Molecular cloning, gene expression analysis and structural modelling of the cellobiohydrolase I from...

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Enzyme and Microbial Technology 46 (2010) 74–81

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Enzyme and Microbial Technology

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olecular cloning, gene expression analysis and structural modelling of theellobiohydrolase I from Penicillium occitanis

atma Bhiri a, Ali Gargourib, Mamdouh Ben Ali c, Hafedh Belghithb,onia Blibecha, Semia Ellouz Chaabounia,∗

Unité Enzymes et bioconversions, Ecole Nationale d’Ingénieurs de Sfax, route soukra km 3.5, BP «1173», 3038 Sfax, TunisiaLaboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP «K», 3038 Sfax, TunisiaLaboratoire d’ Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax, route Sidi Mansour, BP «K», 3038 Sfax, Tunisia

r t i c l e i n f o

rticle history:eceived 16 March 2009eceived in revised form 3 October 2009ccepted 6 October 2009

eywords:enicillium occitanis

a b s t r a c t

The filamentous fungus Penicillium occitanis produces a complete set of cellulolytic enzymes neededfor efficient solubilization of native cellulose. Cellobiohydrolase I (CBHI), the most abundant cellulolyticenzyme produced by this micro-organism, has been purified and characterized.

In this report, the cDNA encoding this enzyme, isolated from a cDNA bank of P. occitanis, and theequivalent genomic sequence have been cloned. DNA sequencing revealed that the cbh1 gene is intronlessand has an open reading frame of 1587 bp encoding a putative polypeptide of 529 amino acids. Thispolypeptide has a predicted molecular mass of 52.5 kDa and consists of a fungal cellulose binding module

DNA and genomic librariesellobiohydrolaseenomic organisationellobiose inhibitionD structure

(CBM) and a catalytic module, linked together by a serine–threonine-rich region.Northern blot analysis showed that cbh1 mRNA expression is partially constitutive since, besides being

highly induced by cellulose, it is slightly repressed by glucose.Comparative investigation of different cellobiohydrolases I 3D structures by molecular modelling

showed that poor hydrogen bonding, together with a more open configuration of the active site accountthe r

. Introduction

Cellulose is an insoluble polysaccharide composed of long lin-ar chains of �-1,4-linked glucose units. It is the most abundantenewable biomass on earth since its microbial breakdown cre-tes the potential for the production of energy [1]. Cellulolyticnzymes can be divided into three types: endo-�-1,4-glucanase (EC.2.1.4), exo-�-1,4-glucanase (cellobiohydrolase, EC 3.2.1.91), and-glucosidase (EC 3.2.1.21). They are collectively known as cellu-

ases and act in a synergistic manner to facilitate complete cleavagef �-1,4-glycosidic bonds of the cellulose to produce glucose [1].

Cellulases are used in waste recycling processes and in therocessing of cellulose-rich raw materials for food, detergent,aper and textiles industries. Recently, cellulases gained signifi-

Abbreviations: cbh1, cellobiohydrolase I; CBM, cellulose binding module; GHF,lycoside hydrolase family.∗ Corresponding author. Tel.: +216 74 274 418; fax: +216 74 275 595.

E-mail address: semia.chaabouni@enis.rnu.tn (S. Ellouz Chaabouni).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.10.002

elative insensitivity of P. occitanis CBHI to product inhibition.© 2009 Elsevier Inc. All rights reserved.

crystalline cellulose. These exo-acting enzymes possess tunnel-likeactive sites, which can only accept a substrate chain via its terminalregions [3]. Thus, CBH enzymes act by threading the cellulose chainthrough the tunnel, where successive cellobiose units are removedin a sequential manner.

Cellobiohydrolases I (CBHI) are modular enzymes consisting ofa minimum of one catalytic module and one cellulose binding mod-ule (CBM) connected by a proline/serine/threonine-rich linker [3,4].CBHs from different microbial sources belong to families 6 and 7of glycoside hydrolases [5]. The most characterized members ofthe family 7 are cellobiohydrolase Cel7A from Trichoderma ree-sei and cellobiohydrolase Cel7D from Phanerochaete chrysosporium.The structure of both CBHs consists of two �-sheets that pack face-to-face to form a �-sandwich [6,7]. The cellobiohydrolase Cel7Afrom T. reesei is composed of long loops, on one face of the sandwich,that form a cellulose binding tunnel of 50 Å. The catalytic residuesare glutamate 212 and 217, which are located on opposite sides ofthe active site, separated by an intervening distance consistent witha double-displacement retaining mechanism [3]. The mechanism of

The fungus Penicillium occitanis has been shown to possess ahigh capacity for the production of cellulases [8,9] having high cel-

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ulose degradation efficiency [10]. Two cellobiohydrolases (CBHInd CBHII) and two �-glucosidases have been purified from thisungus and their properties were characterized [11]. Comparedith other P. occitanis cellulases, the amount of CBHI secreted

s much higher (50% of total proteins). CBHI of P. occitanis wasescribed as an enzyme producing cellobiose from cellulose. Thisnzyme shares some biochemical properties with the cellobio-ydrolases of glycoside hydrolases family 7 [11]. CBHI was alsoeported to act synergistically with cellobiohydrolases II and to benhibited by cellobiose [11]. But, compared to P. chrysosporium, T.eesei and Talaromyces emersonii cellobiohydrolases I, P. occitanisBHI exhibited less pronounced product inhibition [11–13]. Inter-stingly, this enzyme showed a mannanase activity using the Locustean Gum as substrate (unpublished data).

Cellobiohydrolases genes have been cloned and characterizedrom a variety of fungal sources [14–17]. However, there areo reports of gene sequences coding for extracellular cellulolyticnzymes from P. occitanis.

As an initial step toward elucidating the genetic basis for theroduction of cellulases from this organism, we report here theloning and the characterization of the cbh1 cDNA and its corre-ponding gene from a genomic bank of P. occitanis. We also describehe similarity between the deduced CBHI protein and other fungalellobiohydrolases.

In order to explain the origin of the resistance to cellobiosenhibition, exhibited by this enzyme, structural modelling of the. occitanis CBHI has been performed based on the X-ray crystallo-raphic structure of T. emersonii cellobiohydrolase I.

. Materials and methods

.1. Strains

P. occitanis CL100 and Pol6 were provided by Professor Tiraby, CAYLA Company,oulouse—France. The Pol6 strain is a hypercellulolytic mutant selected by Jain et al.8] after eight rounds of mutagenesis from the CL100 mother strain. The CT1 strains a hyperpectinolytic and a fully constitutive mutant selected after a single roundf mutagenesis from the same parental strain [18]. Escherichia coli strain: Top 10 F′

(F′ lacIq Tn10 (TetR)) mcrA �(mrr-hsdRMS-mcrBC) �80lacZ�M15 �lacX74 recA1raD139 �(ara-leu)7697 galU galK rpsL (StrR)endA1 nupG; Invitrogen) was used ashost for the pUC18 and pMOSblue T cloning vectors.

.2. Vectors

pMOSblue T-vector (Amersham) was used for the cloning of PCR fragments;MOS Elox plasmid (Amersham) was used for the construction of the cDNA library;MOS Elox derived from the excision of the �MOS Elox after infection of the BM 25.8train and pUC18 was used for the construction of the genomic library.

.3. Media and growth conditions

Potato dextrose agar (PDA, Merck Co.) was used for the propagation and thetorage of the fungal strains. The liquid medium of Mandels and Weber [19] modifiedy Ellouz Chaabouni et al. [9] was also used. The carbon source is 2% cellulose (AvicelH 101 Fluka, Switzerland) or 2% glucose. The cultures were grown in Erlenmeyerasks (100/500 ml) at 30 ◦C. Luria broth medium was used for the cultivation ofacterial strains.

.4. Amplification of the cbh1 cDNA

Reverse transcription was performed for 60 min at 37 ◦C on cellulose-inducedolyA+ mRNA as a template with an oligo-dT primer [5′-GGGATCCGCGGCCGC(T15)].he mRNA extracted from glucose grown culture, was used as a control.

Based on the sequences of fungal cellobiohydrolases I present in theatabase, primers were designed for the amplification of the cbh1 cDNA.he primer sequences are as follows: P1: 5′-TGCGGTCTCAACGGCGCCCTCTA-3′ ,2: 5′-ATGGACGCCGACGGTGG-3′ and P3: 5′-GAGATGGATATCTGGGAGGCCAA-′

l Technology 46 (2010) 74–81 75

2.5. Construction and screening of the cDNA library

The cDNA library was constructed using 4 �g of mRNA according to the manufac-turer’s instructions of the cDNA Synthesis System kit (Amersham). Library screeningwas done with the RT-PCR amplified fragment described previously, as a probe.

2.6. Construction and screening of the genomic library

Total genomic DNA of CL100 was prepared according to Aifa et al. [20], partiallydigested with Sau3AI and fractionated by sucrose gradient (10–40%) according toHopwood et al. [21]. The fragments sizing from 6 to 9 kb length were isolated andcloned in pUC18 vector linearized at the BamH1 site. The recombinant clones wereselected on Luria broth plates containing 100 �g/ml of ampicillin and 12.5 �g/ml oftetracycline.

2.7. Northern and Southern blots

RNA, prepared according to Aifa et al. [20], was denatured with formamideand size fractionated by electrophoresis in 1.5% agarose formaldehyde gel. Restric-tion enzyme digestions, Northern and Southern hybridizations were performed asdescribed by Sambrook et al. [22] using N+-Hybond.

2.8. Primer extension

Poly(A)+ RNA were isolated as described in the Quick Prep Micro mRNAPurification Kit (Amersham). Two picomoles of a 5′�32P labelled primer (5′-GGTTTCAGCAGTATAAGT-3′) located at 87 nucleotides from the initiating ATG of thecbh1 gene, were mixed with 4.5 �g of polyA+ cellulose-induced RNA in the presenceof 5× annealing buffer (25 mM Tris, pH 8.3; 375 mM KCl and 5 mM EDTA), denaturedat 75 ◦C for 2 min, incubated at 45 ◦C for 30 min and then gradually cooled down to37 ◦C.

After an ethanol precipitation step, the extension was performed in 20 �l finalvolume containing 15 units of AMV (Amersham) in 1× reverse transcriptase buffer(50 mM Tris–HCl, pH 8.3; 50 mM NaCl; 8 mM MgCl2 and 1 mM DTT), 5 mM DTT,1.5 mM dNTP. After 1 h at 42 ◦C, the reaction was stopped by addition of a solutioncontaining 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylenecyanol FF.

2.9. Sequencing and sequence analysis

The nucleotide sequence was carried out on both strands with both Thermose-quenase Cycle Sequencing Kit (Amersham) and the BigDye Terminator version. 3.1Cycle Sequencing Kit using an automated ABI Prism 3100-Avant Genetic Analyser(Applied Biosystems Inc.).

The cbh1 gene sequence reported in this article has been submitted to the Gen-Bank under accession number AY690482.

2.10. Amino acid sequence analysis and homology modelling

Multalin software was used to generate the alignment of CBHI sequences [23].Rendering of the alignment figure including the prediction of the secondary struc-tures was performed with the ESPript program [24]. Putative signal sequences wereidentified using the SignalP prediction software [25].

2.11. Computer-aided model building of the tertiary structure of the CBHI

The automated protein structure homology-modelling server, SWISS-MODEL[26] was used to generate the 3D model. The Deep View Swiss PDB Viewer soft-ware from EXPASY server (available at http://www.expasy.org/spdbv) was used tovisualize and analyze the atomic structure of the model. Molecular modelling of P.occitanis CBHI was analyzed based on the X-ray crystallographic structure of thecellobiohydrolase of T. emersonii (pdb accession code 1Q9H). Finally, PyMOL [27],the Molecular Graphics System was used to render figures.

3. Results and discussion

3.1. Isolation of the cbh1 cDNA and analysis of its sequence

In order to clone the cbh1 cDNA of P. occitanis, we performedRT-PCR reactions on poly(A)+ RNA extracted from the hypercellu-lolytic Pol6 mutant grown on cellulose and glucose using differentprimer combinations: P1–P4, P2–P4, P3–oligo-dT, P2–P5 and P2–P6

The amplified fragments were tested in Southern blot by aCBHI probe obtained by PCR from a Trichoderma species. Only thecellulose-induced RNA allowed the amplification of 1 kb fragmentusing the P3 and the oligo-dT primers, which strongly hybridized

76 F. Bhiri et al. / Enzyme and Microbial Technology 46 (2010) 74–81

Fig. 1. (A) Analysis of the RT-PCR products. Reactions were performed with induced (lines 1, 2, 3, 4 and 5) and uninduced (lines 1′ , 2′ , 3′ , 4′and 5′) poly(A)+ RNA; using the fol-l d 3′);( rs use3 P4 (5(

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owing primers: P1–P4 (lines 1 and 1′); P2–P4 (lines 2 and 2′); P3–oligo-dT (lines 3 anb) Hybridization with the Tricoderma reesei cbh1 probe. (B) Localization of the prime′), P2 (5′-ATGGACGCCGACGGTGG-3′), P3 (5′-GAGATGGATATCTGGGAGGCCAA-3′),5′-AAGGCATTGCGAGTAGTAGTCGTT-3′).

ith the CBHI probe (Fig. 1A). This fragment was then cloned inMOSblue T cloning vector and sequenced. The identity of theragment as a CBHI sequence was then established by nucleotidicequencing and Blast study.

The cbh1 amplified fragment was used as a probe to screen aDNA library (2 × 103 clones) constructed in the � MOS Elox phagicector. The complete nucleotide sequence of the cbh1 cDNA wasetermined by combining partial sequences obtained from variousositive overlapping clones. The nucleotide sequence of cbh1 con-ains a single open reading frame (ORF) consisting of 1587 bp andoding for a putative polypeptide of 529 amino acids. By compar-son with the N-terminal amino acid region of other known CBHIequences, a putative leader peptide of 25 amino acids is predictedsing the SignalP software. The secretory precursor is thought toe processed at a specific cleavage site between the A 25 and Q 26esidues, resulting in the formation of a mature enzyme composedf 504 amino acids with a molecular weight of 52.5 kDa. The dif-erence between the calculated molecular weight (52.5 kDa) andhat of the purified enzyme (60 kDa) [11] is probably due to post-ranslational modifications of the protein. In fact, the CBHI wasound to be a glycoprotein containing about 20% of carbohydrates,hich is in agreement with the results obtained.

The codon usage in the P. occitanis cbh1 showed that this geneontains 55.14% (G + C). Like in the pectin lyase gene (pnl1) of theame fungus [28], cytosine residues are preferred at the third posi-ion of codons and used in 49.15% of the cases (data not shown).s in other cellulase genes [14,29,30], there is a bias against NTAodons.

.2. Isolation of the cbh1 gene

In order to isolate the whole cbh1 gene and to study its reg-lation, a genomic bank covering approximately the totality ofhe fungal genome was prepared in pUC18 plasmid using DNAxtracted from the wild type strain (CL100) of P. occitanis. Approxi-ately 13,000 clones of this library were replicated and hybridizedith the CBHI probe obtained previously (see above): 80 clonesere positives. The presence of the cbh1 insert in these clones was

hecked by Southern blot hybridization and by sequencing. The

P2–P5 (lines 4 and 4′) and P2–P6 (lines 5 and 5′). (a) Staining with ethidium bromide.d for the RT-PCR. The primer sequences are: P1 (5′-TGCGGTCTCAACGGCGCCCTCTA-′-GGGATAGGTGCTGTCGAGCCACAACA-3′), P5 (5′-CCICCA/GCAT/CTGICC-3′) and P6

genes [16,31,32] whereas all the other fungal cbh1 genes sequencedso far have their nucleotide sequences interrupted by introns atvarying positions [33,34].

3.3. Structure of the 5′-non-coding region of cbh1 gene

The localization of the 5′-ends of the transcripts by primerextension analysis revealed heterogeneity in the transcription startsites. Three major transcription start sites located at 29, 32 and 35nucleotides upstream of the ATG were identified (Fig. 2). The corre-sponding 5′-untranslated mRNA sequences are thus 29, 32 and 35nucleotides long. Multiple start sites seem to be common amongfilamentous fungal genes [35], whereas the start point of highereukaryotic genes is usually 30 nucleotides from the TATA-box[36].

Cellulase genes of T. reesei are repressed in the presence of glu-cose by the Catabolite Repression Element (CREI) which togetherwith CREA of the Aspergilli is the only known repressor of cellulaseand hemicellulase genes [17]. Putative regulatory CRE binding sitesin the cbh1 promoter have been identified and investigated in bothTrichoderma and Penicillium species [16,17,37]. The P. occitanis cbh1upstream sequence was found to contain up to seven CRE consen-sus binding sites, suggesting their participation in repression of thisgene in response to glucose.

Cellulase promoters are also positively regulated by Activatorsof Cellulase Expression (ACEI and ACEII) factors [38,39]. Putativesequences involved in the binding of the ACEI and ACEII were alsoidentified in the P. occitanis cbh1 promoter [37].

3.4. Copy number of cbh1 gene

To estimate the copy number of the cbh1 gene in P. occitanis, thegenomic DNA was digested with various restriction enzymes (AvaI,AccI, SacI, BglI, SpeI, HindIII and EcoRV). There are no internal sitesfor AvaI, SacI, BglI, SpeI and HindIII in the probe sequence but onesite for EcoRV and AccI.

The digested genomic DNA was subjected to gel electrophoresis,blotted to hybond membrane and then hybridized with the wholesequence of the cbh1 cDNA used as probe. A single intense band was

F. Bhiri et al. / Enzyme and Microbial Technology 46 (2010) 74–81 77

Fig. 2. Nucleotide and deduced amino acid sequence of the cbh1 gene. The TATA-box is underlined. The AATAAA polyadenylation signal and the CA-polyadenylation site arei the Rt rs ofl o indP GenBa

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n bold and underlined. The peptides used to design the primer sequences tested inranscription initiation sites are indicated by stars. Motifs for the binding of Activatoetters) are shown. The Catabolite Repression Element (CREA) binding sites are alsotential N-glycosylation sites are boxed. This sequence has been submitted to the

he endoglucanase I gene, which shares an identity of 45% with thebh1 gene of T. reesei [40].

Previous works suggested the existence of a single-copy cbh1ene in several Trichoderma strains [41] or multiple copies of thebh1 gene for P. chrysosporium and P. janthinellum [16,33].

.5. Analysis of the cbh1 gene expression

Northern hybridization of RNA isolated from the hypercellu-olytic mutant Pol6 was performed using the cbh1 cDNA as a probe.he RNA from the hyperpectinolytic mutant CT1 and the parentaltrain CL100 was also extracted and used as controls in the samexperiment.

T-PCR are shown by arrows. The putative signal sequence is underlined. PresumedCellulase Expression: ACEI (lower-case letters) and ACEII (italicized and underlinedicated (bold capitalized and underlined letters). The catalytic residues are circled.nk under accession number AY690482.

Fig. 4 shows that a transcript of 1.9 kb hybridizes with thisprobe. The estimated size of the cbh1 mRNA is in good agreementwith that of the isolated cDNA. As shown in the same figure, thecbh1 RNA expression is induced by cellulose and repressed by glu-cose in CL100 and CT1 strains. However, the Pol6 mutant showeda very high induction of cbh1 transcripts on cellulose and a sig-nificant expression on glucose. The cbh1 expression on glucose iseven stronger than that observed on cellulose grown CL100 or CT1strains. Such a result has not been yet described in other filamen-

78 F. Bhiri et al. / Enzyme and Microbial Technology 46 (2010) 74–81

Fig. 3. Southern blot hybridization of Penicillium occitanis DNA. Genomic DNA(20 �g) was cut with AvaI (1), AccI (2), SacI (3), BglI (4), SpeI (5), HindIII (6) andEcoRV (7) and then analysed on 1% agarose gel and probed with the whole CBHIsp

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the key residues involved in the interaction between CBM andcellulose. The structure suggests that aromatic moieties of three

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equence. There are no internal sites for AvaI, SacI, BglI, SpeI and HindIII in therobe sequence but one site for EcoRV and AccI.

.6. Amino acid sequence data of P. occitanis CBHI

Analysis of the deduced amino-peptide sequence using a Blast

ig. 5. Protein sequence analysis and secondary structure alignment with ESPript progrll sequences are printed in white on a red background. Residues identical or with a conyellow background. A low consensus value (50%) was used in the multiple sequence a

onsensus line. A residue that is weakly conserved appears as a lower-case letter in the consensus line. Clusters of homologous symbols are indicated in the consensus line: IV (!alaromyces emersonii cellobiohydrolase (1Q9H) 3D structure is indicated at the top of thnd TT, respectively. The catalytic residues (E234, D236 and E239) are shown by green arrn identity of 93%, 71%, 62%, 61%, 57% and 57% with Penicillium funiculosum (AJ312295)culeatus (O59843), Trichoderma reesei (E00389) and Phanerochaete chrysosporium (M222For interpretation of the references to color in this figure caption, the reader is referred t

Fig. 4. Northern analysis of total RNA extracted from the CL100 wild type strain, thehypercellulolytic Pol6 and hyperpectinolytic CT1 mutants grown on cellulose (C) orglucose (G) for 3 days. The loaded quantity of RNA (20 �g/lane) was controlled bystaining the gel with ethidium bromide prior to blotting.

The C-terminal domain of P. occitanis CBHI showed a high degreeof similarity with the “Cellulose Binding Modules” (CBMs) of family1. The three-dimensional structure of T. reesei CBHI–CBM belong-ing to family 1 and determined by NMR spectroscopy provides

conserved Tyr side chains of the CBM (Y524, Y525 and Y498) formhydrophobic interactions with the cellulose molecule, and the sidechains of three amino acids (Q500, N522, and Q527) stabilize the

am of cellobiohydrolases from glycoside hydrolase family 7. Residues identical inservative substitution in at least four of the seven sequences are printed in red onlignment. A residue that is highly conserved appears as an uppercase letter in theonsensus line. A position with no conserved residue is represented by a dot in the

), BDENZQ (#), LM ($) and FY (%). The secondary structure element from the knowne alignment. �-Helices, �-helices, �-sheets and strict �-turns are denoted �, �, �ows. The CBM and linker sequences are boxed. Penicillium occitanis CBHI exhibited

, Talaromyces emersonii (pdb 1Q9H), Penicillium janthinellum (Q06886), Aspergillus20), respectively. The sequence numbering refers to the Penicillium occitanis CBHI.o the web version of the article.)

F. Bhiri et al. / Enzyme and Microbial Technology 46 (2010) 74–81 79

Fig. 6. (A) Structure model of the Penicillium occitanis CBHI catalytic module. (red) Catalytic residues; (yellow) disulphide bridges. (B) Surface representation of the structuresof CBHI of Trichoderma reseei (a), Phanerochaete chrysosporium (b) and Penicillium occitanis (c). The active site and the catalytic residues are shown in red and yellow,r w. ThP on of to

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espectively. The substrate analog (a nanomer of glucose residues) is drawn in yellohanerochaete chrysosporium and Penicillium occitanis, respectively. (For interpretatif the article.)

ellulose–CBM interaction by forming hydrogen bond with thelucose residues in the cellulose chains [6,42]. In the cellobiohy-rolases I CBM, all these aromatic residues are conserved (Y524,525 and Y498). The presence of tyrosyl or other aromatic residues

n the binding face is typical for carbohydrate–protein interactions.owever, the aromatic residue, at the position 498, is present as a

ryptophan in the Penicillium species and P. chrysosporium (Fig. 5).his residue is demonstrated to have a high affinity towards cel-ulose in the endoglucanase I (EGI) compared to the CBHI of T.eesei. The mutation Y498W in the CBHI increased its CBM affin-ty but did not change its enzymatic activity [43]. The CBHI–CBMs preceded by a putative linker peptide of about 37 amino acids,ich in serine and threonine residues. The linker peptide probablynsures an optimal interdomain distance between the catalytic andhe cellulose binding modules and/or protects the enzyme againstroteolytic attack since it is a flexible and highly O-glycosylatedegion [44].

Koch et al. [16] reported that the CBHI-type enzymes are char-cterized by 22 conserved cysteine residues and a catalytic sitenvolving two glutamic acid residues. The fact that these residuesre conserved in P. occitanis CBHI supports the likelihood that thisnzyme is a CBHI-type cellulase.

Differences in access to the active site appear to influence the

e apparent (Ki) values for cellobiose are 0.02, 0.18 and 2 mM for Trichoderma reseei,he references to color in this figure caption, the reader is referred to the web version

[11–13]. Such Ki value with P. occitanis CBHI has not previouslybeen reported for enzymes in family 7 of glycoside hydrolases.

The strong cellobiose inhibition of T. reesei cellobiohydrolase Iwas clearly reduced (Ki = 0.3 mM) by the deletion of the tip of theloop forming the active site tunnel roof [13].

Despite the sequence conservation between T. reesei cellobiohy-drolase I and P. occitanis CBHI in this region (G267-Y274), the latterenzyme exhibited less pronounced product inhibition without anyneed to delete this region.

3.7. Structure prediction of the catalytic domain of P. occitanisCBHI

In order to investigate the CBHI resistance to cellobiose inhibi-tion at a molecular level, a 3D model of CBHI was constructed, onthe basis of the X-ray crystallographic structure of the cellobiohy-drolase of the thermophilic fungus T. emersonii (pdb accession code1Q9H). CBHI from P. occitanis shares 71% of identity with that of T.emersonii.

As shown in Fig. 6A, P. occitanis CBHI folds into an antiparal-lel �-sheet jellyroll architecture typical of the characteristic foldof GH7 family. This fold is present in an increasingly large num-ber of proteins and was first described for the plant lectins such

80 F. Bhiri et al. / Enzyme and Microbial

Table 1Distances (Å) between some sub-sites and the substrate analog in the active sites ofPhanerochaete chrysosporium, Trichoderma reesei and Penicillium occitanis cellobio-hydrolases. The residue numbering corresponds to that of Penicillium occitanis.

Residues Phanerochaetechrysosporium

Trichoderma reesei Penicillium occitanis

Asp 195 4.38 3.96 4.53Tyr 167 2.63 2.58 2.72

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Glu 234 4.39 4.48 4.5Trp 63 3.05 2.75 3.30Asn 163 3.12 2.73 3.24

f the protein where the substrate is bound. Many of the sidehains in the �-sheets are hydrophobic, and interactions betweenhese residues appear to hold the �-sandwich in position. With thexception of four �-helices and two pairs of short �-strands, theest of the protein consists almost entirely of loops connecting the-strands.

The loops extending from the �-sandwich are stabilized byhe presence of nine disulphide bonds which are located betweenesidues 44–50, 75–96, 86–92, 160–425, 194–232, 198–231,52–278, 260–265 and 283–359. The cleft on the concave side ofhe molecule defines the oligosaccharide substrate binding site. Its lined with mainly aromatic residues on its walls and with acidicesidues at the bottom. The catalytic residues are located in theame �-strand where there is a strict alternation of polar and non-olar side chains, the first pointing toward the surface of the proteinhere they are able to interact with the substrate, the latter toward

he hydrophobic interior.Three catalytic acidic amino acids are conserved between the

embers of glycoside hydrolase family 7 [35]. These catalyticesidues were observed in the P. occitanis CBHI (E234, D236 and239). Their positions in P. occitanis CBHI are identical to thosen T. reesei cellobiohydrolase I, suggesting that they should per-orm the same function in these enzymes. In the acid/base reaction

echanism, Glu234 should be the nucleophile and Glu239 the pro-on donor. Asp236 is likely to be involved in maintenance of theppropriate pKa values for the other catalytic residues, assuringhe correct ionization state of the active site during catalysis, as haseen suggested for T. reesei cellulases [46].

Comparison of the active site of cellobiohydrolase I from T.eesei, P. chrysosporium and P. occitanis showed that all the interac-ions made with cellobiose are conserved, and the three moleculeshared together the same sub-sites. This observation takes moremportance when the interaction distances between the analog ofubstrate and each sub-site are compared. In fact, among the threenzymes, the distances are larger in P. occitanis CBHI compared to P.hrysosporium and T. reesei cellobiohydrolases I, suggesting a morepen configuration of the active site. The differences in distancesre estimated to be about 0.02–0.6 Å between P. occitanis CBHI and. reesei cellobiohydrolase I. As a consequence, the affinity betweenellobiose and the active site may be affected (Table 1).

Thus, poor hydrogen bonding, together with the more open CBHIctive site configuration (Fig. 6B), provides a reasonable explana-ion for why cellobiose binds less tightly to this enzyme than to T.eesei and P. chrysosporium cellobiohydrolases making it less sen-itive to product inhibition.

. Conclusion

In this paper, the isolation of the cDNA encoding cellobiohydro-

Southern analysis of genomic DNA indicated that there isnly a single intronless gene encoding for the cellobiohydrolase

[

Technology 46 (2010) 74–81

I in this strain. Unlike the hyperpectinolytic mutant CT1 andthe parental strain CL100, the hypercellulolytic Pol6 mutant wasshown to be partially constitutive since its cbh1 mRNA, besidesbeing highly induced by cellulose, is slightly repressed by glu-cose.

In many aspects, the P. occitanis CBHI protein is a typical cel-lulase: it has a modular structure with a CBM in the C-terminusjoined to the N-terminus catalytic core by a linker rich in serineand threonine residues. Comparative modelling of the structureof P. occitanis, P. chrysosporium and T. reesei cellobiohydrolases Isuggested that the resistance to product inhibition exhibited byP. occitanis CBHI is due to the poor hydrogen bonding to cel-lobiose associated with a more open configuration of the activesite.

Acknowledgements

We express our gratitude to Prof. G. Tiraby and Dr. H. Durand(Cayla Company, France) for kindly supplying the Penicillium occi-tanis strains used in this work. N. Fradi is thanked for supplying theTrichoderma CBHI probe. We also gratefully acknowledge Prof. R.Ellouz for his continual support and his interest for the subject. Thiswork was supported by Ministère de l’Enseignement Supérieur, dela Recherche Scientifique et de la Technologie.

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