Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

Homologous expression and characterization of Cel61A (EG IV) ofTrichoderma reesei

Johan Karlsson1, Markku Saloheimo2, Matti Siika-aho2, Maija Tenkanen2, Merja Penttila2 andFolke Tjerneld1

1Department of Biochemistry, Lund University, Sweden; 2VTT Biotechnology, Espoo, Finland

There are currently four proteins in family 61 of the

glycoside hydrolases, from Trichoderma reesei, Agaricus

bisporus, Cryptococcus neoformans and Neurospora crassa.

The enzymatic activity of these proteins has not been

studied thoroughly. We report here the homologous

expression and purification of T. reesei Cel61A [previously

named endoglucanase (EG) IV]. The enzyme was expressed

in high amounts with a histidine tag on the C-terminus and

purified by metal affinity chromatography. This is the first

time that a histidine tag has been used as a purification aid

in the T. reesei expression system. The enzyme activity was

studied on a series of carbohydrate polymers. The only

activity exhibited by Cel61A was an endoglucanase activity

observed on substrates containing b-1,4 glycosidic bonds,

e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose

(HEC) and b-glucan. The endoglucanase activity on CMC

and b-glucan was determined by viscosity analysis, by

measuring the production of reducing ends and by following

the degradation of the polymer on a size exclusion chromato-

graphy system. The formation of soluble sugars by Cel61A

from microcrystalline cellulose (Avicel; Merck), phosphoric

acid swollen cellulose (PASC), and CMC were analysed on

a HPLC system. Cel61A produced small amounts of oligo-

saccharides from these substrates. Furthermore, Cel61A

showed activity against cellotetraose and cellopentaose. The

activity of Cel61A was several orders of magnitude lower

compared to Cel7B (previously EG I) of T. reesei on all

substrates. One significant difference between Cel61A and

Cel7B was that cellotriose was a poor substrate for Cel61A

but was readily hydrolysed by Cel7B. The enzyme activity

for Cel61A was further studied on a large number of

carbohydrate substrates but the enzyme showed no activity

towards any of these substrates.

Keywords: Trichoderma reesei; endoglucanase; cellulase;

histidine tag.

Cellulose is the most abundant polymer on earth, and canbe utilized in many ways, one example being its con-version to ethanol, which can then be used as a fuel [1].This use is beneficial to the environment due to the netreduction of carbon dioxide emitted into the atmosphere,in contrast to the case of fossil fuels. The major costin this type of enzymatic bio-ethanol process is theproduction of the cellulases used [2]. As applications ofcellulases in industry are increasing, e.g. in detergent and intextile treatments [3], an increased knowledge about thecellulases is therefore important.

The majority of the known cellulases are produced bybacteria and fungi [4,5]. One of the most studied cellulolyticsystems is that of the fungi Trichoderma reesei. T. reesiproduces at least two cellobiohydrolases (CBHs), Cel6A(CBH II) [6,7] and Cel7A (CBH I) [8,9] (EC 3.2.1.91), andfive endoglucanases (EGs), Cel5A (EG II) [10], Cel7B(EG I) [11], Cel12A (EG III) [12,13], Cel45A (EG V) [14]

and Cel61A (EG IV) [15] (EC 3.2.1.4). Here, we haveadopted the nomenclature for classification of the catalyticdomains into families based on sequence [16]. All of theT. reesei cellulases, except Cel12A, are modular enzymesand consists of a catalytic core domain and a cellulosebinding module (CBM) that are connected by a highlyglycosylated linker region. Cel12A consist only of a cata-lytic domain [13]. Some of the cellulases have been shownto cooperate in the hydrolysis of cellulose, i.e. they act insynergy [4,5]. The 3D structure of the catalytic coredomains of Cel6A [17], Cel7A [18], Cel7B [19] and Cel12A[20], and the cellulose binding module of Cel7A [21] andCel7B [22] has been solved.

Currently, there are four enzymes in family 61 of theglycoside hydrolases, from T. reesei [15], Agaricus bisporus[23], Cryptococcus neoformans [24] and Neurospora crassa(GenBank accession number AL389890). The endogluca-nase activity of Cel61A (EG IV) of T. reesei was shownwhen it was expressed in Saccharomyces cerevisiae.Cel61A (originally called Cel1) of A. bisporus has beenreported to have no detectable glucanase activity [25,26],whereas the activity of Cel61A (originally called Cel1) ofC. neoformans has not been studied. Only the ORF for theN. crassa protein has been published; thus no enzymeactivity has been determined. Cel61A of T. reesei consists ofa CBM, a linker region and a core domain. The CBM wasidentified by its high homology to the CBMs of the otherT. reesei cellulases. The linker was classified due to itsrichness in serines, threonines and prolines, which are acommon feature for cellulase linkers [15].

Correspondence to F. Tjerneld, Department of Biochemistry,

Lund University, PO Box 124, S-221 00 Lund, Sweden.

Fax: þ 46 46 2224534, Tel.: þ 46 46 2224870,

E-mail: [email protected]

(Received 21 August 2001, accepted 18 October 2001)

Abbreviations: CMC, carboxymethylcellulose; HEC,

hydroxyethylcellulose; PASC, phosphoric acid swollen cellulose; CBH,

cellobiohydrolase; EG, endoglucanase; CBM, cellulose binding

module; SEC, size-exclusion chromatography; DNS, dinitrosalicylic

acid reagent.

Eur. J. Biochem. 268, 6498–6507 (2001) q FEBS 2001

In this work, we describe the purification and enzymaticcharacterization of a recombinant Cel61A of T. reeseicontaining a histidine tag on the carboxy terminus. Theprotein was purified on a Ni-chelating column. This is thefirst cellulase purified by this method when expressed inT. reesei. We present a thorough investigation of the activityof Cel61A on a number of substrates. Viscosity and reducingends assays were used to screen for Cel61A activity on arange of carbohydrate polymers. Hydrolysis of CMC and b-glucan was studied by size-exclusion chromatography(SEC). Product formation of Cel61A after hydrolysis ofpolymeric carbohydrates and cello-oligosaccharides wasanalysed by anion-exchange HPLC with pulsed ampero-metric detection. For comparison, the activity of Cel7B(EG I) of T. reesei was determined with several substrates.

M A T E R I A L S A N D M E T H O D S

Cloning procedures

Strains, plasmids and primers. The Escherichia coli strainDH5a was used as a plasmid host and the T. reesei strainRut-C30 for expression of Cel61A. The plasmids pMS54[15] and pAMH110 [27] and the following DNA primerswere used, primer MS3323, antisense: 50-ACCCGCCGTGATGCCCTCTAGTGGTGGTGGTGGTGGTGGTTAAGGCACTGGGCGTAGT-30, primer MS3324, sense: 50-ACTACGCCCAGTGCCTTAACCACCACCACCACCACCACTAGAGGGCATCACGGAGGGT-30, primer 808, antisense:50-ATCGCATTTCCTACCCC-30, primer egl4 504: sense50-CCCACCCCAGACTCTGTA-30.

Construction of the histidine tag. The site-directed muta-genesis was performed as described by Ho et al. [28]. Forthe first PCR, primers MS3324 and 808 were used withplasmid pMS54 [15] as template, Fig. 1. The second PCRused primers MS3323 and egl4 504 with pMS54 as template.The third PCR used primers 808 and egl4 504 with the twoproducts from the previous PCRs as template. The DNAfragment obtained (the product from the third PCR)contained the nucleotide sequence from nucleotide 964to the 30 end of egl4 with CACCACCACCACCACCACinserted just prior to the stop codon, between nucleotides1078 and 1079. This fragment and plasmid pMS54 was cutwith restriction enzymes BssHII and XhoI. The fragmentwas ligated into pMS54, which had been cut with the samerestriction enzymes. The resulting plasmid was called pJK2.The egl4 gene was cut out from pJK2 with restrictionenzymes EcoRI and XhoI, blunt-ended and cloned betweenthe NdeI and KspI sites of the plasmid pAMH110 [27]. Theresulting plasmid, pJK3, thus coded for Cel61A with sixhistidines on the C-terminus under the control of the cbh1promotor and terminator. A correct mutation and ligationwas confirmed by sequencing the 30 end of the egl4 cDNA inpJK3.

T. reesei transformation methods. The plasmid pJK3 wasused to transform T. reesei Rut-C30 as described by Penttilaet al. [29]. The plasmid pJK3 was opened with the restric-tion enzymes Eco RI and Sph I to facilitate the incorporationof the gene in the chromosome. T. reesei was cotransformedwith the plasmid pRLMex30, which carries a hygromycinresistance cassette [30]. The transformed cells were selected

by first streaking on plates containing 1.5% KH2PO4, 0.5%(NH4)2SO4, 2% glucose, 1.8% granulated agar, 0.1% Triton,0.06% MgSO4, 0.08% CaCl2, 0.0005% FeSO4·7H2O,0.00016% MnSO4·H2O, 0.00014% ZnSO4·7H2O, 0.00037%CoCl2·6H2O and 0.01% hygromycin. Stable colonies werestreaked on potato dextran plates containing 3.9% potatodextran to produce spores. The selection of Cel61A-producing strains was performed by cultivating in shakingflasks at 28 8C in a minimal medium [31] with 4% lactose ascarbon source and inducer. The cultivation was inoculatedwith 107 spores. The cultivation was stopped by filtering thecells with a glass-fibre filter (Millipore, Bedford, MA,USA). The best transformant was selected by running a dotblot or SDS/PAGE and a Western blot on the culture filtrateas described below.

Bioreactor cultivation

T. reesei was grown in a 15-L fermentor in medium con-taining 4% lactose, 0.4% peptone, 0.1% yeast extract, 0.4%KH2PO4, 0.28% (NH4)2SO4, 0.06% MgSO4·7H2O, 0.08%CaCl2·2H2O, 0.0005% FeSO4·7H2O, 0.00016% MnSO4·H2O,0.00014% ZnSO4·7H2O, 0.00037% CoCl2·6H2O. The culti-vation was inoculated with mycelia grown in shaking flasksfor 4 days; 2 days at 29 8C followed by two days at 22 8C.The fermentor cultivation was performed at 18 8C and at pH6 for 5 days. The fungus grew slowly at this temperature andpH but the degradation products of Cel61A seen at normalgrowth conditions were drastically decreased.

Purification conditions

The pH of the T. reesei culture filtrate was adjusted to 7.2with 0.5 M NaH2PO4, and the salt concentration adjusted to0.5 M with 4 M NaCl. The pH and salt-adjusted culturefiltrate (250 mL) was then applied to a 250-mL columnpacked with chelating Sepharose FF (Pharmacia, Uppsala,Sweden). The chelating column had previously beencharged with 250 mL 0.1 M NiSO4, and was subsequentlyequilibrated with 50 mM sodium phosphate and 0.5 M NaClat pH 7.2. A stepwise elution profile was chosen, with thefirst step being 20 mM imidazole, in which some con-taminants were eluted. These contaminants were also seenin the culture filtrate from Rut-C30. Cel61A was then elutedwith 200 mM imidazole. Imidazole was dissolved in 50 mM

sodium phosphate and 0.5 M NaCl at pH 7.2. The fractionscontaining Cel61A were pooled and concentrated in anAmicon ultrafiltration unit with PM10 membrane (Amicon,Millipore, Bedford, Ma, USA). The concentrate was appliedto a SEC column, a 10-L Sephacryl S-100 H (Pharmacia,Uppsala, Sweden) with 50 mM Na-acetate, 0.1 M NaCl atpH 5.0 as mobile phase. The fractions from the SECseparation were analysed for Cel61A by performing SDS/PAGE and Western blotting as described below; fractionscontaining Cel61A were pooled and concentrated.

Electrophoresis and Western blotting

NuPAGETM precast gels were used in XcellTM Mini-cellelectrophoresis equipment and were silver stained with theSilverXpressTM kit (Novex, San Diego, CA, USA). Nitro-cellulose membrane was from Bio-Rad (Hercules, CA,USA). Western blots were performed according to

q FEBS 2001 Characterization of Cel61A (Eur. J. Biochem. 268) 6499

manufacturers recommendations (Qiagen) with antibodiesagainst Cel61A [15] and Ni-nitrilotriacetic acid conjugate(Qiagen, Chatsworth, CA, USA).

Characterization

The following substrates were used in the characterizationexperiments. Carboxymethylcellulose (CMC), hydro-xyethylcellulose (HEC), and polygalacturonic acid methylester were from Fluka (Buchs, Switzerland). Barley b-glucan,lichenan, glucomannan (konjac), arabinoxylan (from rye),pullulan, and pachyman were from Megazyme (Bray,Ireland). Locust bean gum, arabinogalactan (from larchwood), laminarin, polygalacturonic acid, chitin, chitosan,p-nitrophenyl-b-D-xylopyranoside, p-nitrophenyl-b-D-glucopyranoside, p-nitrophenyl-a-L-arabinofuranoside,

p-nitrophenyl-b-D-galactopyranoside, and p-nitrophenyl-b-D-mannopyranoside were from Sigma (St Louis, MI,USA). Starch, Avicel, cellopentaose, cellotetraose, andcellotriose were from Merck (Darmstadt, Germany).Pustulan was from Calbiochem (La Jolla, CA, USA).Phosphoric acid swollen cellulose (PASC) was preparedfrom Avicel according to Wood [32].

Analysis for decrease in viscosity

The viscosity analysis was performed as described by Autioet al. [33]. The substrates were dissolved in 50 mM Na-acetate at pH 5.0. The substrate concentration was 1.2%(w/v) for CMC, 1.4% (w/v) for HEC, 1.5% (w/v) forb-glucan, 1% (w/v) for pullulan, 1% (w/v) for lichenan,1.5% (w/v) for polygalacturonic acid methyl ester, 1% (w/v)

Fig. 1. A schematic figure of the construction of

the plasmid pJK3. The plasmid pJK3 encodes for

Cel61A between the cbh1 promotor and terminator

and was used for transformation of T. reesei strain

Rut-C30.

6500 J. Karlsson et al. (Eur. J. Biochem. 268) q FEBS 2001

for locust bean gum, 1.5% (w/v) for polygalacturonic acid,and 1% (w/v) for arabinoxylan. 1.8 mL of the substrate wasadded to a u-tube, which was thermostated in a water-bath at40 8C. Enzyme solution (0.2 mL) was added and mixedwith the substrate. The Cel61A concentration was 1 mM andthe Cel7B concentration was 0.5 nM. The flow time, t,through the capillary was measured at 10-min intervals over30 min The viscosity, n, was calculated using the formula:

nexperimental ¼ ðtexperimental 2 tbufferÞ/tbuffer

For each experiment, a plot was made with incubation time(min) vs reciprocal viscosity (1/n ). The slope was calculatedwhich had the unit viscosity units, VU. The slope is onlylinear in the initial stage of the hydrolysis of the polymer andusually for a specific polymer concentration. As the linearrange was determined for b-glucan and arabinoxylan alone,the results for the other polymers can therefore only bequalitatively analysed.

Production of reducing ends

The reducing ends were analysed using the dinitrosalicylicacid reagent (DNS) [34]. Glucose was used for the standardcurve. Cel61A (1 mM) and Cel7B (0.5 nM) were incubatedwith the substrates for 4–24 h. The substrate concentrationwas 10 g·L21, except for b-glucan which was 5 g·L21. Allexperiments were performed in 50 mM Na-acetate atpH 5.0.

Carbohydrate polymer hydrolysis

Avicel, PASC, and CMC were hydrolysed with 1 mM

Cel61A for 26 h or 0.25 mM Cel7B for 1 h at 40 8C. Theconcentration of Avicel and PASC was 5 g·L21 and theCMC concentration was 10 g·L21. The hydrolysis of Aviceland PASC was terminated by filtering the sample with a0.2-mM filter. The filtrate was analysed for oligosaccharideswith HPLC as described below. The hydrolysis of CMC wasnot terminated but the reaction mixture was analysedimmediately after the incubation was completed.

Oligosaccharide hydrolysis

Cellotriose, cellotetraose and cellopentaose were hydro-lysed with 1 mM Cel61A at 40 8C for 4 h. The enzymeconcentration was lower and the hydrolysis time was signi-ficantly shorter for Cel7B; 1 nM at 5 min for cellotetraoseand cellopentaose and 50 nM at 4 min for cellotriose. Theproduct formation was analysed with a HPLC system asdescribed below.

Oligosaccharide analysis

Soluble sugars, glucose, cellobiose, cellotriose, cellotetraoseand cellopentaose were analysed with a HPLC system with ananion exchange Carbopac PA100 column and pulsed ampero-metric detection (Dionex, Sunnyvale, CA, USA). The oligo-saccharides were eluted isocratically with 50 mM NaOH andwith a simultaneous linear gradient of Na-acetate from 50 to300 mM. The detector was set with the following pulsepotentials and durations: E1 ¼ 0.05 V, 200 ms (sampling);E2 ¼ 0.75 V, 200 ms (cleaning); E3 ¼ 2 0.15 V, 400 ms

(reduction), and a response time of 1 s. Glucose, cellobiose,cellotriose, cellotetraose and cellopentaose were used asstandards.

SEC

CMC, 10 g·L21, and b-glucan, 5 g·L21, were hydrolysedwith 1 mM Cel61A at room temperature. The hydrolysis ofthe polymers was analysed on a size exclusion column (TSKgel GMPWXL; TosoHaas, Stuttgart, Germany) with arefractive index detector ERC-7515A (ERMA Inc., Tokyo,Japan). The polymer and hydrolysis products were elutedwith 50 mM Na-acetate at pH 5.0 as buffer at a flow rate of0.5 mL·min21.

Adsorption to Avicel

The adsorption of Cel61A to Avicel was studied by incu-bating 5 mM Cel61A with 10 g·L21 Avicel for 4 h at 4 8Cwith continuous mixing. The experiment was stopped byfiltering the substrate, with adsorbed enzyme, using a0.2-mm filter. The Cel61A concentration in the filtrate wasdetermined by measuring the absorbance at 280 nm.

Glucuronidase activity

The glucuronidase activity was assayed as described bySiika-aho et al. [35] with 20-O-(4-O-methyl-glucuronic acid)-b-D-xylobiose as substrate. The substrate (2.5 mg·mL21) wasincubated with 0.5 mM of Cel61A for 10 min at 50 8C.

Activity on p-nitrophenol conjugates

The activity against p-nitrophenyl-b-D-xylopyranoside,p-nitrophenyl-b-D-glucopyranoside, p-nitrophenyl-a-L-arabinofuranoside, p-nitrophenyl-b-D-galactopyranoside,and p-nitrophenyl-b-D-mannopyranoside was assayed asdescribed by Bailey et al. [36]. Cel61A (1 mM) was incu-bated with the appropriate substrate (1 mM) for 10 min at50 8C.

Other enzymes

Cel7B was purified from a culture filtrate of T. reeseiQM9414. The culture filtrate was buffer-exchanged to20 mM ammonium acetate at pH 7.0 in a Sephadex G25column (Pharmacia, Uppsala, Sweden). The purificationwas performed in two anion-exchange steps. Firstly, in aSource Q (Pharmacia, Uppsala, Sweden) with a lineargradient of ammonium acetate at pH 7.0 from 20 mM to1 M. The second step was performed as above except thatthe pH was decreased to 6.5. The purification was performedat room temperature using a FPLC system (Pharmacia,Uppsala, Sweden). The Cel7B preparation was pure asjudged from a silver stained SDS/PAGE gel as describedabove.

R E S U L T S

Cloning, expression and cultivation

A Cel61A recombinant protein, where six histidines wereinserted on the C-terminal of the CBM, was successfully


constructed by overlap extension PCR, Fig. 1. The geneencoding the enzyme was between the cbh1 promotor andterminator. This expression vector was transformed intoT. reesei Rut-C30. The cultivation of T. reesei expressingthe recombinant Cel61A was performed at 18 8C and atpH 6. The low temperature and high pH were chosen due tosome proteolysis of the fusion protein when the fungus wascultivated under normal conditions, 28 8C and pH 4.5. Thecultivation was successful, and the enzyme was produced insignificant amounts into the culture filtrate. There were noreliable methods to determine the Cel61A concentration inthe culture filtrate, however, 120 mg of Cel61A could bepurified from 1 L of culture filtrate. The total proteinconcentration in the culture filtrate was 1.7 g·L21.

Purification of Cel61A

The culture filtrate was pH and salt adjusted prior to beingapplied to the chelating column, which was charged withNi2þ. Fractions which contained Cel61A were eluted with200 mM imidazole (data not shown). The fractions contain-ing Cel61A were concentrated and then further purified on aSEC column. The SEC step was used to separate Cel61Afrom some low molecular mass contaminants, which wereprobably degradation products from Cel61A. The purity ofthe enzyme preparation was determined using silver stainedSDS/PAGE and Western blots with antibodies againstCel61A and the His-tag, Fig. 2. The SDS/PAGE showed twomajor bands from the culture filtrate (Fig. 2A, lane 1). Theband at 55 kDa was Cel61A and at 66 kDa most probablyCel7A, which is the major protein produced by T. reesei onlactose. Cel61A was reasonably pure after the first step(Fig. 2A, lane 3), and no impurities could be observed afterthe final purification step (Fig. 2A, lane 5). The Western blotagainst Cel61A showed some contaminants after the firstpurification step (Fig. 2B, lane 3), which were absent afterthe final step (Fig. 2B, lane 5). The impurities could also beobserved after the first purification step on the Western-blotagainst the His-tag, Fig. 2C. These contaminants could notbe observed after the final purification step (Fig. 2C, lane 5).

Characterization of Cel61A

Activity on carbohydrate polymers. The endo-activity ofCel61A was tested by the viscosity assay and reducing endsassay on a number of substrates, Table 1. The hydrolysiswas performed with 1 mM Cel61A at 40 8C. The activity ofCel7B was analysed on some of the substrates for com-parison. Cel61A showed viscosity activity, i.e. reduced theviscosity, on CMC, HEC and b-glucan. Cel7B had activityon the same substrates and in addition, significant activitywas found on arabinoxylan. The xylanase activity for Cel7Bhas been shown previously [37,38]. The specific activity forCel61A was approximately 104 times lower than for Cel7Bon these substrates. The activity was further tested withreducing ends assays on several substrates. Cel61A showedlow activity on CMC, b-glucan and lichenan. On thesesubstrates the activity for Cel7B was several orders ofmagnitude higher. Cel61A had no detectable activity on anyof the other studied substrates.

The production of oligosaccharides by 1 mM Cel61A at40 8C was studied on a number of substrates, (see Table 2).Cel61A produced mainly cellobiose on microcrystalline

(Avicel) and amorphous (PASC) cellulose substrates. Thecellobiose concentration was higher with PASC as substratecompared to Avicel. Small amounts of glucose andcellotriose were produced on both substrates. Cellobiosewas the main product with CMC as substrate, together witha low amount of glucose. For comparison, the productformation was studied for Cel7B on the same substrates.Overall, the product formation for Cel7B was similar toCel61A even though Cel7B had significantly higher specificactivity.

Activity on cello-oligosaccharides. The activity of Cel61Aon cellotriose, cellotetraose and cellopentaose was investi-gated, Table 3. Cellopentaose was hydrolysed to cellobioseand cellotriose, with small amounts of glucose and cello-tetraose. The main product from the hydrolysis ofcellotetraose was cellobiose even though glucose andcellotriose were also produced. Hydrolysis of cellotrioseproduced only small amounts of glucose and cellobiose.For comparison, Cel7B was also used to hydrolyse

Fig. 2. SDS/PAGE and Western blot of culture filtrate and from

purification of Cel61A. (A) SDS/PAGE. (B) Western blot against

Cel61A. (C) Western blot against the histidine-tag. Lane 1, culture

filtrate; lane 2, pH- and salt-adjusted culture filtrate; lane 3, main

fraction from the peak containing Cel61A from the first purification

step; lane 4, pooled fractions containing Cel61A from the first

purification step; lane 5, fraction containing Cel61A from the second

purification step; lane 6, molecular mass standards.


oligosaccharides. The hydrolysis rate was several orders ofmagnitude higher for Cel7B than for Cel61A, as judged bythe much lower enzyme concentration and hydrolysis timeneeded for Cel7B. One significant difference between theenzymes was that cellotriose was hydrolysed by Cel7B butcellotriose was a poor substrate for Cel61A.

SEC studies of product formation

The decrease in polymer molecular mass due to Cel61Aactivity was studied by size exclusion chromatography onCMC and b-glucan (Fig. 3). The hydrolysis was performedat room temperature with 1 mM Cel61A. The molecularmass was reduced for both CMC and b-glucan by theCel61A activity. The peak at 11 mL elution volume wasfrom the solvent. The Cel61A activity on both substrateswas similar in the sense that the molecular mass of thepolymers was reduced without any major production offragments with low relative molecular mass. Even after 15 hof hydrolysis, the molecular mass of CMC was above thelargest dextran standard used, 80 kDa. The column has a

separation range up to 1 MDa. The relative molecular massof b-glucan was decreased to 80 kDa after 3 h andcontinued to decrease to approximately 50 kDa after 15 h.

Adsorption to Avicel

The adsorption of Cel61A to cellulose was studied byincubating Cel61A with microcrystalline cellulose substrate(Avicel). After 4 h incubation at 4 8C, 55% of Cel61A wasadsorbed to Avicel. We have verified that the relatively lowadsorption is not due to proteolytic degradation leading toremoval of the CBM. The purified Cel61A has the correctmolecular mass (shown by SDS/PAGE) and the enzymebinds to a Ni-chelating column thus showing that the proteinis intact as the histidine tag is on the CBM.

Screening of Cel61A activity on low molecular masschromogenic substrates

The activity of Cel61A on a number of p-nitrophenolchromogenic substrates was studied (see Materials and

Table 1. Screening of hydrolytic activity for T. reesei Cel61A. Carbohydrate polymer hydrolysis by Cel61A and Cel7B analysed by viscosity and

reducing ends assays. Hydrolysis at 40 8C with 1 mM Cel61A and 0.5 nM Cel7B. ND, not determined.

Substrate

Cel61 Cel7B

Viscosity

(VU·mol21)

Reducing ends

(M·mol21·h21)

Viscosity

(VU·mol21)

Reducing ends

(M·mol21·h21)

Carboxymethyl cellulose 3.3 £ 106 11 5.8 £ 1010 1.4 £ 105

Hydroxyethyl cellulose 2 £ 106 0 2 £ 1010 2.1 £ 104

b-Glucan 1.8 £ 106 32 2.1 £ 1010 2 £ 105

Lichenan ND 21 ND 4.3 £ 104

Laminarin ND 0 ND 0

Pachyman ND 0 ND 0

Arabinoxylan 0 ND 1.7 £ 109 ND

Glucomannan 0 ND ND ND

Locust bean gum 0 ND ND ND

Arabinogalactan ND 0 ND ND

Polygalacturonic acid 0 ND ND ND

Poly galcturonic acid methyl ester 0 ND ND ND

Pullulan 0 ND ND ND

Starch ND 0 ND ND

Chitin ND 0 ND ND

Chitosan ND 0 ND ND

Table 2. Hydrolysis of carbohydrate polymers by Cel61A and Cel7B analysed by HPLC for determination of product formation. The

hydrolysis was performed at 40 8C with 5 g·L21 Avicel and PASC and 10 g·L21 CMC. The hydrolysis was with 0.25 mM Cel7B for 1 h and 1 mM

Cel61A for 26 h.

Substrate Enzyme

Glucose

(mM)

Cellobiose

(mM)

Cellotriose

(mM)

Cellotetraose

(mM)

Cellopentaose

(mM)

Avicel Cel61A , 5 5 , 2 – –

PASC Cel61A , 5 19 2 – –

CMC Cel61A 16 57 – 3 2

Avicel Cel7B 108 193 , 2 – –

PASC Cel7B 636 823 24 – –

CMC Cel7B 321 257 – – –


methods). The substrates were selected to investigate whetherthe enzyme had b-xylosidase, b-glucosidase, a-arabinosi-dase, a-galactosidase or b-mannosidase activity. Cel61A didnot show any detectable activity in the screening of thep-nitrophenyl conjugates. A possible a-glucuronosidaseactivity was analysed by use of 20-O-(4-O-methyl-glucuronic

acid)-b-D-xylobiose as substrate. Cel61A did not show anydetectable glucuronosidase activity.

D I S C U S S I O N

In this study, we have performed a homologous expressionof Cel61A as a fusion protein with a histidine tag, under astrong promotor. We decided to express the protein inT. reesei Rut-C30 due to its ability to express high levels ofcellulases [39]. Furthermore, a homologous expression ofCel61A increased the probability for a correct glycosylationof the enzyme. T. reesei Cel5A, Cel7B and Cel12A havepreviously been expressed in S. cerevisiae [13,40,41]; Cel5A,Cel6A, Cel7A, Cel7B and Cel45A have been expressed inAspergillus oryzae [42] and Cel7B has also been expressed inYarrowia lipolytica [43]. The major drawback in theseheterologous expression experiments was the difference inglycosylation. Cel12A was glycosylated when expressed inS. cerevisiae, which is remarkable because it is not glyco-sylated in its native form [13]. Cel7B was heavily over-glycosylated both when expressed in S. cerevisiae andY. lipolytica [41,43]. Heterologous expression in A. oryzaeseems to be a better option from this point of view, as only aslight overglycosylation was reported [42]. The over-glycosylation did not affect the specific activity for theS. cerevisiae-expressed Cel7B [41], however, no comparativestudies with the wild-type enzyme were reported. Previouscomparative studies of Cel6A expressed in S. cerevisiae andnative enzyme have shown that the specific activity wasdecreased in the heterologously expressed Cel6A [44].

Thus, in heterologous expression systems it has beenpossible to obtain active cellulases, but to our knowledge,there has not yet been reported a specific activity close to thatof the native enzyme. A characterization of a heterologouslyexpressed enzyme might therefore not be valid for com-parisons to the wild-type enzyme. We chose a homologousexpression system in T. reesei for production of Cel61A forcharacterization of its enzymatic properties. To facilitate thepurification of Cel61A from the other cellulases the histidinetag was constructed on the C-terminus of Cel61A. This isthe first time that a histidine tag has been used as apurification aid in the T. reesei expression system.

Cloning and purification

The fusion protein, Cel61A with six histidines on theC-terminus, was expressed in significant amounts and was

Fig. 3. Hydrolysis of CMC and b-glucan by Cel61A analysed by

SEC. (A) 10 g·L21 CMC was hydrolysed by Cel61A, 1 mM, at 20 8C.

(B) 5 g·L21 b-glucan was hydrolysed by Cel61A, 1 mM, at 20 8C. The

degradation of the substrate was analysed at certain time intervals; 0, 3,

9, and 15 h.

Table 3. Hydrolysis of oligosaccharides by Cel61A and Cel7B analysed by HPLC for determination of product formation. The starting

concentration of the oligosaccharide is in parentheses. The hydrolysis was performed at 40 8C for 1 h for Cel61A and for 5 min for Cel7B on

cellotetraose and cellopentaose and 4 min on cellotriose. The Cel61A concentration was 1 mM and the Cel7B concentration was 1 nM for

cellotetraose and cellopentaose and 50 nM for cellotriose.

Substrate Enzyme

Glucose

(mM)

Cellobiose

(mM)

Cellotriose

(mM)

Cellotetraose

(mM)

Cellopentaose

(mM)

Cellopentaose Cel61A 5.5 24.5 26.6 4 97 (120)

Cellotetraose Cel61A 8.5 38.9 10.5 136 (150) –

Cellotriose Cel61A 5.9 6 199 (200) – –

Cellopentaose Cel7B , 5 9.6 19.7 5.1 174 (200)

Cellotetraose Cel7B – 16.5 17.2 317 (345) –

Cellotriose Cel7B 102.3 65.3 189 (244) – –


easily purified using conventional methods. The obtainedmolecular mass obtained with SDS/PAGE (55 kDa) of thefusion protein is in agreement with the molecular massreported for the wild-type enzyme [15]. This indicates thatthe purified fusion protein is intact, i.e. that the CBM has notbeen cleaved off. Also, the molecular mass determinationindicates that the fusion protein is glycosylated to a similardegree as the wild-type protein. When cultivating T. reeseiexpressing recombinant Cel61A under normal conditionsfor cellulase production (28 8C and pH 4.5) some degra-dation products were detected (results not shown). It ispossible that the addition of the histidine tag on theC-terminus of Cel61A makes it more susceptible toproteases than the wild-type enzyme. This problem wasovercome by cultivating at lower temperature (18 8C) andhigher pH (pH 6). The cultivation became slower but theproteolysis of Cel61A was clearly decreased.

Characterization of hydrolysis properties

Cel61A showed activity on substrates containing b-1,4glucosidic bonds, i.e. CMC, HEC, b-glucan and lichenan(Table 1). The Cel61A activity on these substrates wasseveral orders of magnitude lower than that observed forCel7B. Cel61A expressed in S. cerevisiae showed activityagainst Avicel, PASC, CMC and b-glucan [15]. It is difficultto compare the activity of Cel61A reported here withCel61A expressed in S. cerevisiae due to the fact that theenzyme concentration was not determined in the latter case.The product pattern, i.e. formation of oligosaccharides, wassimilar for Cel61A compared to Cel7B, e.g. cellobiose wasmainly formed with Avicel as substrate.

Size exclusion chromatography analysis of the productsfrom Cel61A hydrolysis of CMC and b-glucan revealed atrue endoglucanase activity, Fig. 3. The molecular massdecreased during the hydrolysis of both polymericsubstrates. However, the activity was still very lowcompared to Cel7B, which was able to hydrolyse thesubstrates to higher extent at a significantly lower enzymedosage [45].

It could be argued that the low endoglucanase activityobserved for Cel61A was due to a small contamination ofcellulases in the Cel61A preparation. It has been reportedthat two Cel6A preparations purified by affinity chroma-tography had slightly different contaminations by Cel5Aand Cel7B [46]. These minute contaminations changed thehydrolytic activity of the preparations significantly. A re-purification of Cel61A was performed to clarify if contami-nation could explain the endoglucanase activity. AfterCel61A was bound to the chelating column it was washedwith approximately 40 column volumes of starting bufferbefore the enzyme was eluted with 200 mM imidazole.Thus, a repeat of the first purification step, with the additionof an extended wash procedure, was performed (seeMaterials and methods). As no proteins were eluted fromthe culture filtrate of the nontransformed Rut-C30 with200 mM imidazole, we expect that the major cellulases donot bind to the chelating column. However, if a minuteamount of the other cellulases do bind and are eluted withCel61A, the extended washing procedure would decreasethe contamination of the Cel61A preparation. The activityfor the re-purified Cel61A was analysed on b-glucan usingthe DNS assay and on cellopentaose by analysing the

products by HPLC. The re-purified Cel61A had the samespecific activity as the original Cel61A preparation. Inaddition, as no activity was found on arabinoxylan forCel61A (Table 1), we can conclude that Cel7B was notpresent in the Cel61A preparation as T. reesei Cel7B hasxylanase activity [37,38]. Furthermore, the product patternfor Cel61A on CMC was different from that observed forCel5A, Cel12A and Cel45A [47]. Also, a test for contami-nating activities has been made using a chromophoricsubstrate. Both the original Cel61A preparation and the re-purified Cel61A showed activity against p-nitrophenyl-b-D-cellotrioside and they had the same specific activity.The obtained activity was orders of magnitude lower thanwhat was observed for T. reesei Cel5A. Thus, we have beenable to show that the other known endoglucanases ofT. reesei are not present in the purified Cel61A preparation,and we conclude that the observed endoglucanase activity isa Cel61A activity.

Ab Cel61A (Cel1) from A. bisporus expressed inS. cerevisiae has been reported to have no activity onCMC, Avicel or filter paper [26]. No details about the assayswere given, therefore it might be possible that the AbCel61A activity was below the detection limit. However,one should be careful when comparing glycoside hydrolasesfrom different organisms within the same family becausethey do not have to have the same activity, e.g. glycosidehydrolase family 5 that contains cellulases, mannanases andxylanases among others.

The function of Cel61A

From Table 1 we conclude that Cel61A does not show thefollowing activities: b-1–3-glucanase, b-1,6-glucanase,mannanase, xylanase, b-1,3-galactosidase, amylase, pectinaseor chitinase. Furthermore, Cel61A is not a glucuronidase.The enzyme clearly has endoglucanase activity as shownby the ability to perform endo-type cleavages of b-1,4glycosidic bonds in carbohydrate polymers. However, theCel61A endoglucanase activity is relatively low comparedwith Cel7B. The question remains: what is the biologicalfunction of Cel61A? This enzyme is induced together withthe other cellulases in T. reesei [15]. This implies thatCel61A is involved in carbohydrate polymer hydrolysis. Thespecific endoglucanase activity for Cel7B is several ordersof magnitude higher than for Cel61A. It is therefore unlikelythat the fungus would produce Cel61A for its endoglucanaseactivity when it is already producing more efficientendoglucanases. In addition, Cel61A has been included inexperiments searching for defibrillating activity togetherwith several T. reesei cellulases. No defibrillating activitywas observed for Cel61A. We observed that Tr Cel61Aadsorbs to cellulose (Avicel) and Armesilla et al. [25] havereported that Ab Cel61A adsorbs to microcrystallinecellulose. The Ab Cel61A is also induced when the fungusis growing on cellulose and repressed in glucose medium[48]. Even though no enzymatic activity has been found forAb Cel61A, this clearly indicates that the enzyme has afunction in carbohydrate polymer hydrolysis. It is possiblethat both Tr Cel61A and Ab Cel61A are active against somespecific parts of more complex natural cellulosic substrates.However, further studies are needed to reveal the function ofthe glycoside hydrolase family 61 enzymes.


A C K N O W L E D G E M E N T S

Michael Bailey is thanked for the large scale cultivation. This research

was supported by grants from the Nordic Energy Research Programme.

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Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei

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