Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris
© Kirikyali et al.; licensee Springer 2014
Received: 5 July 2014
Accepted: 7 August 2014
Published: 31 August 2014
β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min−1 mg−1 respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.
KeywordsAspergillus oryzae Xylose β-xylosidase Enzyme kinetics Protein expression
Xylanolytic and cellulolytic enzymes encoded by filamentous fungi have been employed in several industrial applications for improving digestibility in animal feed, production of sweeteners, pharmaceuticals, additive chemicals for biofuel production and for the replacement of hazardous chemicals in textile and paper manufacture (Michelin et al. ). As a consequence it has been a necessity for enzymes involved in cellulose and hemicellulose hydrolysis to be individually identified and characterised in order to utilise them in the process of converting waste agricultural materials into valuable products with greater efficiency.
Hemicellulose is comprised of a linear main chain β-1,4 linked D-xylose backbone with short lateral side chains of different lower molecular weight sugar residues (Dyk and Pletschke ). Enzymatic hydrolysis of hemicellulose commences with the removal of side chains that block the sites where xylanases cleave the xylan backbone. Endo-1,4-β-xylanase enzymes cleave the glycosidic bonds in a selective manner depending on the chain length, degree of branching of substrate molecules and the presence of alternative carbohydrate moieties (Polizeli et al. ). Cleavage of the xylan backbone yields xylooligosaccharides and the final trimming is carried out by β-xylosidase, whereby short chain oligosaccharides and xylobiose are hydrolysed from the non-reducing termini to release xylose monomers (Polizeli et al. ; Teng et al. ).
Among xylanolytic enzymes, endo-xylanases and β-xylosidases have attracted attention as they commence and complete the breakdown of hemicellulose fraction respectively (Kulkarni et al. ). For comprehensive hydrolysis β-xylosidases play an important role in the removal of xylooligosaccharides from the catalytic environment, which assists by the elimination of the end-product inhibitors of endo-xylanases.
The gene encoding for XylA was previously identified by Kitamoto et al. () and was reported to be responsible for the rapid browning of soy sauce. In addition Kitamoto et al. () were interested in the antisense inhibition of XylA expression in order to hinder the translation in Aspergillus oryzae KBN616 to produce a mutant strain that could be used in Japanese food industry. However, XylA is a potentially efficient candidate for the facilitation of hydrolysis of hemicellulose applications in industrial processes. The work presented here reports the expression of a β-xylosidase from Aspergillus oryzae in Pichia pastoris and the kinetic characterisation of the recombinant enzyme.
Materials and methods
Construction of expression vector
The β-xylosidase encoding gene (xylA) was kindly provided by Noriyuki Kitamoto (Aichi Industrial Technology Institute, Japan). The gene sequence appears in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB013851. The gene was sub-cloned into pCR®2.1 and subsequently into the Pichia pastoris expression vector pPpHis4_GAP_BglII (TU Graz) under control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter.
Transformation and expression of recombinant β-xylosidase in Pichia pastoris
Plasmid DNA (10 μg) containing the xylA gene sequence was linearized with Bgl II for integration at the GAP locus and was transformed into P. pastoris GS115 using Invitrogen Pichia Easycomp kit (as per manufacturers’ guidelines). Positive transformants, displaying the His+ phenotype on Regeneration Dextrose medium (RD) agar plates (1 M sorbitol, 2% dextrose, 1.34% yeast nitrogen base, 4×10−5% biotin, 0.005% amino acids (L-glutamic acid, L-methionine, L-lysine, L-leucine, L-isoleucine) and 1.5% agar), before transfer onto RD plates containing 50 μg ml−1 bromo-4-chloro-3-indolyl β-D-xylopyranoside (X-xyl) (Sigma-Aldrich, UK) and incubated at 30°C for 2 days. Functional expression of the β-xylosidase under the control of GAP promoter was tested by cleavage of xylopyranoside from the synthetic indicator X-xyl.
A single colony displaying the highest level of blue precipitate was sub-cultured from the RD plate onto a YPD plate (1% yeast extract, 2% peptone, 2% glucose, 2% agar) and incubated at 30°C for 3 days. A seed culture was raised using a single colony inoculated into 50 ml YPD liquid medium, incubated at 28°C overnight. One hundred millilitres of YPD broth was then inoculated to 1 OD600nm and incubated for 72 h at 28°C. One ml of culture was removed every 24 h to test expression levels using the synthetic 4-nitrophenyl β-xylopyranoside (PNPX) as described in section 2.4. Following incubation the cells were harvested by centrifugation at 4000 × g for 10 min.
Purification of recombinant β-xylosidase
Following centrifugation the culture supernatant was concentrated using a Sartorius Sartocon Slice then diafiltered with 10 volumes of Tris-salt buffer (10 mM Tris, 50 mM NaCl, pH 7.5). The concentrate was then stabilised using 30% (w/v) sucrose based on protein concentration and frozen for long term storage at −20°C. Prior to enzymatic assays the 30% (w/v) sucrose was removed from the recombinant enzyme concentrates using a Vivaspin concentrator (GE Healthcare, UK) with a 10 kDa molecular weight cut off membrane filter and the filtrate was washed with Tris-salt buffer (10 mM Tris, 50 mM NaCl, pH 7.5).
Enzyme assays using synthetic substrates
Assays for β-xylosidase activity were performed by measuring the pNP released from p-nitrophenyl glycoside synthetic substrates 4-nitrophenyl-β-D-xylopyranoside (PNPX), 4-nitrophenyl-β-D-glucopyranoside (PNPG) and 4-nitrophenyl-α-L-arabinofuranoside (PNPAf) in a final volume of 4 ml for 20 min in 50 mM sodium phosphate buffer pH 6.0 at 50°C. Reactions were terminated by the addition of 1 M Na2CO3 and the amount of released pNP was measured at OD410nm. One unit (U) of β-xylosidase activity is defined as the amount of enzyme required to release 1 μmol of pNP per min under assay conditions. Kinetic parameters (Km and Vmax) were determined by the measurement of activity against pNPX using different substrate concentrations (0.5 - 12 mM) using the standard assay procedure. Enzyme assays were performed in triplicate and are presented as mean values with standard error.
Enzyme assays using xylooligosaccharides
Activities against xylobiose, xylotriose and xylotetraose were determined at varying substrate concentrations (0.25 – 4 mg ml−1) in a final volume of 1 ml for 10 min in 50 mM sodium phosphate buffer pH 6.0 at 50°C. All assays were carried out in triplicate and were terminated by the addition of 1 M Na2CO3. Reaction products were separated according to molecular size by HPLC (Dionex ICS-3000 SP) with CarboPacTM PA20 column (3 × 150 mm) and a gradient of 10 – 50 mM sodium hydroxide was applied for 20 min at a flow rate of 1 ml min−1. The products were quantified on the basis of standard peak areas from various concentrations of control xylose, xylobiose, xylotriose and xylotetraose solutions. Enzyme assays were performed in triplicate and are presented as mean values with standard deviations.
To investigate the effect of end product xylose on catalytic activity, reactions were carried out in the presence of various xylose concentrations from 1 mM to 80 mM using synthetic substrate concentrations of either 1 mM or 4 mM pNPX. To further confirm the type of inhibition, kinetic constants were determined from experiments carried out using fixed inhibitor concentrations of 5 mM xylose at varying substrate concentrations from 0.25 mM to 8 mM under standard assay conditions.
The effects of monosaccharide sugars (20 mM glucose, mannose, galactose, arabinose, fructose and xylose), metal ions and chemicals (10 – 20 mM LiCl, KCl, ZnCl2, SDS, EDTA and DTT) on enzyme activity were tested using 50 mM sodium phosphate buffer pH 6, 1 mM pNPX and 2 μg of enzyme at 50°C for 10 min in a final volume of 4 ml and measured optical density of released p-nitrophenyl at OD410nm.
Determination of protein concentration
Protein concentrations were determined by the standard assay procedure using Pierce Coomassie® Plus Protein Assay Reagent. Sample diluents were used as the blank and the absorbance measured at OD595nm. All assays were performed in triplicate and the OD595nm readings of unknown sample were compared against BSA protein standard series which covered the range of concentrations between 50 and 1500 μg ml−1.
Determination of molecular mass by SDS-PAGE
SDS-PAGE was performed using 8% polyacrylamide gels according to the method described by Laemmli (). Protein bands were stained with colloidal Coomassie Blue. Bands from SDS-PAGE were excised and were subjected to trypsin digestion prior to mass spectrometry analysis.
Analyses of samples were carried out by LC-ESI-tandem MS on a Q-TOFII mass spectrometer fitted with a nanoflow ESI (electrospray ionization) source (Waters Ltd, UK). Peptides were separated on a PepMap C18 reverse phase, 75 μmi.d., 15-cm column (LC Packings) and delivered on-line to the MS via a CapLC HPLC system. Sequence interpretation for individual peptides was performed using the PepSeq MASCOT tool of the MassLynx™ 4.0 software package (Waters).
Characterisation of recombinant β-xylosidase
Determination of optimal conditions
The optimum pH of enzymatic activity was assayed in phosphate buffer system of varying pH values from 2–9 in the presence of 2 mM PNPX. The enzyme displayed activity within a narrow pH range, with an optimum of pH 4.5 and at least 65% activity from pH 3 – 6; less than 5% activity was observed at pH 7 – 9 (Figure 2B).
Substrate specificity and kinetic analysis
The substrate specificity of the recombinant enzyme was determined using various 4-nitrophenyl glycoside synthetic substrates and xylooligosaccharides. Recombinant XylA hydrolysed 4-nitrophenyl β-xylopyranoside efficiently but had trace hydrolytic activities against 4-nitrophenyl-β-D-glucopyranoside or 4-nitrophenyl-α-L-arabinofuranoside. Specific activities were determined as 150, 2 and 0.9 U mg−1 for PNPX, PNPAf, PNPG respectively.
Kinetic analysis of synthetic and natural substrates determined in 50 mM sodium phosphate buffer (pH 6.0) at 50°C
Vmax(μmol min−1 mg−1)
kcat/Km (mM−1 s−1)
1 ± 0.3
250 ± 0.001
pNPX + 20 mM Xylose
2.9 ± 0.5
250.5 ± 23
pNPX + 20 mM Fructose
0.1 ± 0.06
14.5 ± 3
2.6 ± 0.3
25.5 ± 0.1
3.07 ± 0.3
21.3 ± 0.3
0.62 ± 0.4
14.5 ± 0.003
The degradation of various xylooligosaccharides (X2, X3 and X4) by recombinant XylA was analysed by HPLC. Xylose was released from all substrates and the rate of xylose released decreased with increasing chain length of the xylooligosaccharide. Table 1 shows reductions in the catalytic conversion parameters Vmax and kcat with respect to the increasing chain length of the xylooligosaccharides. The relative affinity of XylA towards the natural substrate xylotetraose (X4) was significantly greater than xylobiose (X2) or xylotriose (X3) with respect to the Km values.
Effect of carbohydrates on catalytic activity
Effect of metal ions and chemical compounds on enzyme activity
The effects of various metal ions and reagents on β-xylosidase activity were assayed at 10 mM and 20 mM concentrations (Figure 3B). Mosrt notably the addition of Zn2+ (10 mM) enhanced enzyme activity by 80%. The detergent SDS at 20 mM reduced the catalytic activity by 40%.
A β-xylosidase encoding gene (xylA) from Aspergillus oryzae KBN616 was expressed in a soluble, active form under control of the constitutive GAP promoter in Pichia pastoris. The predicted presence of a native signal sequence (SignalP) was confirmed through secretion of the mature protein by the expression host. XylA was predicted to have a molecular mass of 86.4 kDa and 12 potential N-linked glycosylation sites. Kitamoto et al. () previously identified purified the native enzyme from A. oryzae culture supernatant, in which the enzyme produced a single protein band with an apparent molecular mass of 110 kDa on SDS-PAGE. However the molecular mass range of XylA determined by SDS-PAGE from P. pastoris was 153 to 165 kDa indicating differences in post-translational modification consistent with the predicted glycosylation sites. Heterologous proteins expressed in P. pastoris are subject to glycosylation and several plant cell wall degrading enzymes expressed in P. pastoris are reported to be hyper-glycosylated, including β-xylosidase from Paecilomyces thermophila (Juturu and Wu ), cellobiose dehydrogenase from Neurospora crassa (Zhang et al. ) and endo-xylanase from Actinomadura sp. S14 (Sriyapai et al. ).
The biochemical properties of the recombinant β-xylosidase closely match the native enzyme with respect to the observed optimal pH range (pH 4.5 – 5) and temperature (55°C) for enzyme activity. These are comparable to other fungal xylosidases (Saha ; La Grange et al. ; Wakiyama et al. ; Zanoelo et al. ; Rasmussen et al. ), which exhibit optimal activities between pH 3–5 at 60°C.
The recombinant enzyme was most active against p-nitrophenyl-β-D-xylopyranoside (PNPX), with minimal activities towards 4-nitrophenyl-β-D-glucopyranoside (PNPG) and 4-nitrophenyl-α-L-arabinofuranoside (PNPAf). Activity towards a broad range of synthetic substrates by other fungal β-xylosidases has been reported, although maximum activity is generally towards PNPX (Margolles-Clark et al. ; Ohta et al. ; Wakiyama et al. ; Katapodis et al. ). The exception to this is Aspergillus awamori X-100 β-xylosidase, which is reported to exhibit a greater kcat against PNPAf (Eneyskaya et al. ). The recombinant enzyme exhibited kinetic constants for the hydrolysis of PNPX of 1.0 mM and 353 μmol min−1 mg−1 for Km and Vmax respectively. A range of kinetic constants have been reported for the hydrolysis of PNPX by fungal β-xylosidases but these values are similar to those reported for purified β-xylosidases from Sporotrichum thermophile (Katapodis et al. ) and Fusarium proliferatum (Saha ). The hydrolysis of various xylooligosaccharides (X2, X3 and X4) was monitored by HPLC. In the presence of individual xylooligosaccharides, xylose was detected as an initial product of catalysis indicating that the recombinant β-xylosidase is an exo-cutting enzyme. The kcat values for the xylooligosaccharides decrease with increasing chain length in the order of X2 (36.0 sec−1) > X3 (30.1 sec−1) > X4 (20.5 sec−1). However, the reduction in the Km value for xylotetraose (X4) results in a greater catalytic efficiency towards this substrate. The observed changes in the kinetic constants (Km and kcat) with respect to xylooligosaccharide chain length show similar patterns to those reported for the β-xylosidases originating from Talaromyces emersonii, Trichoderma reesei and Aspergillus nidulans (Rasmussen et al. ; Dilokpimol et al. ). In contrast Neurospora crassa β-xylosidase is reported to show a reduction in catalytic efficiency towards xylotetraose compared to shorter chain xylooligosaccharides (Kirikyali and Connerton, ). This is largely due to a relative reduction in the affinity of the N. crassa enzyme towards the xylotetraose substrate (>Km value).
Similar to the data reported for the β-xylosidases from Penicillium sclerotiorum (Knob and Carmona ), Talaromyces thermophilus (Guerfali et al. ) and Paecilomyces thermophila (Yan et al. ) the presence of 20 mM concentrations of the metal ions Li+, K+ or Zn2+ had no effect on enzyme activity. The β-xylosidase of Talaromyces thermophilus has been reported to retain 44% activity in the presence of 10 mM of the detergent SDS (Guerfali et al. ), and similarly XylA retained catalytic activities of 75% and 60% respectively at 10 and 20 mM SDS.
Xylose has been determined to be a competitive inhibitor of recombinant A. oryzae β-xylosidase. In the presence of 20 mM xylose with varying substrate concentrations the Km was altered with no corresponding effect on Vmax. This is consistent with competitive inhibition in which the inhibitor interferes with the catalytic properties of enzyme by affecting substrate binding affinity by conferring a Ki of 2.7 mM. In this respect the recombinant enzyme displays similar characteristic to the β-xylosidases from A. niger (Ki 2.9 mM) (Gomez et al. ) and T. Reesei (Ki 2.4 mM) (Rasmussen et al. ). However, xylose tolerant β-xylosidases have been reported to exist with Ki values up to 200 mM (Yan et al. ; Zanoelo et al. ). Fructose has the novel characteristics of an uncompetitive inhibitor in which the inhibitor interacts with the enzyme-substrate complex to prevent product formation. In this case the binding of xylooligosaccharide to the active site of β-xylosidase creates a binding site for fructose. High substrate concentrations of substrate will increase the occupancy of the active site and the binding sites for fructose, and therefore the effective inhibition. The functional and physiological consequences of this finding should be considered when the enzyme has to function in the presence of mixed substrates.
This work has been supported by project funds from BBSRC and Biocatalysts Ltd. The authors would like to thank Noriyuki Kitamoto (Aichi Industrial Technology Institute, Japan) for kindly providing the XylA gene. We also would like to thank our technicians, Lorraine Gillet, Nicola Cummings for their advice and David Coles for his assistance with HPLC and Dr Susan Liddell for her assistance with Mass Spectrometry.
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