Bioresource Technology 102 (2011) 9710–9717

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Bioresource Technology

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Purification and biochemical characterization of a moderately halotolerant NADPHdependent xylose reductase from Debaryomyces nepalensis NCYC 3413

Sawan Kumar, Sathyanarayana N. Gummadi ⇑Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India

a r t i c l e i n f o

Article history:Received 5 April 2011Received in revised form 29 June 2011Accepted 11 July 2011Available online 22 July 2011

Keywords:Debaryomyces nepalensisXyloseXylose reductasePurificationHalotolerant

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.07.030

⇑ Corresponding author. Tel.: +91 44 2257 4114; faE-mail address: gummadi@iitm.ac.in (S.N. Gumma

a b s t r a c t

A Xylose reductase (XR) from the halotolerant yeast, Debaryomyces nepalensis NCYC 3413 was purified toapparent homogeneity. The enzyme has a molecular mass of 74 kDa with monomeric subunit of 36.4 kDa(MALDI-TOF/MS) and pI of 6.0. The enzyme exhibited its maximum activity at pH 7.0 and 45 �C (21.2 U/mg). In situ gel digestion and peptide mass fingerprinting analysis showed 12–22% sequence homologywith XR from other yeasts. Inhibition of the enzyme by DEPC (diethylpyrocarbonate) confirmed the pres-ence of histidine residue in its active site. The enzyme exhibited high preference for pentoses over hex-oses with greater catalytic efficiency for arabinose than xylose. The enzyme also showed absolutespecificity with NADPH over NADH. The enzyme retained 90% activity with 100 mM of NaCl or KCl and40% activity with 1 M KCl which suggest that the enzyme is moderately halotolerant and can be utilizedfor commercial production of xylitol under conditions where salts are present.

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1. Introduction

Lignocellulose represents the largest natural source of hexosesand pentoses and xylose constitutes the predominant carbonsource in hydrolyzed lignocelluloses (Gírio et al., 2010). Yeastsmetabolize xylose by first reducing it to xylitol via xylitol reductase(XR) (EC 1.1.1.21) (Yablochkova et al., 2003). Subsequently, xylitolis oxidized to D-xylulose by a NAD+-linked xylitol dehydrogenasewhich enters into pentose phosphate pathway. Xylulose-5-phos-phate can then be metabolically converted into ethanol by NADH-linked alcohol dehydrogenase via transketolase reactions and gly-colysis (Ostergaard et al., 2000). Xylitol is a five carbon sugar alco-hol. It has sweetness comparable to that of sucrose which makes it apotential sugar substitute in wide range of diabetic food products.On industrial scale, xylitol is produced from hemicellulose derivedxylose through chemical hydrogenation process requiring metalcatalysts and extreme reaction conditions. However, this processis not cost effective owing to the cost intensive and complex processof xylitol recovery (Dieters, 1975). Alternatively, xylitol can be pro-duced using microbes or immobilized xylose reductase which cata-lyzes the conversion of xylose to xylitol. In both the cases, thexylitol production depends on the properties of XR such as kinetics,coenzyme specificity, catalytic efficiency etc. Hence, this enzymehas been the focus of many biochemical and structural studies.

Based on their coenzyme specificity, XRs can be classified asNADPH specific XRs and XRs that can utilize both NADPH and NADH

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x: +91 44 2257 4102.di).

with physiological catalytic efficiency. Due to this difference, someyeast strains such as, Debaryomyces hansenii, Candida tropicalis,Kluyveromyces marxianus, Pichia guilliermondii, Candida parapsilosisproduces xylitol during xylose metabolism (Kim et al., 1997; Yab-lochkova et al., 2003; Sampaio et al., 2005), while some other yeastssuch as Candida shehatae, Pichia stipitis, Pachysolen tannophilus con-vert xylose to ethanol (Sanchez et al., 2002). The catalytic efficiencyof purified XR from these strains is not very high. Therefore, screen-ing of new strains producing xylose reductase with high catalyticefficiency and activity at a broad range of pH, temperature and salttolerance is very important for increasing the xylitol productivity.

Debaryomyces nepalensis NCYC 3413 was isolated from rottenapple and the strain was found to tolerate up to 2 M NaCl and3 M KCl (Gummadi and Kumar, 2006; Gummadi et al., 2007). Itcould grow at pH ranging between 3.0 and 10.0 and at temperaturesranging between 8 and 42 �C (Gummadi and Kumar, 2008; Kumaret al., 2008). The strain accumulated intracellular polyols (glycerol,sorbitol, and arabitol) to withstand salt stress condition (Kumar andGummadi 2009). In the previous study, we reported that the straincould assimilate glucose and xylose, the major constituents of lig-nocellulose hydrolysate and produce up to 72 ± 3 g/l of xylitol asa key metabolite by feeding a mixture of 100 g/l xylose and 100 g/l glucose (Kumar and Gummadi, 2010). It was also shown that xyli-tol was obtained when high XR activity was observed. Since a saltand metal-tolarant XR would be useful for xylitol production fromsubstrates potentially contaminated with different metal ions andsalts, the current study was undertaken to determine the metaland salt tolerance of XR from Debaryomyces nepalensis. The sub-strate specificity of the enzyme was also analyzed.

S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717 9711

2. Methods

2.1. Materials

Hexose and pentose sugars, SDS, Coomassie brilliant blue R-250,acrylamide and buffer components were purchased from Sisco Re-search Laboratories and Himedia Laboratories, India. Materials forprotein chromatography were purchased from Amersham Pharma-cia Biotech, USA. Amicon ultracentrifugal filter units (MW cut off –10 kDa) were purchased from Amicon Bioseparations, MiliporeCorporation (Bedford, USA). Eletrophoretic reagents and proteinmolecular weight standards were from BioRad Laboratories (Her-cules, USA). Coenzymes and gel filtration standards were pur-chased from Sigma–Aldrich, UK. All the other chemicals usedwere of analytical grade procured in India.

2.2. Strain and growth condition

D. nepalensis NCYC 3413, non-pathogenic ascomycetous yeastwas maintained on solid YEPP plates containing yeast extract10 g/l, peptone 20 g/l, pectin 5 g/l and agar 20 g/l, incubated at30 �C. Inoculum was prepared in YEPD medium having the follow-ing composition: yeast extract 10 g/l, peptone 20 g/l and glucose20 g/l. A single colony from overnight-grown culture plate wastransferred to YEPD medium (50 ml) and incubated for 12 h at30 �C and 180 rpm. About 2% (v/v) of this inoculum was added to50 ml of semi synthetic media supplemented with 150 g/l xyloseand incubated at 30 �C and 180 rpm for 60 h. The semi-syntheticmedia had the following composition; xylose, 150 g/l; (NH4)2SO4,3 g/l; MgSO4, 0.1 g/l; K2HPO4, 6 g/l; Na2HPO4, 3 g/l; yeast extract,1 g/l; CaCl2�2H2O, 147 mg/l; FeCl3, 10 mg/l; MnSO4�H2O, 3.4 mg/l;ZnSO4�7H2O, 4.3 mg/l; CuSO4�5H2O, 0.25 mg/l; citric acid, 6.9 mg/l.

2.3. Preparation of cell lysates

Cells were harvested by centrifugation (8000g; 10 min; 4 �C).The pellet was washed twice with 50 mM phosphate buffer (pH7.0) and resuspended in TEM buffer (20 mM Tris–HCl (pH 7.5),0.5 mM EDTA and 0.5 mM b-mercaptoethanol) to final concentra-tion of 20 g cell dry weight/l. The cell suspension was passed threetimes through a cell disruptor (Constant Systems Ltd., Warwick,UK) preset at 4 �C/35 kpsi (13 ml/shot) for lysis. The crude cell ex-tract was obtained after removing cell debris by centrifugation at8200g (Eppendorf 5810 R) for 30 min at 4 �C. The cell supernatantwas used as enzyme source for further purification. Protein wasestimated by the Lowry’s method with bovine serum albumin asa standard (Lowry et al., 1951).

2.4. Analytical methods

Xylose and xylitol were determined based on their retentiontimes with an Agilent HPLC system equipped with refractive indexdetector (Agilent 1100) and Aminex HPX-87H column (Bio-Rad,Richmond, USA) at 50 �C with 0.01 N H2SO4 solution as the mobilephase at a flow rate of 0.5 ml per minute.

2.5. Xylose Reductase assay

Unless stated otherwise, the activity of the XR enzyme wasdetermined spectrophotometrically by monitoring the change inA340 in 500 ll reaction mixture containing 50 mM potassium phos-phate buffer (pH 7.2), 0.15 mM NADPH as a reductant, and 160 mMxylose as substrate at 30 �C. An amount of enzyme was added asrequired to ensure the constancy of D(A340)/D(time) for the reac-tion time of at least 1 min. The activity was calculated over the

linear portion of the curve with molar extinction coefficient of6220 M�1cm�1 for NADPH. One enzyme unit (U) was defined asthe amount of enzyme catalyzing the oxidation of 1 lmol ofNADPH per minute at the assay conditions (Kumar and Gummadi,2010). The kinetics of XR activity were determined by varying theinitial substrate concentration and measuring the initial velocity inthe standard assay mixture as described above at optimal pH andtemperature.

2.6. Enzyme purification

All purification procedures were carried out at room tempera-ture (25 �C) with BIO-RAD FPLC system. The absorbance of theeluted protein was measured at 280 nm by UV detector. Prior touse, cell lysates and buffers were filtered through membrane filter(pore size, 0.45 lm). Buffers were degassed in sonication bath for20 min. The cell lysate (25 ml, � 2.2 mg protein/ml) was appliedon DEAE–Sepharose column (1 ml HiTrap™ IEX FF Columns, GEhealthcare), previously equilibrated with TEM buffer (20 mMTris–HCl (pH 7.5), 0.5 mM EDTA and 0.5 mM b-mercaptoethanol),at a flow rate of 0.5 ml/min. The column was washed with fivebed volumes of the TEM buffer. The bound protein was eluted fromthe column by a linear gradient of 0–0.5 M NaCl prepared in TEMbuffer. The active fractions were pooled and concentrated by Ami-con ultracentrifugal filter units (MW cut off – 10 kDa). Buffer ex-change was performed by dialysis against 50 mM potassiumphosphate buffer (pH 7.2) using a dialysis membrane (10 kDa MWcut off, Himedia Laboratories, India). Dialyzed fractions werepooled and (NH4)2SO4 was added to a final concentration of1.5 M. The concentrated protein fractions were applied to phenylSepharose 6 fast flow (1 ml HiTrap™ HIC columns, GE healthcare,USA) pre-equilibrated with 1.5 M (NH4)2SO4 prepared in 50 mMpotassium phosphate buffer (pH 7.2). Elution was performed by alinear gradient of 1.5–0 M (NH4)2SO4 in 50 mM potassium phos-phate buffer (pH 7.2) at a flow rate of 0.5 ml/min. The active frac-tions from the hydrophobic interaction chromatography werepooled, dialyzed against 50 mM potassium phosphate buffer (pH7.2) and concentrated to a protein concentration of approxi-mately1.5–2 mg/ml by ultracentrifugal filter units. The concen-trated protein was then applied to Superdex G-200 (120 mlcolumn) pre-equilibrated with 10 mM Tris–Cl buffer containing50 mM NaCl (pH 7.5) and eluted at a flow rate of 0.5 ml/min. Theactive fractions were pooled, concentrated and dialyzed against50 mM potassium phosphate buffer (pH 7.2) and concentrated toa protein concentration of approximately 1.0–1.5 mg/ml by ultra-centrifugal filter units and the 100 ll aliquots were stored at�20 �C. The purified protein fraction was used for characterizationstudies.

2.7. Mass spectrometry analysis of the purified protein

The purified protein fraction was analyzed on SDS–PAGE andvisualized by Coomassie blue R-250 stain using standard tech-niques (Sambrook et al., 1989). The single Coomassie-stained pro-tein band was excised from the gel, the gel pieces were destainedwith 100 mM NH4HCO3/acetonitrile (1:1 v/v), and the protein wasdigested with trypsin gold (Promega Corporation) by overnightincubation in digestion buffer at 37 �C followed by addition of10 ll of 10% trifluoroacetic acid (TFA). The digest was briefly cen-trifuged at 5000g for 30 s to pellet the gel slices. The gel pieceswere washed again with 120 ll of 50% acetonitrile/2.5% trifluoro-acetic acid to elute the remaining peptides, followed by spin at5000g for 30 s. The supernatants were pooled in 1.5 ml microcen-trifuge tubes and the eluted peptides were concentrated using avacuum evaporator (He et al., 2003; Shevchenko et al., 2006). Sam-ples for MALDI (matrix-assisted laser desorption/ionization) were

Fig. 1. SDS–PAGE analysis of step wise purification of xylose reductase from D.nepalensis with12% polyacrylamide gel. Lanes 1–5 (left to right): lane 1, proteinstandards; lane 2, crude cell extract; lane 3, DEAE–Sepharose fraction; lane 4,Phenyl–Sepharose fraction; lane 5, size exclusion fraction (purified protein). Eachlane of SDS–PAGE was loaded with 6 lg of the corresponding protein fraction.

9712 S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717

prepared by mixing one portion of protein digest with nine por-tions of MALDI matrix (a-Cyano-4-hydroxycinnamic acid) contain-ing 10 mM ammonium mono basic phosphates (Shevchenko et al.,2006; He et al., 2003). Mass spectrometric data were acquiredusing Applied Biosystems 4800 MALDI TOF/TOF (Applied Biosys-tems, Foster City; CA). The data interpretation was carried outusing the GPS Explorer software (Applied Biosystems, Framing-ham, MA). The obtained peptide masses were then searchedagainst NCBI database by using Mascot peptide mass fingerprintingsearch program (Matrix Science, Boston; MA) (He et al., 2003).

2.8. Isoelectric focusing

The isoelectric point of the purified XR was determined usingpre-cast 4–7 pH linear gradient, 7 cm IPG strip (BioRad, USA), inprotean IEF cell (BioRad, USA) following the manufacturer’sinstructions. The re-hydration of the IPG strips was performed at50 V, 20 �C for 12 h. Focusing was performed at 250 V for 15 min,4000 V for 1 h and final focusing was performed till the end of total10,000 Vh at 4000 V. The strips were stained with Coomassie bril-liant blue R-250 to visualize the band of XR after electrophoresis.

2.9. Effect of pH and temperature

The effects of pH and temperature on the activity of XR weredetermined by standard assay procedures as described in Sec-tion 2.5. To study the effect of pH on enzyme, the activity was mea-sured at 30 �C, using 50 mM sodium acetate (pH 4.0–5.5);potassium phosphate (pH 6.0–8.0); Tris–HCl (pH 8.5); and gly-cine–NaOH (pH 9.0–9.5). To determine the optimum temperaturefor the enzyme activity, the experiment was performed by recircu-lating water through water bath connected to the UV–Vis spectro-photometer (Lambda 25 UV/VIS spectrophotometer, Perkin Elmer)with a jacketed cuvette holder. The XR activity was measured atvarious temperatures ranging between 10 and 60 �C at optimalpH obtained from pH effect studies.

2.10. Enzyme stability studies

Deactivation studies of the purified xylose reductase were car-ried out by incubating 1.5 lg of the purified enzyme at differentcombinations of pH and temperature. The selected pHs were 6.0and 7.0, and at each pH the deactivation was carried out at 25,35 and 45 �C. Aliquots of the enzyme at different time points weretaken and the residual activity was measured under standard assayconditions. The half life of the enzyme was calculated as describedby Naidu and Panda (2003).

2.11. Effect of metal ions and additives

The effect of metal ions (Mg2+, Ca2+, Mn2+, Fe2+, Zn2+, Cu2+, Ni2+,Na+, K+) and of dithiothreitol (DTT), b-mercaptoethanol, glycerol,phenylmethylsulfonyl fluoride, EDTA), were determined by per-forming enzyme activities in the presence of varying concentrationof these compounds with 1.5 lg of enzyme in 50 mM phosphatebuffer (pH 7.0). The residual activity of enzyme was then deter-

Table 1Purification of xylose reductase from the cell extract of D. nepalensis.

Purification step Total protein (mg) Total activity (U)

Cell free lysate 48.8 19.3DEAE–Sepharose 5.9 11.3Phenyl–Sepharose 1.98 10.1Superdex-200 1.2 2.7

mined under the standard assay conditions. Activity of enzymewithout any additive was considered to be 100%.

2.12. Effect of histidine modifying reagent on XR activity

The effect of diethyl pyrocarbonate (DEPC), a histidine modifier,on XR activity was determined in 50 mM phosphate buffer (pH 7.0)by incubating 1.5 lg of enzyme with different concentration ofDEPC for 30 min at 25 �C. The residual activity of enzyme wasdetermined under the standard assay conditions. Activity of en-zyme without DEPC was considered to be 100%.

3. Result and discussion

3.1. Purification of XR from D. nepalensis

Xylose reductase produced by D. nepalensis was purified fromcrude cell extract prepared from cells grown on xylose. Kumarand Gummadi (2010) showed that the expression of XR is induc-ible in D. nepalensis by xylose in the medium. It has also beenshown that XR activities in P. tannophilus and P. stipitis are inducedin xylose-grown but not in glucose-grown cells (Bicho et al., 1988).Most of the XR purification procedures described in the literaturefocused on a combination of dye-ligand affinity chromatographyand anion-exchange chromatography (Neuhauser et al., 1997;Mayr et al., 2003; Lee et al., 2003; Panagiotou and Christakopoulos2004). However, we have established a three step easy purificationprocedure using a combination of anion exchange, hydrophobicinteraction and size exclusion chromatography that allows purifi-cation of XR to apparent electrophoretic and chromatographichomogeneity (Table 1, Fig. 1). Anion exchange (DEAE–Sepharose)

Specific activity (U/mg) Purification fold Yield (%)

0.4 1 1001.9 4.9 58.75.1 13.1 52.65.7 14.5 13.9

Fig. 2. Effect of pH and temperature on activity and stability of xylose reductase purified from D. nepalensis. (A) pH dependence of xylose reductase activity at 30 �C, (B) effectof reaction temperature, (C) pH stability, and (D) temperature stability of xylose reductase. Relativity activity of 1 unit corresponds to specific activity of 17.1 and 23.6 U/mg ofpurified protein at pH 6.0 and pH 7.0, respectively measured under optimized enzyme assay conditions. The values reported are mean of triplicate measurements.

Table 2Substrate specificity of xylose reductase purified from D.nepalensis.

Substrate (150 mM) Specific activity (U/mg)

D-Xylose 22.3 ± 0.5 (100)*

L-Arabinose 28 ± 0.2 (126)

D-Ribose 9.2 ± 0.2 (41)

D-Glucose 3.5 ± 0.26 (14.5)

D-Galactose 4.6 ± 0.13 (21)

D-Fructose 0.40 ± 0.13 (1.3)

D-mannose ND**

* Values in parenthesis represent relative specific activity (in%).** ND - not detectable.

Table 3Kinetic parameters of xylose reductase from D. nepalensis.

Substrate Km (mM) Kcat (s�1) Kcat/Km (s�1 mM�1)

Xylose 80 ± 7 12.7 ± 1.3 0.16Arabinose 76.3 ± 6 29.5 ± 2 0.39Ribose 174 ± 16 19.5 ± 1.9 0.12NADPH 0.08 ± 0.01 11.0 ± 0.8 137.5

The kinetic parameters were determined by non-linear regression. The valuesreported are mean of three individual experiments. Km is Michaelis constant, Kcat isthe catalytic constant, and Vm is the maximum specific activity of enzyme.

S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717 9713

followed by hydrophobic interaction chromatography (Phenyl–Se-pharose) resulted in a significant purification (�13-fold). The finalsize-exclusion chromatography (Superdex-200) removed contami-nants as observed in SDS–PAGE (Fig. 1A). The protein was purifiedto homogeneity with �15-fold purification, 14% activity recoveryand 2.5% protein recovery (Table 1). Analysis by SDS–PAGE demon-strates the apparent homogeneity of the purified protein (Fig. 1).

3.2. Molecular mass determination

The molecular weight of purified enzyme as determined bySDS–PAGE was 36 kDa (Fig. 1). MALDI-TOF/MS analysis of the puri-fied protein yielded a value of 36,429 Da for the molecular mass ofXR subunit, which was closely similar to the value determined by

SDS–PAGE (Supplementary Fig. 1A). However, the functionalmolecular mass was found to be 74 kDa, as determined by gel fil-tration chromatography on a Superdex-200 column, suggestingthe enzyme to be a dimer (Supplementary Fig. 1B, C). XR fromsome of the common xylose metabolizing yeasts such as P. stipitis(Verduyn et al., 1985) and C. tropicalis (Yokoyama et al., 1995) andfungus C. flavus (Mayr et al., 2003) have also been reported to behomodimeric with 35 ± 1.0 kDa monomeric subunits. However,XR from others yeasts such as P. tannophilus (Ditzelmuller et al.,1984) and Saccharomyces cerevisiae (Kuhn et al., 1995) aremonomeric.

3.3. Biochemical characterization of purified XR

The activity of purified XR from D. nepalensis was examined as afunction of pH and temperature (Fig. 2). Maximum activity was

Table 4Properties of D. nepalensis XR and its comparison with other yeasts and fungi.

Organism Mr, native (kDa) Mr, subunit (kDa) Coenzyme pI Km (xylose) (mM) Km (NADPH) (mM) Optimal pH Reference

D. nepalensis 74 36.4 NADPH 6 80 0.08 7.0 This workS. cerevisiae 36.8 35 NADPH 6.6 27.9 0.013 5.0 Kuhn et al. (1995)P. tannophilus 38 38 NADPH 4.87 162 0.06 7.0 Ditzelmuller et al. (1984)P. stipitis 65 34 NADPH > NADH 5.76 42 0.009 6.0 Verduyn et al. (1985)C. tropicalis 58 36.5 NADPH 4.15 30 to 37 0.09 to 0.014 6.0 Yokoyama et al. (1995)C. parapsilosis 69 36.4 NADH > NADPH 5.19 31.5 0.036 6.0 Lee et al. (2003)N. crassa 53 38.4 NADPH > NADH – 34 0.018 5.5 Woodyer et al. (2005)

(–) indicates that the corresponding data is not available.

CLUSTAL 2.1 multiple sequence alignment

Candida_sp._GCY_2005 MTTS---STIKLNSGYEMPIVGFGCWKVTNETAADQIYNAIKIGYRLFDG 47 Candida_tropicalis MSTTPTIPTIKLNSGYEMPLVGFGCWKVTNATAADQIYNAIKTGYRLFDG 50 Debaryomyces_hansenii -------MSIKLNSGYDMPLVGFGCWKVDNDTCAATIYNAIKVGYRLFDA 43 Candida_shehatae -MSPSPIPAFKLNNGLEMPSIGFGCWKLGKSTAADQVYNAIKAGYRLFDG 49 Candida_tenuis -MSAS-IPDIKLSSGHLMPSIGFGCWKLANATAGEQVYQAIKAGYRLFDG 48

:**..* ** :******: : *.. :*:*** ******.

Candida_sp._GCY_2005 AQDYGNEKEVGEGINRAIKDGLVKREELLITSKLWNNFHDPKNVELALDK 97 Candida_tropicalis AEDYGNEKEVGEGINRAIKEGLVKREELFITSKLWNNFHDPKNVETALNK 100 Debaryomyces_hansenii AQDYGNCKEIGEGINKALDEGLVARDELFITSKLWNSYHDPKNVELALKK 93 Candida_shehatae AEDYGNEQEVGEGVKRAIDEGIVTREEIFLTSKLWNNYHDPKNVETALNK 99 Candida_tenuis AEDYGNEKEVGDGVKRAIDEGLVKREEIFLTSKLWNNYHDPKNVETALNK 98

*:**** :*:*:*:::*:.:*:* *:*:::******.:******* **.*

Candida_sp._GCY_2005 TLSDLNLGYLDLFLIHFPIAFKFVPIEEKYPPGFYCGDGNNFHYENVPLL 147 Candida_tropicalis TLSDLNLDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDNFHYEDVPLL 150 Debaryomyces_hansenii VLSDMKLDYLDLFLIHFPIAFKFVPIEERYPPGFYCGDGDKFHYENVPLA 143 Candida_shehatae TLKDLKVDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGDNFVYEDVPIL 149 Candida_tenuis TLADLKVDYVDLFLIHFPIAFKFVPIEEKYPPGFYCGDGNNFVYEDVPIL 148

.* *:::.*:******************:**********::* **:**:

Candida_sp._GCY_2005 DTWKALEKLVQKGKIRSIGISNFTGALIYDLIRGATIKPSVLQIEHHPYL 197 Candida_tropicalis DTWKALEKLVEAGKIKSIGISNFTGALIYDLIRGATIKPAVLQIEHHPYL 200 Debaryomyces_hansenii DTWKAMEKLTKSGKVKSIGISNFSGALIYDLLRSAEIKPAVLQIEHHPYL 193 Candida_shehatae ETWKALEKLVKAGKIRSIGVSNFPGALLLDLFRGATIKPAVLQVEHHPYL 199 Candida_tenuis ETWKALEKLVAAGKIKSIGVSNFPGALLLDLLRGATIKPAVLQVEHHPYL 198

:****:***. **::***:***.***: **:*.* ***:***:******

Candida_sp._GCY_2005 QQPKLIEYVQKQNIAITAYSSFGPQSFLELESKRALDTPTLFEHKTIKSI 247 Candida_tropicalis QQPKLIEYVQKAGIAITGYSSFGPQSFLELESKRALNTPTLFEHETIKLI 250 Debaryomyces_hansenii QQPRLVEYVQSQNIAITGYSSFGPQSFLELKHSKALDTPTLFEHKTIKSI 243 Candida_shehatae QQPKLIEYAQKVGITVTAYSSFGPQSFVEMNQGRALNTPTLFEHDVIKAI 249 Candida_tenuis QQPKLIEFAQKAGVTITAYSSFGPQSFVEMNQGRALNTPTLFAHDTIKAI 248

***:*:*:.*. .:::*.*********:*:: :**:***** *..** *

Candida_sp._GCY_2005 AEKHGKTPAQVLLRWATQRNIAVIPKSNNPARLAQNLSVVDFDLSKEDIQ 297 Candida_tropicalis ADKHGKSPAQVLLRWATQRNIAVIPKSNNPERLAQNLSVVDFDLTKDDLD 300 Debaryomyces_hansenii ANKNKKTPAQVLLRWASQRNIAVIPKSNNPDRLLQNLEVNDFDLSKEDFE 293 Candida_shehatae AAKHNKVPAEVLLRWSAQRGIAVIPKSNLPERLVQNRSFNDFELTKEDFE 299 Candida_tenuis AAKYNKTPAEVLLRWAAQRGIAVIPKSNLPERLVQNRSFNTFDLTKEDFE 298

* * * **:*****::**.******** * ** ** .. *:*:*:*::

Candida_sp._GCY_2005 EISALDIGVRFNDPWDWDNIPIFV 321 Candida_tropicalis NIAKLDIGLRFNDPWDWDNIPIFV 324 Debaryomyces_hansenii EISKLDQELRFNNPWDWDKIPIFA 317 Candida_shehatae EISKLDINLRFNDPWDWDNIPIFV 323 Candida_tenuis EIAKLDIGLRFNDPWDWDNIPIFV 322

:*: ** :***:*****:****.

Fig. 3. Multiple alignment of xylose reductase protein sequence as performed by CLUSTAL W showing Multiple alignments of the XR sequences among D. hansenii, C.tropicalis, Candida sp. GCY 2005 and other xylose reductases as performed by CLUSTAL W. The amino acid residues underlined and highlighted in bold represents the sequenceof the purified XR as analyzed by PMF. The amino acid residue as indicated by the arrow shows the conserved tetrad of the active site residues which consist of Tyr, Lys, Asp,His.

9714 S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717

observed at pH 7.0. The enzyme demonstrated activity at a broadpH range with 80% of maximal activity between pH 6.0 and 8.0. Itretained 40% of its maximal activity at pH 5.0 and 9.0 (Fig. 2A). Dit-zelmuller et al. (1984) also reported XR with optimum pH at 7.0 inP. tannophilus. However, XR from a large number of yeasts areknown to exhibit maximum activity at pH in the range of 5.5–6.0(Yokoyama et al., 1995; Lee et al., 2003; Woodyer et al., 2005).

The optimal temperature for XR was at 45 �C (Fig 2B). The en-zyme activity increased linearly from 30 to 45 �C, resulting in4.5-fold increase in XR activity. This result is consistent with thoseobtained with XR from C. tropicalis (Zhang et al., 2009) and Neuros-pora crassa (Woodyer et al., 2005) which also showed maximalactivity at 45 �C. The D. nepalensis enzyme was highly stable atpH 6.0 and at temperatures 25 and 35 �C for 6 h. Under these

Table 5Peptide fingerprint analysis identifying the proteins as xylose reductase.

Match Accession number Amino acid region Queries matched Mass fingerprints

Debaryomyces hansenii Q6BLI9 35–52 6 K.VGYRLFDAAQDYGNCK.E59–69 K.ALDEGLVAR.D76–86 K.LWNSYHDPK.N99–116 K.LDYLDLFLIHFPIAFK.F227–239 K.ALDTPTLFEHK.T249–257 K.KTPAQVLLR.W

Candida sp. GCY 2005 Q5I599 8–25 4 K.LNSGYEMPIVGFGCW163–181 R.SIGISNFTGALIYDLIR.G231–243 K.V R.ALDTPTLFEHK.T253–262 K.TPAQVLLR.W

Candida tropicalis MYA-3404 XP_002546515.1 11–28 2 K.LNSGYEMPLVGFGCWK.V166–184 K.SIGISNFTGALIYDLIR.G

Mascot results for protein identification by peptide mass fingerprinting of xylose reductase tryptic digest as analyzed by MALDI-TOF/TOF-MS.

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conditions enzyme exhibited no significant loss in its activity(�95% of its residual activity) (Fig. 2C). The enzyme retainedapproximately 70% and 55% of its activity after 6 h at pH 7.0 andat 25 and 35 �C, respectively (Fig. 2D). However at 45 �C, the en-zyme retained only 33% of its activity at pH 7.0 after 45 min andit lost its activity completely within 30 min at pH 6.0 (Fig. 2Cand D). In addition, at 45 �C, the half life (t1/2) of the enzyme atpH 6 and 7 was 5 and 33 min, respectively. These results show thatthe purified XR is more sensitive to temperature than pH. The iso-electric point of the purified XR was 6.0 and thus different fromthose of XRs from most other yeasts whose isoelectric point rangesbetween 4.1 and 5.2 (Lee et al., 2003; Panagiotou and Christakopo-ulos, 2004; Yokoyama et al., 1995).

Table 6Effect of divalent metal ions activity of xylose reductase from D. nepalensis.

Metal ion Concentration (mM) Specific activity (U/mg)

Control 0 23.1 ± 1.5 (100)*

Mg2+ 0.5 21.7 ± 0.8 (93.8)Mn2+ 0.5 22 ± 1.4 (94.9)Zn2+ 0.5 21 ± 1.2 (90.6)Ca2+ 0.5 19.7 ± 1.2 (85)Ni2+ 0.5 14.6 ± 0.7 (63.2)Fe2+ 0.5 13.2 ± 1.9 (57.2)Cu2+ 0.25 0.43 ± 0.03 (1.8)

* Values in parenthesis represent relative specific activity (in%).

3.4. Substrate and coenzyme specificity

HPLC analysis of the reaction mixture showed a decrease in xy-lose concentration accompanied by xylitol synthesis. This con-firmed that the purified XR catalyzed the reduction of xylose toxylitol. The broad substrate specificity of purified XR is apparentfrom Table 2. The enzyme reduced pentoses with higher activitythan hexoses with maximal activity of 28 U/mg towards arabinose.The enzyme showed poor activity with glucose (3.5 U/mg) and gal-actose (4.6 U/mg). No detectable activity was observed with D-mannose as a substrate (Table 2). This observation is consistentwith the XR isolated from P. stipitis (Verduyn et al., 1985), C. trop-icalis (Yokoyama et al., 1995), C. parapsilosis (Lee et al., 2003). Thevalue of Km for D-xylose and L-arabinose as determined by theLineweaver–Burk plot are 80 and 76.3 mM (Table 3). The catalyticefficiency Kcat/Km of the purified enzyme was highest for arabinose(Kcat/Km = 0.39 s�1 mM�1) which also displayed the highest affinitytowards the enzyme (Km = 76.3 mM). The Kcat/Km for xylose and ri-bose was 41% (0.12 s�1 mM�1) and 31% (0.16 s�1 mM�1), respec-tively as compared to that of arabinose. XR from P. stipitis and C.tropicalis, also displayed higher activity with D-arabinose as com-pared to xylose and other substrates examined (Verduyn et al.,1985; Yokoyama et al., 1995).

Unlike many other xylose metabolizing yeasts, D. nepalensisproduced xylitol as major metabolite instead of ethanol (Sanchezet al., 2002). This may be due to the difference in coenzyme spec-ificity of XR. The XR from ethanol producing strains utilizes bothNADPH and NADH as coenzyme (Ditzelmuller et al.,1984; Verduynet al., 1985; Wang et al., 2007), while those producing xylitol weremore specific to NADPH (Yokoyama et al., 1995). Study on coen-zyme specificity of purified XR from D. nepalensis showed thatthe enzyme is highly specific for NADPH (Km = 0.08 mM) with aturn over number of 11 s�1 (Table 3). The reduction of xylose didnot occur in the presence of NADH as cofactor under any condition

(data not shown). A high specificity towards NAPDH leads to ashortage of NAD+ required for the oxidation of xylitol. Hence, D.nepalensis accumulated xylitol. The enzyme was not able to oxidizexylitol with NAD+ or NADP+ as oxidants. Table 4 displays the char-acteristics of the purified XR from different yeasts and fungi andcompared with D. nepalensis used in the present study.

3.5. Peptide-Mass fingerprinting

The monoisotopic masses obtained for individual peptides werein the range of 800–3400 Da (Supplementary Fig. 1) and the dataanalysis revealed high similarity with xylose reductases from otheryeasts (Fig. 3). A sequence coverage of 22% was achieved with xy-lose reductase from D. hansenii (Genebank- Q6BLI9_DEBHA), whilexylose reductase from C. tropicalis (GenBank: BAA19476) and Can-dida sp. GCY 2005 (GenBank: Q5I599_9ASCO) had a sequence iden-tity of 12% and 18%, respectively with the tryptic digested peptidefragments of the XR from D. nepalensis (Table 5). The structure andthe catalytic mechanism of xylose reductase from C. tenuis havebeen completely revealed (Kratzer et al., 2006). Multiple align-ments of the XR sequences from the above mentioned PMF match,C. tenuis and another xylose metabolizing yeast, C. shahatae re-vealed an overall homology in the conserved tetrad of the activesite residues. Alignment with other XR sequences showed thatXR from D. nepalensis also contain the same conserved catalytic tet-rad previously identified in C. tenuis by Kratzer et al., 2006 (Fig. 3).

3.6. Effect of DEPC on XR activity

The His-residue of the catalytic tetrad of XR plays a vital role todirect the orientation of substrate in the active site pocket of XR(Kavanagh et al., 2002). Hence, the effect of DEPC, a histidine mod-ifier was examined on the XR activity (Veronica and Barber, 2001).The result showed that XR activity was strongly inhibited by DEPC.Purified XR showed a residual activity of 11.7 U/mg (55% relative to

Fig. 4. Effect of (A) chemical additives, and (B) salt on xylose reductase activity. Xylose reductase activity was measured under standard condition at optimum pH andtemperature. The values reported are the mean of triplicate measurements.

9716 S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717

control) and 1.2 U/mg (5% relative to control) in the presence of 1and 5 mM DEPC. The enzyme was almost completely inhibited(>97%) in the presence of 10 mM DEPC confirming that His residueis essential for its catalysis.

3.7. Effect of metal ions and other additives

Of the various divalent metal ions tested, the activity of purifiedXR was significantly reduced (>97%) with a residual activity of0.43 U/mg in the presence of 0.25 mM Cu2+. The enzyme was mod-erately inhibited by �60% in presence of Fe2+ and Ni2+. A residualactivity of 13.2 and 14.6 U/mg was observed with Fe2+ and Ni2+,respectively, at a concentration of 0.5 mM in the reaction mixture.The metal ions, Mg2+, Zn2+, Mn2+ and Ca2+ showed a residual activ-ity of 21 ± 1 U/mg (�90% relative activity) at a concentration of0.5 mM in the reaction mixture (Table 6). Cecconi et al. (1998) re-ported that Cu+2 induce a site specific oxidation of aldose reductasewhich leads to enzyme inactivation. Cardoso et al. (2008) sug-gested a reductive mechanism to be responsible for enzyme inac-tivation mediated by Fe+2 in presence of NADPH. Based on thesereports, we hypothesize that Fe2+ and Cu2+ mediated inactivationof XR from D. nepalensis involved the same mechanism. Cu+2 alsoinhibited C. parapsilosis XR (Lee et al., 2003).

No significant decrease in XR activity was observed withincreasing concentration of glycerol, b-mercaptoethanol and EDTAup to 10 mM (Fig. 4A). The XR activity was not significantly inhib-ited by EDTA (up to 10 mM) suggesting that it is a metal free en-zyme. EDTA has a protective effect on XR against the inhibitionby Fe2+ and Cu2+. The residual activity was determined for the en-zyme pre-incubated with EDTA and added to the reaction mixturecontaining 0.5 mM Fe2+ and 0.25 mM Cu2+. In an another experi-ment, the activity was determined for the enzyme pre-incubatedwith 0.5 mM Fe2+ or 0.25 mM Cu2+ and added to the reaction mix-ture containing EDTA. XR activity was completely restored in boththe cases. Lee et al. (2003) also showed similar phenomenon ofEDTA protection with the XR from C. parapsilosis.

A large dose dependent decrease in activity was observed whenDTT or PMSF were added to the reaction mixture showing a resid-ual activity of 17.5 ± 0.6 U/mg (�70% relative to control) at 1 mMconcentration of DTT or PMSF. The enzyme was almost completelyinhibited (>95%) in presence of 10 mM PMSF, however it retained aresidual activity of 8.6 ± 0.7 U/mg (�33% relative to control) withDTT at a concentration 10 mM (Fig. 4A). XR from C. shehatae wasalso reported to be inhibited by DTT (Wang et al., 2007). However,the addition of sulfhydryl compounds, DTT or b-mercaptoethanol

enhanced the enzyme activity of XR isolated from C. tenuis (Neuha-user et al., 1997).

3.8. Effect of salt (NaCl/KCl) on XR activity

As compared to divalent ions, the purified XR was highly stablein the presence of monovalent ions (Na+ and K+). Azuma et al.(2000) reported an increase in xylitol production by C. tropicalisin presence of 4% NaCl (0.68 M) due to the increase in xylose reduc-tase. However, NaCl at a concentration of 0.1 mM reduced the XRactivity by about 25% in C. tropicalis (Su et al., 2010). Purified XRfrom D. nepalensis retained 90% of its activity on incubation with100 mM of both Na+ (NaCl) and K+ (KCl) ions (Fig. 4B). The XR activ-ity gradually decreased with further increase in salt concentration.A residual activity of 3.7 ± 0.5 U/mg (�15%) was observed when500 mM of NaCl was added to the reaction mixture. Unexpectedly,about 40% of XR activity was retained with KCl at 1 M concentra-tion in the reaction mixture (Fig. 4B). These observations suggestthat XR purified from D. nepalensis is moderately halotolerant.The halotolerant proteins are highly enriched in acidic amino acidsand contain relatively low levels of basic and hydrophobic aminoacids (Dennis and Shimmin, 1997). Complete sequence of the pro-tein is needed to calculate the % composition of total acidic and ba-sic amino acid residues. Hence, the moderately halotolerantbehavior of XR from D. nepalensis could not be explained basedon the present information available on its protein sequence.

4. Conclusions

The xylose reductase from D. nepalensis was purified and subse-quently characterized. The purified enzyme acts on several sub-strates and is strongly specific for NADPH as cofactor, whichfacilitates xylitol production. The enzyme operates at a wide rangeof pH and temperature. More importantly, it showed a remarkablyhigh activity in the presence of NaCl and KCl and hence, it is mod-erately halotolerant. The ease of purifying this enzyme coupledwith high stability and catalytic efficiency may prove useful indeveloping an enzyme based bioprocess for xylitol production.

Acknowledgement

This work is supported by research grant from Department ofBiotechnology, Government of India. Author(s) thank Dr. N. Manojand Mr. Mrityunjay Singh, IITM, for the help in XR purification and

S. Kumar, S.N. Gummadi / Bioresource Technology 102 (2011) 9710–9717 9717

characterization. Author(s) thank Ms. Anju Kashyap for her help inediting the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.07.030.

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