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Enzyme and Microbial Technology 57 (2014) 63–68

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

j o ur na l ho mepage: www.elsev ier .com/ locate /emt

eterologous expression and kinetic characterisation of Neurosporarassa �-xylosidase in Pichia pastoris

. Kirikyali, I.F. Connerton ∗

ivision of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK

r t i c l e i n f o

rticle history:eceived 16 July 2013eceived in revised form 30 January 2014ccepted 5 February 2014vailable online 14 February 2014

a b s t r a c t

To degrade plant hemicelluloses fungi employ �-xylosidases to hydrolyse xylooligosaccharides, releasedby endo-xylanases, into xylose. We have expressed the �-xylosidase from Neurospora crassa in Pichiapastoris under the control of alcohol oxidase 1 (AOX1) promoter. The recombinant enzyme is optimallyactive at 50 ◦C and pH 5.0 with Km and Vmax values of 8.9 mM and 1052 �mol min−1 mg−1 respectivelyagainst 4-nitrophenyl �-xylopyranoside. Xylose is a non-competitive inhibitor with a Ki of 1.72 mM. The

eywords:eurospora crassaylose-Xylosidasenzyme kinetics

enzyme is characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of �-1,4 linked xylooligosaccharides (X2, X3 and X4) but also capable of transxylosilation. Catalytic conversionof X2, X3 and X4 decreases (Vmax and kcat) with increasing chain length.

© 2014 Elsevier Inc. All rights reserved.

rotein expression

. Introduction

Biological processing of plant biomass has become a significantoncern in the quest for renewable energy. A pre-requisite is thefficient extraction and hydrolysis of cellulose and hemicelluloseractions of agricultural lignocellulosic biomasses. Robust meth-ds such as treatments with concentrated acids, alkali, hydrogeneroxide, steam explosion, hot water treatment, CO2 explosionnd organic solvent treatments have been employed to accom-lish this [1]. However the use of harsh chemicals for hydrolysis canause problems during post-treatment processes, for example, sol-bilised sugars can become contaminated with chemical residueshat limit their subsequent use. Enzymes remain an attractive alter-ative for the clean degradation of plant cell materials. However, tofficiently bioconvert plant biomass from various sources requireshat customisable multi-functional enzyme cocktails are availableor commercial use.

Lignocellulosic materials are composed of cellulose, hemi-ellulose, pectin, proteins and an aromatic polymer lignin. Theemicellulose fraction of lignocellulosic residues has attractedttention due to its abundance in nature and its heteropolymeric

tructure. Unlike cellulose, hemicellulose comprises of linear mainhain of �-1,4 linked d-xylose residues with short lateral sidehains of different sugar residues [2,3]. However, in order to make

∗ Corresponding author. Tel.: +44 115 95 16 161; fax: +44 115 95 16 162.E-mail address: ian.connerton@nottingham.ac.uk (I.F. Connerton).

ttp://dx.doi.org/10.1016/j.enzmictec.2014.02.002141-0229/© 2014 Elsevier Inc. All rights reserved.

use of lignocellulosic material as a substrate requires the synergisticaction of a diverse range of enzymes for complete depolymerisationinto free monomeric sugars [3,4].

Enzymatic hydrolysis of hemicellulose commences with theremoval of side chains that block the sites where xylanases cleavethe xylan backbone. Endo-1,4-�-xylanase enzymes do not cleavethe xylan backbone randomly but cleave the glycosidic bonds in aselective manner depending on the chain length, degree of branch-ing and the presence of alternative carbohydrate moieties [5].Cleavage of the xylan backbone yields xylo-oligosaccharides thatare substrates for �-xylosidase, which hydrolyses the short chainoligosaccharides and xylobiose from their non-reducing termini torelease xylose monomers [5,6].

Research into bioconversion has evolved into multidisciplinaryactivities that include studies to enhance enzyme utility with par-allel genetic improvements of agricultural feedstocks. In this studywe report the expression of a novel �-xylosidase from Neurosporacrassa, in Pichia pastoris and kinetic characterisation of recombinantenzyme for industrial applications intended for the degradation ofhemicellulose fraction of plant cell wall materials into monosac-charide constituents.

2. Materials and methods

2.1. Strains, culture medium and growth conditions

Wild type N. crassa strain ST A (74 A) was maintained on Vogel’s agar with2% (w/v) sucrose as carbon source. Conidia were inoculated into 100 ml singlestrength liquid Vogel’s medium containing sucrose (20 g/L) using sterile technique

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4 N. Kirikyali, I.F. Connerton / Enzyme a

nd incubated at 30 ◦C in a shaking incubator at 220 rpm for 4 days. Mycelia wereecovered by vacuum filtering and stored at −20 ◦C prior to genomic DNA extraction.

Escherichia coli TOP10 were used as bacterial host for DNA manipulations withhe pCR®2.1-TOPO® cloning vector provided within the TOPO TA Cloning® kit fromnvitrogen. Transformants were selected on LB plates (10 g/L tryptone, 5 g/L NaCl,

g/L yeast extract, 15 g/L agar, pH 7.5) containing 50 �g/ml kanamycin (Invitrogen,SA) overnight at 37 ◦C.

P. pastoris strain GS115 (his4) was used as a host to produce recombinantnzymes, which was transformed with the expression vector pPIC3.5K (Invitro-en). P. pastoris GS115 cultures were grown in BMGY (1% yeast extract, 2% peptone,00 mM potassium phosphate pH 6.0, 1.34% yeast nitrogen base, 4 × 10−5% biotinnd 1% glycerol) liquid cultures at 30 ◦C in a shaking incubator at 220 rpm prioro transformation and expression of recombinant proteins. Following transforma-ion cells were recovered on Regeneration Dextrose medium (RD) agar plates (1 Morbitol, 2% dextrose, 1.34% yeast nitrogen base, 4 × 10−5% biotin, 0.005% aminocids (l-glutamic acid, l-methionine, l-lysine, l-leucine, l-isoleucine), 0.004% his-idine and 1.5% agar). Methanol utilisation phenotypes (His+Mut+ and His+MutS)f transformants were determined on Minimal Methanol (MM) (1.34% yeast nitro-en base, 4 × 10−5% biotin, 0.5% methanol and 1.5% agar) plates. P. pastoris GS115ransformants were subcultured on Minimal Dextrose (MD) (1.34% yeast nitrogenase, 4 × 10−5% biotin, 2% dextrose, 1.5% agar) plates incubated at 30 ◦C. Recombi-ant enzyme expression was carried out in Buffered Methanol complex (BMMY)1% yeast extract, 2% peptone, 100 mM potassium phosphate pH 6.0, 1.34% yeastitrogen base, 4 × 10−5% biotin and 0.5% methanol) liquid medium at 30 ◦C.

.2. Isolation of genomic DNA and cloning of NCU09923.3

The flash frozen mycelial mats were crushed using a mortar and pes-le then 250 �l of the liquefied pulp transferred into microcentrifuge tubes,nd genomic DNA extracted using the ethanolic perchlorate method oftevens and Metzenberg [7]. Specific primers were designed based on the′ and 3′ terminal nucleotide sequences of NCU09923.3 as reported byhe Broad Institute (www.broadinstitute.org/annotation/genome/neurospora). Theorward primer 5′-CATATGAAGTCGTCTTGGGCTTC-3′ and reverse primer 5′-ATAAGCTTTTACACCACCACTTGACCAC-3′ were used for PCR amplification. Themplification was performed in a final volume of 50 �l using a BioRad C1000TM Ther-al Cycler (BioRad, USA). The PCR master mix comprised of 1× PCR buffer (10 mM

ris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin), 10 mM dNTPs, 50 ng ofemplate DNA, 100 pmol/�l of each primer and 2.5 units of Taq DNA polymeraseSigma–Aldrich, UK). Amplification parameters were 96 ◦C for 3 min; 35 cycles eachonsisting of 94 ◦C for 30 s, 62 ◦C for 45 s, 72 ◦C for 150 s, followed by a final extensionf 72 ◦C for 5 min. PCR products were separated by 1% (w/v) agarose gel electrophore-is with 10 mg ml−1 ethidium bromide and visualised using a UV transilluminatorBioRad, USA). A 2.3 kbp PCR product was excised and purified from 1% (w/v) agaroseel using Wizard SV Gel and PCR Clean-Up system (Promega), cloned into pCR®2.1-OPO® vector (Invitrogen, USA) and transformed into TOP10 E. coli following theanufacturers’ protocols. Plasmids were then purified using Qiaprep Spin Miniprep

it (Qiagen).

.3. Construction of expression vector

The translation of NCU09923.3 was predicted to have a secretion signal usinghe SignalP 4.0 server. The entire coding region was excised EcoRI fragment fromCR®2.1-TOPO® vector and ligated into the EcoRI site within the multiple cloning sitef the P. pastoris vector pPIC3.5K downstream to alcohol oxidase 1 (AOX1) promoter.he plasmid was recovered in TOP10 E. coli and the gene orientation confirmed byNA sequencing.

.4. Transformation and expression of recombinant ˇ-xylosidase in P. pastoris

Prior to transformation, the recombinant plasmid pPIC3.5K-NCxyl and the par-nt expression vector pPIC3.5K (as a control) were linearised with BglII enzyme inrder to target recombination events at AOX1 locus of P. pastoris. P. pastoris GS115ompetent cells were prepared following the procedures described for spheroplas-ing in the Pichia Expression Kit manual (Invitrogen). For transformation, 8–10 �gf linearised vector was mixed with 100 �l of the prepared spheroplasts and trans-ormants were recovered on Regeneration Dextrose (RD) plates at 30 ◦C for 5 days.ositive transformants, displaying the His+ phenotype on RD plates were transferredn Minimal Methanol (MM) plates to test their methanol utilisation phenotype.olonies from MM plates were transferred onto MM plates containing 50 �g/ml-bromo-4-chloro-3-indolyl �-d-xylopyranoside (X-xyl) (Sigma–Aldrich, UK) and

ncubated at 30 ◦C for 2 days. Functional expression of the �-xylosidase under theontrol of alcohol oxidase promoter was tested by cleavage of xylopyranoside fromhe synthetic indicator X-xyl (Sigma–Aldrich, UK).

A single colony, exhibiting the highest level of expression during small scale

xpression and most abundant blue precipitate on MM plates spread with X-xylas inoculated from corresponding MD plate into 20 ml of BMGY liquid medium and

rown overnight at 30 ◦C in a shaking incubator at 220 rpm. From the overnight cul-ure 2 ml aliquots were inoculated into 6 individual 1 L fresh BMGY liquid medium,n 2 L Erlenmeyer flasks, and incubated until the OD600 nm reached 18–20 at 30 ◦C in a

icrobial Technology 57 (2014) 63–68

shaking incubator at 220 rpm. Cells were harvested by centrifugation at 2500 × g for10 min at room temperature and the pelleted cells resuspended in 50 ml of BMMYbefore transfer into 3 L BMMY in Erlenmeyer flasks. The cultures were incubated at30 ◦C for 5 days at 220 rpm and 100% methanol was added to a final concentrationof 0.5% every 24 h to maintain induction. For each day 1 ml of culture was removedto test expression levels using synthetic substrate 4-nitrophenyl �-xylopyranoside(PNPX). Following incubation the cells were harvested by centrifugation at 4000 × gfor 10 min and the culture supernatants were subjected to ammonium sulphateprecipitation.

2.5. Purification of recombinant ˇ-xylosidase

Culture supernatant was recovered by centrifugation at 4000 × g for 10 min.Ninety percentage (w/v) of solid ammonium sulphate was added to the super-natant and allowed to dissolve overnight at 4 ◦C. The precipitated proteins werecollected by centrifugation at 20,000 × g for 40 min at 4 ◦C. The recovered pelletswere dissolved in 120 ml of Tris–salt buffer (10 mM Tris–HCl, 50 mM NaCl, pH 6.0)and stored at 4 ◦C. Aliquots of 5 ml were loaded onto HiPrepTM 26/60 SephacrylTM

S-200 gel filtration column (2.6 cm × 10 cm) (GE Healthcare, UK) associated with anAKTA FPLC system with elution at a flow rate of 1 ml min−1 with Tris–salt buffer(10 mM Tris–HCl, 50 mM NaCl, pH 6.0). Fractions were collected and assayed foractivity against 4-nitrophenyl �-xylopyranoside (PNPX) using a spectrophotometricmethod at OD410 nm. Active fractions were pooled and desalted through a PD 10Column (Amersham Pharmacia Biotech, UK). The desalted recombinant enzymesolution was concentrated by Vivaspin concentrator (GE Healthcare, UK) with a3 kDa molecular weight cut off membrane filter and stored at 4 ◦C until furtheranalysis.

2.6. Enzyme assays using synthetic substrates

Assays for �-xylosidase activity was performed by measuring the p-nitrophenyl(pNP) released from p-nitrophenyl glycoside synthetic substrates 4-nitrophenyl-�-d-xylopyranoside (PNPX), 4-nitrophenyl-�-d-glucopyranoside (PNPG) and 4-nitrophenyl-�-l-arabinofuranoside (PNPAf) in a final volume of 4 ml for 20 min in50 mM sodium phosphate buffer pH 6.0 at 50 ◦C. All enzyme assays were carriedout in triplicate and the data presented are mean values with standard deviation ofthree independent experiments. Reactions were terminated by the addition of 1 MNa2CO3 and the amount of released pNP was measured at 410 nm. One unit (U) of�-xylosidase activity is defined as the amount of enzyme required to release 1 �molof pNP per minute under assay conditions. Kinetic parameters (Km and Vmax) weredetermined by the measurement of activity against pNPX using different substrateconcentrations (0.5–15 mM) by the standard assay procedure.

2.7. Enzyme assays using xylooligosaccharides

Activities against xylobiose, xylotriose and xylotetraose were performed at vary-ing substrate concentrations (0.25–4 mg ml−1) in a final volume of 1 ml for 10 minin 50 mM sodium phosphate buffer pH 6.0 at 50 ◦C. All assays were carried out intriplicate and were terminated by the addition of 1 M Na2CO3. Reaction productswere separated according to molecular size by Dionex ICS-3000 SP High PressureLiquid Chromatography (HPLC) using a CarboPacTM PA20 column (3 mm × 150 mm)and a gradient of 10–50 mM sodium hydroxide applied for 20 min at a flow rate of1 ml min−1. The products were quantified on the basis of peak areas against standardconcentrations of xylose, xylobiose, xylotriose and xylotetraose. Triplicate data arepresented as the mean and standard deviation of three independent experiments.

2.8. Inhibition studies

To investigate the effect of end product xylose on catalytic activity, reactionswere carried out in the presence of various xylose concentrations from 1 mM to80 mM using synthetic substrate concentrations of either 1 mM or 4 mM pNPXfor the construction of Dixon plots. Kinetic constants were determined usingLineweaver–Burk plots from experiments carried out using a fixed inhibitor concen-tration of 5 mM xylose at varying pNPX concentrations (0.5–8 mM) under standardassay conditions as described in Section 2.6.

The effects of monosaccharide sugars (1 mM glucose, mannose, galactose, arab-inose, fructose and xylose), xylitol, metal ions and chemicals (1–20 mM LiCl, KCl,ZnCl2, SDS, EDTA and DTT) on enzyme activity were tested using 50 mM sodiumphosphate buffer pH 6, 1 mM pNPX and 2.4 �g of enzyme at 50 ◦C for 10 min in afinal volume of 2 ml and the release of p-nitrophenyl measured at 410 nm.

2.9. Determination of protein concentration

Protein concentrations of samples were determined by the standard assay pro-cedure using Pierce Coomassie® Plus Protein Assay Reagent. Sample diluents wereused as the blank and measured the absorbance at 595 nm. All assays were quantifiedusing BSA protein standards (50 and 1500 �g/ml).

N. Kirikyali, I.F. Connerton / Enzyme and Microbial Technology 57 (2014) 63–68 65

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0

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B

Fig. 2. The effect of temperature (A) and pH (B) on purified recombinant �-xylosidase. (A) The optimal temperature was measured in 50 mM sodium phosphate

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ig. 1. SDS-PAGE of recombinant �-xylosidase. Lane M, pre-stained Novex-nvitrogen protein marker. Lane 1, recombinant �-xylosidase after purification byel filtration.

.10. Determination of molecular mass by SDS-PAGE

In order to estimate the molecular mass, SDS-PAGE was performed using 8%olyacrylamide gel according to the method described by Laemmli [8]. Proteinsere stained with colloidal Coomassie Blue and the protein bands excised from

DS-PAGE were subjected to trypsin digestion prior to mass spectrometry analysis.

.11. Mass spectrometry

Analyses of samples were carried out by LC-ESI–tandem MS on a Q-TOFII masspectrometer fitted with a nanoflow ESI (electrospray ionisation) source (Waterstd., UK). Peptides were separated on a PepMap C18 reverse phase, 75 �m i.d., 15-m column (LC Packings) and delivered on-line to the MS via a CapLC HPLC system.equence interpretation for individual peptides was performed using the PepSeqASCOT tool of the MassLynxTM 4.0 software package (Waters).

. Results

.1. Purification and characterisation of recombinant-xylosidase

The �-xylosidase gene is contained within an open readingrame of 2325 nucleotides with no introns, which encodes a pro-ein 775 amino acids. A putative signal peptide was identifiedy SignalP software, thus the mature protein is predicted to be55 amino acids with a molecular mass of 81.8 kDa. The recombi-ant enzyme was recovered from P. pastoris culture supernatantt approximately 32 mg L−1 and purified as shown in Table 1.he program NetNGly 1.0 predicts 9 potential N-linked glycosyl-tion sites and the program NetOGlyc 4.0 predicts 10 potential-linked glycosylation sites for �-xylosidase. The molecular massstimated from SDS-PAGE is between 120 and 180 kDa, which is

onsistent with a glycosylated product (Fig. 1). The protein wasxcised from SDS-PAGE and confirmed to be N. crassa NCU09923ylan 1,4 �-xylosidase based on tryptic peptide masses. The N-erminal sequence was determined by de-novo sequencing using

able 1ummary of the purification of recombinant �-xylosidase secreted by Pichia pastoris.

Total protein (mg) Total activity

Supernatant (3 L) 241 ± 5 9132 ± 412

(NH4)2SO4 (90%) 231 ± 8 9446 ± 921

S-200 gel filtration 98 ± 1 9525 ± 693

Standard deviation of triplicate data.a Total activity measured with 2 mM PNPX.

buffer (pH 6.0) in the presence of 1 mM PNPX at various temperatures for 10 min. (B)The optimum pH was determined by incubating at 50 ◦C for 10 min in the presenceof 1 mM PNPX in sodium phosphate buffer varying pH.

mass spectrometry as IDLPFKTYPDCVNGPLASLK which is identi-cal to the predicted amino acid sequence following removal of thesignal peptide.

3.2. Determination of optimal conditions

The optimum temperature was determined by incubation of therecombinant enzyme in the presence of 50 mM sodium phosphatebuffer pH 6.0 and 1 mM 4-nitrophenyl �-xylopyranoside (PNPX)for 10 min at varying temperatures between 20 ◦C and 90 ◦C. Theenzyme displayed optimum activity at 50 ◦C and retained morethan 60% activity between the ranges of 40 and 60 ◦C (Fig. 2A).

The optimal pH of enzyme was determined by incubation at pHvalues from 2 to 9 in the presence of 1 mM PNPX. The resulting

enzyme activity profile exhibit an optimum of pH 5 and retainscatalytic activity more than 75% between the ranges of pH 4–6(Fig. 2B).

(U)a Specific activity (U/mg) Protein yield (%)

37.9 ± 2.3 10039.2 ± 3.2 95.897.9 ± 8.0 40.4

66 N. Kirikyali, I.F. Connerton / Enzyme and Microbial Technology 57 (2014) 63–68

Table 2Substrate specificities and kinetic analysis of synthetic and natural substrates determined in 50 mM sodium phosphate buffer (pH 6) at 50 ◦C.

Substrate Km (mM) Vmax (�mol min−1 mg−1) kcat (s−1) kcat/Km (mM−1 s−1)

pNPX 8.9 ± 2.0 1052 ± 145.3 1472.2 165.4pNPX + 5 mM xylose 7.8 ± 3.3 270.1 ± 64.9 252.3 0.9pNPG Nd Nd Nd NdpNPA Nd Nd Nd NdXylobiose (X2) 4.1 ± 1.3 10.2 ± 1.2 14.2 3.4

6.9 1.45.8 0.7

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Xylose Concentra�on (mM)

Fig. 3. Dixon plot constructed using varying xylose concentrations against recipro-cal of initial rate data. �, 1 mM and ♦, 4 mM PNPX.

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Xylotriose (X3) 4.9 ± 0.6 4.9 ± 0.4

Xylotetraose (X4) 7.9 ± 1.8 4.1 ± 1.6

Standard deviation of triplicate data; Nd, not detected.

.3. Substrate specificity and kinetic analysis

The substrate specificity of recombinant enzyme was deter-ined using various 4-nitrophenyl glycoside synthetic sub-

trates and xylooligosaccharides. Table 2 shows catalytic activitygainst 4-nitrophenyl �-xylopyranoside but no hydrolytic activityas detected against 4-nitrophenyl-�-d-glucopyranoside or 4-itrophenyl-�-l-arabinofuranoside. Kinetic constants for syntheticnd natural substrates were determined using Lineweaver–Burklots and the resulting values are shown in Table 2. Thenzyme exhibited highest Km and Vmax values of 8.9 mM and052 �mol min−1 mg−1 respectively for the hydrolysis of 4-itrophenyl �-xylopyranoside. The product of the reaction, xylose,

s an inhibitor which alters Vmax but does not modify the Km; sug-esting xylose is acting as a non-competitive inhibitor. A Dixon plotresented in Fig. 3 indicates xylose has a Ki of 1.72 mM.

The degradation of various xylooligosaccharides (X2, X3 and X4)y recombinant enzyme was monitored by analysing the reac-

ion products by HPLC. Xylose was released from all substratesnd the rate of xylose released decreased with the increasinghain length of the xylooligosaccharide. Table 2 shows reduc-ions in the catalytic conversion parameters Vmax and kcat with

0.0 2.5 5.0 7.5 10. 0 12. 5 15. 0 17. 5 21. 0-50

0

50

100

150

200

250

300

350

400

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1 - Xylose - 8.04 7

2 - Xylo biose - 13 .63 7

3 - Xylotriose - 15.93 0

0.0 -50

0

50

100

150

200

250

300

350

n

min

A

ig. 4. HPLC profile of (A) xylobiose and (B) xylotriose hydrolysis. All reactions were perff the control reactions performed in the absence of enzyme under identical conditions arere detectable in the control standards. The transxylosilation products are indicated by

2.5 5.0 7.5 10. 0 12. 5 15. 0 17. 5 21. 0

C

min

1 - 5.26 3 2 - Xylose - 8.30 03 - Xylobiose - 14.11 7

4 - Xylotriose - 17.48 7

5 - Xylotetrose

B

ormed in 50 mM sodium phosphate buffer (pH 6.0) at 50 ◦C for 40 min. The profilese presented as insets to panels A and B. No contaminating xylotriose or xylotetraose

the arrows.

N. Kirikyali, I.F. Connerton / Enzyme and Mi

0

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120

140

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0

20

40

60

80

100

120

140

160

DTTEDTASDSZnCl2KClLiClControl

Rela

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ac�v

ity (%

)

A

B

Fig. 5. Enzyme activity profiles in the presence of (A) carbohydrates and (B) �, 1and �, 10 mM metal ions and chemical compounds within reaction mixtures. Allreactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 1 mMPa

rIb(itht

3

apgt1

3

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with the increasing chain length in the order of X2 (4.18 mM) < X3

NPX at 50 ◦C. Control reactions were performed in the absence of carbohydratesnd chemicals under identical conditions.

espect to the increasing chain length of the xylooligosaccharides.n relation to the hydrolysis of the natural substrates, the recom-inant �-xylosidase displays relative affinities towards xylobioseX2) > xylotriose (X3) > xylotetraose (X4) with respective reductionsn the Km values (Table 2). Unexpectedly xylotriose and xylote-raose was detected whilst xylobiose and xylotriose were beingydrolysed, indicating that the recombinant enzyme is capable ofransxylosilation (Fig. 4).

.4. Effect of carbohydrates on catalytic activity

The effect of monosaccharide sugars (10 mM) on the catalyticctivity of recombinant �-xylosidase was examined using 1 mMNPX as a substrate. None of the sugars tested (arabinose, mannose,alactose, glucose and fructose) exhibited any inhibition or activa-ion on the catalytic activity (Fig. 5A). The enzyme was inhibited by0 mM xylose but not 10 mM xylitol.

.5. Influence of metals ions and reagents on ˇ-xylosidase activity

The influence of metal ions and reagents on recombinant-xylosidase activity was investigated at 1 mM and 10 mM concen-

rations (Fig. 5B). Weak stimulation in activity (<20%) was observedn the presence of monovalent metal ions Li+ and K+ at 10 mM

oncentration. The addition of Zn2+ and SDS (10 mM) adverselynhibited enzyme activity, whereas 10 mM EDTA or DTT enhancedatalytic activity of recombinant enzyme.

crobial Technology 57 (2014) 63–68 67

4. Discussion

In this study the gene encoding �-xylosidase (NCU09923) fromN. crassa was cloned and expressed in P. pastoris. A high levelof secreted recombinant protein (32 mg L−1) was recovered andsubsequently purified from culture supernatant. The protein hasa predicted molecular mass of 83 kDa with 9 potential N-linkedglycosylation and 10 potential O-linked glycosylation sites. Thepurified enzyme produced a range of masses estimated between120 and 180 kDa on SDS-PAGE that is indicative of glycosylationof a single polypeptide chain [9]. The native enzyme propertiesfrom N. crassa culture supernatant have been previously studiedby Deshpande et al. reporting that the purified �-xylosidase has amolecular weight of 83 kDa determined by SDS-PAGE that is simi-lar with the predicted size of the NCU09923.3 isozyme selected inthis study that features a secretion signal [10]. Therefore N. crassamay not perform extensive post-translational modification of thenative �-xylosidase. Issues regarding the differences in molecu-lar mass of predicted and recombinant �-xylosidase expressedin P. pastoris due to post-translational modification have beenreported previously: for example a mass difference of 13 kDa forthe enzyme originating from Paecilomyces thermophila [11]. Thebiochemical properties of the recombinant �-xylosidase closelymatch the native enzyme with respect to the optimal pH and tem-perature, where the native enzyme is also reported to be secretedwith optimum activities at 55 ◦C and pH in the range of 4.5–5. Theseactivity profiles are comparable to other fungal xylosidases sharingsequence similarity in GH family 3 [10–15], which typically exhibitoptimal activities between pH 3–5 and at temperatures around60 ◦C in reference with Carbohydrate Active Enzymes database(CAZy) (www.cazy.org/Glycoside-Hydrolases.html).

Substrate specificity of the purified recombinant �-xylosidasewas tested using 4-nitrophenyl glycoside synthetic substratesand xylooligosaccharides. The enzyme was most active towardsp-nitrophenyl-�-d-xylopyranoside and did not display anyadditional �-l-arabinofuranoside or �-d-glucosidase activitiestowards 4-nitrophenyl-�-d-glucopyranoside and 4-nitrophenyl-�-l-arabinofuranoside, indicating that the N. crassa �-xylosidaseis restricted to one substrate class in a similar fashion to thosereported from Fusarium proliferatum [15] and Humicola grisea var.Thermoidea [16].

The purified recombinant enzyme exhibited Michaelis–Mentenkinetic constants for Km and Vmax against pNPX of 8.9 mM and1052 �mol min−1 mg−1 respectively. The Km value suggests weakeraffinity for pNPX when compared with the Km (0.047 mM) reportedfor the secreted native �-xylosidase from N. crassa [10], implyingthat glycosylation of the recombinant protein by P. pastoris mayhave significantly affected its substrate binding affinity. Of all thefungal �-xylosidase kinetic characteristics reported against pNPX,the highest kcat (6787.6 s−1) was recorded from Humicola insolens[6] and comparatively the enzyme in the present study exhibits thehighest Vmax and the second highest kcat (1472.2 s−1) determined.The degradation of various xylooligosaccharides (X2, X3 and X4)by the recombinant enzyme was monitored by analysing the reac-tion products by HPLC. In the presence of xylooligosaccharides theenzyme produces xylose as an initial product of catalysis indicatingthat the recombinant �-xylosidase is an exo-cutting enzyme. Theinitial rate of xylose release was observed to decrease with increas-ing chain length of the xylooligosaccharide, similar to �-xylosidasesfrom F. proliferatum [15], Talaromyces emersonii, Trichoderma ree-sei [13], Aspergillus japonicus [14] and P. thermophila [17]. TheKm values for the xylooligosaccharides increased simultaneously

(4.91 mM) < X4 (7.93 mM) revealing a reduced affinity for longerchain substrates. In addition the catalytic rate was also affectedby the chain length of the substrate, with kcat values decreasing

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[

8 N. Kirikyali, I.F. Connerton / Enzyme a

n the order of X2 (14.2 s−1) > X3 (6.9 s−1) > X4 (5.8 s−1). Kineticonstants (Km and kcat) were also concluded to alter accordinglyith xylooligosaccharides chain length in �-xylosidases from T.

mersonii, T. reesei and A. nidulans in a similar pattern [13,18].The influence of various metal ions and reagents on recombinant

-xylosidase activity was investigated. The influence of K+ has beenreviously reported to have an inhibitory effect at 4 mM concen-ration on �-xylosidase from P. thermophila [17] but this was notbserved here. However, the addition of Zn2+ and SDS at 10 mMoncentration adversely affected the enzyme in a similar mannero that reported for Penicillium sclerotiorum �-xylosidase [19]. Sen-itivity to SDS suggests that hydrophobic interactions within therotein are important in maintaining the functional conformation.owever, T. thermophilus �-xylosidase was reported to retain 44%ctivity in the presence of 10 mM SDS [4]. Addition EDTA or DTT didot affect enzyme activity at 1 mM but improved activity (≥15%) at0 mM concentrations. Similar effects of EDTA and DTT have beenbserved for �-xylosidases from P. thermophila [20], F. proliferatum15] and P. sclerotiorum [19].

Xylose has been determined to be a non-competitive inhibitorf recombinant N. crassa �-xylosidase. In the presence of 5 mMylose with varying substrate concentrations the Vmax is alteredith no corresponding effect on Km. This is consistent with non-

ompetitive inhibition in which the inhibitor interferes with theatalytic properties of enzyme without affecting substrate bind-ng affinity by conferring a Ki of 1.72 mM. This at variance withhe majority of �-xylosidases reported to date that are reportedo be competitively inhibited by xylose with Ki values between.2 and 10 mM [12,13,15,19,21,23]. However, xylose tolerant �-ylosidases have been reported with Ki values up to 200 mMrom Scytalidium thermophilum and P. thermophila [20,22]. A non-ompetitive inhibitor could affect catalytic activity by binding thenzyme at a site other than that used in catalysis. There may beub-site(s) adjacent to catalytic site, to which xylose may bind tondirectly affect the catalytic rate by causing conformational strain.he relative affinity of product to a sub-site could cause the Ki valueso differ. The recombinant N. crassa �-xylosidase is an exo-enzymehere increasing the xylooligosaccharide chain length adversely

ffects activity. Extended xylooligosaccharides could also occupyub-sites that are detrimental to hydrolysis. However, structuralnalysis is required to provide evidence for this hypothesis. Forractical purposes, xylose tolerant enzymes are preferable due toheir efficiency in degrading xylan containing natural substrates.owever �-xylosidases with low xylose tolerance (Ki of 1–10 mM),

ike the recombinant enzyme in this study, tend to have substantialransxylosylating activities in which xylose is utilised as a substrateo produce xylooligosaccharides in a similar manner to GH family

�-xylosidase from Aspergillus awamori [23].

cknowledgements

This work has been supported by project funds from BBSRCnd Biocatalysts. The authors would like to thank Lorraine Gilletnd Nicola Cummings for their advice, David Coles for his

[

icrobial Technology 57 (2014) 63–68

assistance with HPLC and Dr. Susan Liddell for her assistance withMass Spectrometry.

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