecular weight, demonstrating the presence of higher order N-linked glycans. The deglycosylated rCel7A band appears narrower and sharper than the parent, indicating less mass polydispersity. The mass of the PNGaseF deglycosylated rCel7A is similar to that of Cel7A, providing further evidence that the native Cel7A enzymes are most likely minimally populated at the N-linked glycosylation sites.
Mass spectrometric (MS) analysis
Accurate reconstruction of the mass profile of rCel7A CD was not possible due to the extreme heterogeneity in the masses. Instead, the intact protein was analyzed by MALDI-MS to reveal an average overall molecular weight of 61835 Da, with a 1/2 height peak width of 2400 Da. Subtracting the mass range of the glycosylated linker peptide and CBM (8.2 to 9.9 kDa) (Figure 5B) gives the mass range of the CD as 52.4 to 53.1 kDa. Further subtracting the calculated mass of the non-glycosylated CD results in 6 to 6.7 kDa as the total mass contribution of N-linked glycosylation on this CD. The mass spectrum of the rCel7A CD (Figure 5B) prominently features b-ions corresponding to N-glycan masses that fragmented at the reducing-end of the first N-acetylglucosamine (GlnNAc) unit attached to the protein backbone. Although only up to GlnNAcMan8 is highlighted in Figure 5B, the series of N-linked glycan masses was observed throughout the detection range (400 to 3500 amu), up to GlnNAcMan20. These masses were absent from the mass spectra of Cel7A CD (Figure 5A).
A summary of mass contributions of the glycans on Cel7A and rCel7A.
N-linked glycans on CD
6 – 6.7 kDa
O-linked glycans on linker peptide
8.1 – 9.7 kDa
8.2 – 9.9 kDa
61 – 63 kDa
66 – 69 kDa
Cellobiohydrolase activity of deglycosylated T. reesei Cel7A
Comparing extents of cellulose hydrolysis after 24 hours at 38°C.
1Cellulose Hydrolysis (%)
1Cellulose Hydrolysis (%)
On PASC, the ratio of hydrolysis at 24 h by rCel7A to Cel7A was 0.38 ± 0.06. The hydrolysis ratio did not improve significantly (0.53 ± 0.16) when only the CDs were reacted with PASC. Deglycosylated rCel7A, however, hydrolyzed 86% as much PASC as did the Cel7A (ratio of 0.86 ± 0.05). These results demonstrate that although rCel7A is only half as active as Cel7A on PASC, PNGaseF treatment of rCel7A restores a significant portion of the native activity. Although the data on BC is confounded by the large magnitudes of the standard deviations, there is still a significant improvement in the relative activity of rCel7A on BC due to the PNGaseF treatment.
We found that binding affinity of Cel7A on cellulose was reduced without its linker peptide and CBM at the early stages of digestion. Generally it is felt that the CBM is responsible for the adsorption of cellulases, such as Cel7A to cellulose. The observed rates and extents of hydrolysis were lower for the CD than for the intact Cel7A, suggesting the specific activity (i.e. turnover rate) of the bound enzyme was significantly reduced by the absence of the linker and CBM. It is also important to note that BC prepared as described here has a reported content of microcrystalline cellulose of about 80–85% , which means that the digestion observed here for the CDs (~5% at 120 h) does not represent conversion comparable with that found for the intact enzymes (~25% at 120 h); i.e., the CD conversion is probably due to hydrolysis of amorphous cellulose content in BC. These data support the concept that the linker peptide and CBM are essential for optimum activity of Cel7A. The Cel7A enzyme is thought to act processively from the reducing ends of cellulose (21). The linker peptide and CBM may; therefore, be important for proper processive action of the enzyme.
Heterologous expression of T. reesei Cel7A in A. niger var. awamori compromised its activity on cellulose. Deglycosylation of the rCel7A using PNGaseF demonstrates that N-linked glycan can reduce the specific activity of the enzyme. The higher N-glycan content on rCel7A appeared to be at least partially responsible for this reduction, with the potential for recovering partial native activity by enzymatic deglycosylation. The rCel7A showed higher binding on cellulose but conversion was lower than the native enzyme, suggesting that rCel7A may be more prone to non-productive binding to cellulose. In contrast to Cel7A, the affinity of rCel7A to cellulose was reduced upon elimination of the linker peptide and CBM. It seems thus that the linker peptide and CBM promotes non-productive binding of rCel7A to cellulose. One possible explanation is that the larger N-linked glycan structures on the CD of rCel7A interact with the linker peptide and/or CBM to increase the likelihood of non-productive binding. Another possibility is that the N-linked glycans of rCel7A may interact with the cellulose surface to interfere with the binding and function of the enzyme
Contrary to the activity differences observed with purified Cel7As, previous comparisons of Cel7A and rCel7A in binary saccharification assays with Acidothermus cellulolyticus E1 endoglucanase showed very similar activities on pretreated corn stover (PCS) (1). The contrasts observed with purified monocomponent digestion; therefore, potentially highlight mechanistic differences that can perhaps be overcome in a synergistic mixture, especially where the mixtures generate much higher degrees of conversion of microcrystalline cellulose (i.e., >20% conversion). The binary enzyme saccharification assays on pretreated yellow poplar (PYP); however, showed decreased activity when using rCel7A compared to the native (1), suggesting that substrate properties can also be a strong influencing factor. Synergistic cellulase activities (such as the presence of endoglucanases) may overcome some of the differences in substrate properties. For example, if increased glycosylation interferes with the processivity of rCel7A, causing the enzyme to disengage from the substrate more frequently, and then having sufficient endoglucanase-created reducing-ends could mitigate the effects of this handicap.
In this study, we demonstrated that fungal heterologous expression of T. reesei Cel7A could affect the final N-linked glycosylation of the protein and result in compromised activity on cellulose. If it is necessary to utilize a non-native expression host for T. reesei cellulases, in vitro processing of the protein glycosylation may be required to maximize the activity of these enzymes.
The authors would like to thank Eric Knoshaug for helpful discussions on A. niger var. awamori fermentation conditions as well as constructive criticisms of the manuscript. The MALDI analyses were conducted by the Biomolecular Research Facility at the University of Virginia. This work was funded by the Department of Energy Office of the Biomass Program.
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