BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan J. Normand & E. Bonnin & P. Delavault Received: 22 July 2011 /Revised: 19 October 2011 /Accepted: 2 November 2011 /Published online: 19 November 2011 # Springer-Verlag 2011 Abstract In order to produce a recombinant rhamnogalac- turonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamno- galacturonases. Using 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalac- turonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme. Keywords Rhamnogalacturonase . Acetylated rhamnogalacturonans . Irpex lacteus . Pichia pastoris Introduction Rhamnogalacturonan (RG) is one of the major elements of pectin, which is the major component of dicot cell wall. RG backbone is composed of an alternance of rhamnose and galacturonic acid (GalA) and carries neutral sugar side chains on the rhamnose residues. It can also be substituted by acetyl groups on O-2 and/or O-3 position of GalA (Ishii 1995; Voragen et al. 1995). The degree of acetylation of pectin molecules is defined as the number of acetyl groups present on 100 GalA and varies according to the plant source. Thus, pectins isolated from apple and citrus are slightly acetylated (Axelos and Thibault 1991), whereas pectins from sugar beet and potato tuber are highly acetylated (Voragen et al. 1995). In most of the plants, RG is the most acetylated area of pectins (Ishii 1995; Schols and Voragen 1996; Ralet et al. 2005). The presence of such non-sugar substituent limits the inter-chain interactions and thus the gelling properties of pectin (Williamson 1991). Pectin-degrading enzymes are relevant tools to study pectin structure. Among them rhamnogalacturonases are involved in the degradation of the pectic backbone. Those enzymes are produced by a wide range of microorganisms and most of the known rhamnogalacturonan hydrolases have been purified from ascomycete organisms (Schols et al. 1990; Suykerbuyk et al. 1997). Their activity is J. Normand : E. Bonnin INRA, Unité de Recherche Biopolymères, Interactions, Assemblages, BP 71627, 44316 Nantes Cedex 03, France J. Normand : P. Delavault (*) Laboratoire de Biologie et Pathologie Végétales, EA 1157-IFR 149 QUASAV, UFR Sciences et Techniques, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes Cedex 3, Nantes, France e-mail: [email protected]Appl Microbiol Biotechnol (2012) 94:1543–1552 DOI 10.1007/s00253-011-3705-5
Transcript
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Cloning and expression in Pichia pastoris of an Irpex lacteusrhamnogalacturonan hydrolase tolerantto acetylated rhamnogalacturonan
J. Normand & E. Bonnin & P. Delavault
Received: 22 July 2011 /Revised: 19 October 2011 /Accepted: 2 November 2011 /Published online: 19 November 2011# Springer-Verlag 2011
Abstract In order to produce a recombinant rhamnogalac-turonase from the basidiomycete Irpex lacteus using amolecular approach, PCR primers were designed based ona sequence alignment of four known ascomycete rhamno-galacturonases. Using 5′ and 3′ rapid amplification ofcDNA ends (RACE) experiments, a 1,437-bp full-lengthcDNA containing an open reading frame of 1,329 bp wasisolated. The corresponding putative protein sequence is of443 amino acids and contains a secretion signal sequence of22 amino acids. The theoretical mass of this protein is44.6 kDa with a theoretical isoelectric point of 6.2. Theamino acid sequence shared not only significant identitieswith ascomycete and basidiomycete putative rhamnogalac-turonases but also complete similarity with peptidesobtained from a recently purified rhamnogalacturonasefrom I. lacteus. The recombinant protein was successfullyexpressed in active form in Pichia pastoris. SDS-PAGEassay demonstrated that the recombinant enzyme wassecreted in the culture medium and had a molar mass of56 kDa. This recombinant rhamnogalacturonan hydrolaseexhibited a pH optimum between 4.5 and 5 and atemperature optimum between 40°C and 50°C, whichcorrespond to that of the native rhamnogalacturonase from
I. lacteus. The study of its specificity through reactionproducts analysis showed that it was highly tolerant to thepresence of acetyl groups on its substrate, even more thanthe native enzyme.
Rhamnogalacturonan (RG) is one of the major elements ofpectin, which is the major component of dicot cell wall. RGbackbone is composed of an alternance of rhamnose andgalacturonic acid (GalA) and carries neutral sugar side chainson the rhamnose residues. It can also be substituted by acetylgroups on O-2 and/or O-3 position of GalA (Ishii 1995;Voragen et al. 1995). The degree of acetylation of pectinmolecules is defined as the number of acetyl groups presenton 100 GalA and varies according to the plant source. Thus,pectins isolated from apple and citrus are slightly acetylated(Axelos and Thibault 1991), whereas pectins from sugar beetand potato tuber are highly acetylated (Voragen et al. 1995).In most of the plants, RG is the most acetylated area ofpectins (Ishii 1995; Schols and Voragen 1996; Ralet et al.2005). The presence of such non-sugar substituent limits theinter-chain interactions and thus the gelling properties ofpectin (Williamson 1991).
Pectin-degrading enzymes are relevant tools to studypectin structure. Among them rhamnogalacturonases areinvolved in the degradation of the pectic backbone. Thoseenzymes are produced by a wide range of microorganismsand most of the known rhamnogalacturonan hydrolaseshave been purified from ascomycete organisms (Schols etal. 1990; Suykerbuyk et al. 1997). Their activity is
J. Normand : E. BonninINRA, Unité de Recherche Biopolymères, Interactions,Assemblages,BP 71627,44316 Nantes Cedex 03, France
J. Normand : P. Delavault (*)Laboratoire de Biologie et Pathologie Végétales,EA 1157-IFR 149 QUASAV, UFR Sciences et Techniques,Université de Nantes,2 rue de la Houssinière,44322 Nantes Cedex 3, Nantes, Francee-mail: [email protected]
hampered towards pectin of certain plants having a highdegree of acetylation, indicating that they are sensitive tosubstrate acetylation (Schols et al. 1990; Schols andVoragen 1994; Kofod et al. 1994). Therefore, owning anenzyme able to act on acetylated rhamnogalacturonanwould be a powerful tool to study the structure ofacetylated pectin as well as the acetylation pattern.
Irpex lacteus is a wood-decaying basidiomycete thatproduces numerous cell wall polysaccharide-degradingenzymes. This fungus is used to produce the commercialpreparation Driselase, containing cellulases and xylanasesand used in protoplast preparation. By using acetylatedrhamnogalacturonan extracted from sugar beet pectin as thesubstrate, it was recently shown that Driselase contains arhamnogalacturonase tolerant to acetylated substrate. Thisnew enzyme has been purified and its specificity studiedtowards a range of pectic substrates (Normand et al. 2010).
In this present study, in order to produce easily thisenzyme in high quantity, a molecular strategy wasemployed to express the corresponding cDNA in a hostorganism. As known rhamnogalacturonan hydrolases areglycosylated, enzyme production was carried out inPichia pastoris, a yeast known to produce less hyper-glycosylated recombinant protein than Saccharomycescerevisiae (Cereghino and Cregg 1999), and known notto produce possible contaminating cell-wall-degradingenzymes (Fu et al. 2001). The biochemical characteristicsof the recombinant rhamnogalacturonase as well as itsmode of action towards acetylated substrates were investigatedand compared to those of the native rhamnogalacturonase fromI. lacteus (Normand et al. 2010).
Materials and methods
Strains and vectors
Escherichia coli strain XL1-blue (Stratagene, Cedar Creek,TX, USA) and pGEM-T easy vector (Promega, Madison,WI, USA) were used as the host–vector expression system.I. lacteus strain KY2902 (ATCC 44426) was purchasedfrom LGC-Promochem (Molsheim, France). P. pastorisstrain X-33 and expression vector pPICZαA were pur-chased from Invitrogen (Carlsbad, CA, USA).
I. lacteus culture and RNA isolation
I. lacteus strain KY2902 was cultivated in YM medium(0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1%dextrose) containing sugar beet pectin (1%) at 26°C. Eachday the enzymatic activity on acetylated RG and notacetylated RG was measured in the culture medium. After11 days, total RNAs were extracted from 100 mg of
material using the TRIzol procedure (Invitrogen, Carlsbad,CA, USA).
Isolation and cloning of a full-length I. lacteusrhamnogalacturonan hydrolase cDNA
Degenerated PCR primers were designed following sequencecomparison of four RG-hydrolases from Aspergillus aculeatus(UniProtKB accession no. Q00001), Aspergillus niger (Uni-ProtKB accession no. P87160 and P87161) and Botryotiniafuckeliana (UniProtKB accession no. P87247), which sharehighly similar regions (Fig. 1). A partial cDNA fragment of I.lacteus RG-hydrolase was amplified by RT-PCR. The primersused were RGH3 (5 ′ -GGHYTGGAYGGKATTGATGTCTGG-3′) and RGH6 (5′-GTNGGDATGGTNGGDATNGGDAT-3′), which were designed based on two well-conserved amino acid sequences, GLDGIDVW and IPIP-TIPT, respectively. PCR products were cloned in the pGEM-T easy cloning vector and the final reaction mixture wastransformed into XL1-Blue Competent Cells. The recombi-nant plasmids were sequenced by the GATC Biotech AGCompany (Konstanz, Germany). The sequence of each PCR-amplified inserts was confirmed by sequencing two differentplasmids. To obtain the full-length cDNA, 5′ and 3′ RACEwere performed using the GeneRacer™ kit from Invitrogen(Carlsbad, CA, USA). The primers used in 3′ RACE wereGeneRacer™ 3′ (5′-GCTGTCAACGATACGCTACGTAACG-3′) and irpex 3RACE (5′-CGGTGCAGAACGTGAACTTGGAGAA-3′). The primers used in 5′ RACE wereGeneRacer™ 5′ (5′-CCAACTCCCACGCAA-3′) andirpex 5RACE (5 ′-CGGTGCTCTGGCTACTCCAATACTGA-3′). By aligning the sequences of the 3′ and 5′RACE products, a full-length cDNA sequence of a RG-hydrolase was deduced and obtained through RT-PCR usingthe specific primers (forward primer 5′-AAAGAAGTTC-CACCCACTTCAAAGGA-3′ and reverse primer 5′-TACATTAAATCGTGTCTCATGACGTC-3′) (Fig. 2). Proper se-quence of the full-length I. lacteus RG-hydrolase cDNAwas confirmed by cloning and DNA sequencing as above-mentioned, and the corresponding plasmid was namedpIRPEXFL.
Plasmid construction
The cDNA encoding the mature I. lacteus RG-hydrolase genewas PCR amplified using the specific primers (irpexEcorI:5′-CCGGAATTCCAGCTGTCTGGGTCCGTGGG-3′ andirpexXbaI: 5′-GCTCTAGATTAGGCGCTATCAATAGCAGC-3′) and the recombinant pIRPEXFL as a template.Primers irpexEcorI and irpexXbaI introduced EcoRI andXbaI restriction sites respectively (underlined in the abovesequence). After digestion with EcoRI and XbaI (NewEngland BioLabs, Ipswich, UK), the PCR product was
inserted into the vector pPICZαA (Invitrogen, Carlsbad, CA,USA). Proper construction was confirmed by restrictiondigestion and DNA sequencing and was designated aspPICZαA-RGH.
Transformation and expression of recombinant RGasein P. pastoris
The recombinant plasmid pPICZαA-RGH was linearizedwith PmeI (New England BioLabs, Ipswich, UK), thentransformation of P. pastoris was performed by electro-poration according to the EasySelect Pichia Expression Kit
(Invitrogen, Carlsbad, CA, USA). Recombinant cloneswere selected on YPDS (1% yeast extract, 2% peptone,2% dextrose, 1 M sorbitol, 2% agar) plates containingZeocin (Invitrogen, Carlsbad, CA, USA) at a finalconcentration of 100 μg/mL. Colonies were then streakedonto YPD 100 μg/mL Zeocin plates. This streaking processof single colonies was repeated three times. Single colonieswere then streaked on YPD plates with Zeocin concentra-tion of 100, 200 and 500 μg/mL. After 2 days of incubationat 29°C, each clone was taken from plate of higherconcentration that it grew on and streaked on the samemedium but with 500, 1,000 and 2,000 μg/mL Zeocin.
Fig. 1 CLUSTAL-W alignment of amino acid sequences of RGase Afrom A. aculeatus (UniProtKB accession no. Q00001), A. niger RGaseA and B (UniProtKB UniProtKB no. P87160 and P87161) and B.
fuckeliana (UniProtKB accession no. P87247) and position ofsequences to design degenerated primers RHG1 to RGH6
Colonies growing on 2,000 μg/mL Zeocin were conserved.Then, colonies were inoculated in 50 mL buffered glycerol-complex medium (1% yeast extract, 2% peptone, potassiumphosphate buffer 100 mM, pH 6, 1.34% yeast nitrogen basewith ammonium sulphate and without amino acids, 4×10−5% biotin, 1% glycerol) in baffled flask. After shakingat 29°C, during 24 h, the culture medium was centrifuged(2,000×g, 5 min at room temperature) and the pellet wasresuspended in 500 mL buffered methanol-complex medi-um (buffered glycerol-complex medium within glycerol isreplaced by methanol 0.5%) and kept in 0.5% methanol byaddition of 2.5 mL of 100% methanol every 24 h. Every24 h, 500 μL of culture were taken for further activity
assays. After 4 days of induction, cultures were centrifuged(2,000×g, 5 min, 4°C) and the supernatant was concentrat-ed to 5 mL using PolyEther Sulfone membrane (10 kDa cutoff; Sartorius, Aubagne, France) adjusted to 20 mM acetatebuffer, pH 5.0 and used as the enzyme source.
Enzyme purification
All purification steps were carried out at 4°C. The samplewas applied on two Superdex 75 columns (16×160 mm;GE Healthcare) mounted in series and equilibrated with thesame buffer. The elution was carried out with the samebuffer at a flow rate of 0.3 mL/min and monitored by
gactggagcacgaggacactgacatggactgaaggagtagaaagaagttccacccacttcaaaggagacaacatggcttgcggattctcgcgtcttacggctctgctcgctctttgcagc M A C G F S R L T A L L A L C S atctctggatcgctcgctcagctgtctgggtccgtgggacctacgtcctcgctgtcgtcg I S G S L A Q L S G S V G P T S S L S S aagcagggtaccatctgtaacgttctcaactatggtggctctgtcggttccagtgatatt K Q G T I C N V L N Y G G S V G S S D I ggacctgccattggtaaagcgttcagcgactgtgtcaccaaggccaccaacggtgctacg G P A I G K A F S D C V T K A T N G A T ctctacgtaccccccggtaactacaacatgcagacttggcagacactgaaccacggcact L Y V P P G N Y N M Q T W Q T L N H G T aagtgggcattccaattggacggtgttatcactcgtacaagcactactggaggtaacatg K W A F Q L D G V I T R T S T T G G N M atcgtcatccagaacgccaacgactttgagttcttctcgagcacgggtaagggtgctatc I V I Q N A N D F E F F S S T G K G A I cagggtaacggctaccagtgtcgcaatgctggacctcgtctcatccgtgtagtcacctcg Q G N G Y Q C R N A G P R L I R V V T S aacaactggtctttgcacgacatcatcatggttgactctcccgagttccaccttgtcatt N N W S L H D I I M V D S P E F H L V I caggacggctcgaatggtgaagtgtacaacacggtcattcgcggaggaaacctcggcggc Q D G S N G E V Y N T V I R G G N L G G tctgacggtatcgacgtctggggtacgaactactggatccacgacatcgaagtcaccaac S D G I D V W G T N Y W I H D I E V T N cgcgacgaatgcgtcaccgtcaagtcccccgcgaaccacatccaagtcgagcaaatctgg R D E C V T V K S P A N H I Q V E Q I W tgcaaccagtccggaggctccgccatcggctcgcttggcgccaacactaccatccagaac C N Q S G G S A I G S L G A N T T I Q N gtgctctaccgaaatgtgtacacgaacggaggcaaccagatcttcatgatcaagtctaat V L Y R N V Y T N G G N Q I F M I K S N ggtggaagcgggacggtgcagaacgtgaacttggagaattttattgcgaggaacacggcg G G S G T V Q N V N L E N F I A R N T A tacgggttggatattgatcagtattggagtagccagagcaccgcgcccggcaatggcgtg Y G L D I D Q Y W S S Q S T A P G N G V cagctgaaggacatcaccttctctaactgggatggcttcatcaccgacggtgctcgccgc Q L K D I T F S N W D G F I T D G A R R gcccccatccaagtcctctgcgycgacggagccccctgcacggacatcaacatcaacaac A P I Q V L C X D G A P C T D I N I N N gtcaacctctgggccgccaacaaccaagccaccaacaagtgccgtagtgcgtacggtact V N L W A A N N Q A T N K C R S A Y G T ggcgcctgcctcaagtcgggcagcggcggaagttactcccaggtgacgaagacgatcagc G A C L K S G S G G S Y S Q V T K T I S aagcctcccgctttcactactccggcgacgatgagtggagatttgtcggatggcttcccg K P P A F T T P A T M S G D L S D G F P acgaactcaccgattccgattccaaccatcccaccgtcgttcttccccggcacccagccg T N S P I P I P T I P P S F F P G T Q P ctcaagrctttggccggaaagtaggtgttgttctgggtcctggcaaaacgatgggagtcg L K X L A G K gtgaggtgtcggacgtcatgagacacgatttaatgtatgcaggataaaaagttgaataaa tctcggatatcctttcaaaaaaaaaaaaaaaa
Fig. 2 I. lacteus rhamnogalac-turonase full-length cDNAsequence (GenBank accessionno. FJ213603) and deducedamino acid sequence. In thenucleotide sequence, primersused to obtain the full-lengthcDNA are in bold italic, andstart and stop codons are in boldunderlined. In the amino acidsequence, the signal peptide isunderlined, the putativeN-glycosylation site is in boldunderlined and the putativeO-glycosylation sites are inunderlined bold italic. Thepeptides obtained from thenative rhamnogalacturonase thatare encountered in the deducedprotein sequence of therecombinant rhamnogalacturo-nase (Normand et al. 2010) arehighlighted
following the adsorption at 280 nm. All the fractions weretested for their activity towards non-acetylated RG. Therhamnogalacturonase rich fractions were pooled. Thischromatographic step was repeated on this pool containingrhamnogalacturonase activity.
Enzymatic assays and substrates
Acetylated RG was prepared by enzymatic degradation ofsugar beet pectin (Teknisk SBP Danisco; Bonnin et al. 2008;Normand et al. 2010). The acetylated rhamnogalacturonanhad a degree of acetylation of 45 and a GalA/Rha ratio of1.5. Non-acetylated RG were prepared according to Bonninet al. (2001).
Enzymatic assays for depolymerase activities wereperformed by measuring the release of reducing groups ina reaction mixture containing substrates (5 mg/mL for non-acetylated RG and 2.5 mg/mL for acetylated RG) in 50 mMsodium acetate buffer, pH 5.0 and enzyme sample. Themixture was incubated at 40°C and reducing sugars weremeasured by the method of Nelson (Nelson 1944) adaptedin microplates (Sturgeon 1990) using GalA as the standard.Enzymatic activities were expressed in nkatal (nkat), onenkat being defined as the amount of enzyme that catalysesthe release of 1 nmol of reducing end per second in theconditions described above.
Enzyme analysis
Protein content was determined using BioRad reagent (Marnesla Coquette, France) and bovine serum albumin (A-3912,Sigma; L’Isle d’Abeau, France) as standard (Bradford 1976).
Protein homogeneity and molar mass were evaluated bypolyacrylamide gel electrophoresis under denaturing con-ditions (SDS-PAGE) using silver staining (Gottlieb andChavko 1987). SDS-PAGE was carried out in a Mini-Protean 3 apparatus (BioRad, Marnes la Coquette, France)using a continuous 10–20% polyacrylamide gel in presenceof 0.1% sodium dodecyl sulphate and calibrated usingKaleidoscop prestained molecular weight markers (BioRad,Marnes la Coquette, France).
Influence of pH and temperature
To study the influence of pH and temperature on theenzymatic activity, optimum for activity and stability con-ditions were performed as described in Normand et al. 2010.
Substrate hydrolysis for analysis of the degradationproducts
Enzymatic degradations of acetylated and non-acetylatedRG, and product analysis by high performance anion
exchange chromatography (HPAEC) were performed asdescribed in Normand et al. 2010.
Results
Cloning and sequence analysis of a recombinant I. lacteusRG-hydrolase
Sequences of four known RG-hydrolases from the Asco-mycetes A. aculeatus (UniProtKB accession no. Q00001),A. niger (UniProtKB accession no. P87160 and P87161)and B. fuckeliana (UniProtKB accession no. P87247) werealigned allowing identification of several regions with highsimilarities and design of six primers (Fig. 1). The primerset, RGH3 and RGH6, amplified a 700-bp cDNA productfrom RNA isolated from a 10 days culture wheredepolymerase activities of RG substrates was maximum.Using 5′ and 3′ RACE experiments, a full-length 1,437-bpcDNA was isolated, containing an open reading frame of1,329 bp (Fig. 2). The sequence reported in this paper hasbeen deposited in the GenBank database under accessionno. FJ213603. The deduced amino acid sequence corre-sponds to a 421-amino-acid protein with a 22-amino-acidN-terminal signal sequence (SignalP 3.0; http://www.cbs.dtu.dk/services/SignalP/), with a cleaving site betweenAla22 and Gln23 (Fig. 2).
This sequence was searched against the NCBI proteindatabase (http://www.ncbi.nlm.nih.gov/) and shared 53%identity with the RG-hydrolases A from A. aculeatus and A.niger, and 66% identity with a putative rhamnogalacturonasefrom the basidiomycete Postia placenta (Fig. 3).
Analysis of the deduced protein sequence predicted anisoeletric point of 6.2 and a molecular mass of 44.6 kDa(ProtParam; http://www.expasy.org).
Analyses of the sequence for potential glycosylationsites indicated the presence of 12 O-linked glycosylationsites among them 11 are localised in the C-terminal part ofthe protein (NetOGlyc 3.1; http://www.cbs.dtu.dk/services/NetOGlyc/), and three N-linked glycosylation sites withonly one having a sufficient score (NetNGlyc 1.0; http://www.cbs.dtu.dk/services/NetNGlyc/).
The cDNA was cloned in P. pastoris pPICZαAexpression vector for further production of the recombinantprotein.
Expression and purification
Recombinant colonies that can resist at highest concentra-tion of Zeocin might have multi-copies of integration intothe P. pastoris genome, potentially leading to higher levelsof protein expression (Sunga et al. 2008). Therefore, one ofthe transformants able to grow at 2 mg/mL of Zeocin was
selected. The P. pastoris X-33 transformed with the emptypPICZαA vector was used as a control. Activity level waschecked each day of a culture induced by methanol. After4 days, proteins from the culture medium were collected bycentrifugation and used for enzyme activities and proteinanalysis. No rhamnogalacturonase activity was detected inthe culture medium of the control strain under the sameculture condition. Activity on acetylated rhamnogalacturonanwas 10.5 nkat/mL for a protein expression of 3.64 mg/mL inthe culture supernatant of transformed yeast. SDS-PAGEanalysis followed by silver staining revealed different bands(Fig. 4 (A)). Culture medium was concentrated. To purify therecombinant rhamnogalacturonase, a size-exclusion chroma-tography was performed. Rhamnogalacturonase activity wasmeasured in the resulting fractions and active fractions werepooled. Rhamnogalacturonase activity of the pool was11 nkat/mL. SDS-PAGE analysis followed by silver color-ation still revealed four bands from 18 to 215 kDa (Fig. 4(B)). Contaminant bands were removed after a second size-exclusion chromatography (Fig. 4 (C)). SDS-PAGE analysisshowed the band of interest at 56 kDa. The purified enzyme
had a specific activity of 1,250 nkat/mg when measured onnon-acetylated RG and 480 nkat/mg when measured onacetylated RG.
Enzyme analysis
Influence of pH and temperature was examined towards non-acetylated RG and acetylated RG. The optimum activityoccurred between pH 5.0 and 5.5 for non-acetylated RG andbetween 4 and 4.5 for acetylated RG (Fig. 5a) and in a rangeof temperature from 40°C to 60°C on non-acetylated RG andjust around 50°C on acetylated RG (Fig. 5b).These resultswere close for both substrates. Protein stability was alsotested. After 90-min pre-incubation, the enzyme activity wasstable from pH 3.5 to 6, where 50% to 60% of the activityremained (data not shown). After pre-incubation of theenzyme at different temperatures, the activity presents thesame stability up to 50°C. By increasing the temperature, theactivity decreased dramatically and less than 10% of theinitial activity remained after pre-incubation at 60°C andbeyond (data not shown).
A. aculeatus QLSGSVGPLTSASTKGATKTCNILSYGAVADNSTDVGPAITSAWAACK----SGGLVYIP 56 A. niger QLSGSVGPLTSAHSKAATKTCNVLDYGAVADNSTDIGSALSEAWDACS----DGGLIYIP 56 P. placenta QLSGSVGPTTPLSDKS--VLCNILDYGGKIG-SSDIGPAIQKAFDDCVLTNSSPSTLYVP 57 I. lacteus QLSGSVGPTSSLSSKQG-TICNVLNYGGSVG-SSDIGPAIGKAFSDCVTKATNGATLYVP 58
******** :. * **:*.**. . *:*:*.*: .*: * . . :*:*
A. aculeatus SGNYALNTWVTLTGGSATAIQLDGIIYRTGTASGNMIAVTDTTDFELFSSTSKGAVQGFG 116 A. niger PGDYAMDTWVSLSGGKATAIILDGTIYRTGTDGGNMILVENSSDFELYSNSSSGAVQGFG 116 P. placenta TGDYDMQTWVTLTGGSSWAFRLDGLITRTATTGGNMIAVENAYDFEFYSKNSAGGIQGAG 117 I. lacteus PGNYNMQTWQTLNHGTKWAFQLDGVITRTSTTGGNMIVIQNANDFEFFSSTGKGAIQGNG 118
A. aculeatus YVYHAEG-TYGARILRLTDVTHFSVHDVILVDAPAFHFTMDTCSDGEVYNMAIRGGNEGG 175 A. niger YVYHREGDLDGPRILRLQDVSNFAVHDIILVDAPAFHFVMDDCSDGEVYNMAIRGGNSGG 176 P. placenta YQCRNDG----PRLIRMVTSERWSLHDLILVDSPEFHLVIQQGSGGEVYNLAIRGADIGG 173 I. lacteus YQCRNAG----PRLIRVVTSNNWSLHDIIMVDSPEFHLVIQDGSNGEVYNTVIRGGNLGG 174
A. aculeatus LDGIDVWGSNIWVHDVEVTNKDECVTVKSPANNILVESIYCNWSGGCAMGSLGADTDVTD 235 A. niger LDGIDVWGSNIWVHDVEVTNKDECVTVKGPANNILVESIYCNWSGGCAMGSLGADTDITD 236 P. placenta SDGVDVWGENYWIHDVEVTNRDECVTVKSPASNILVERIWCNQSGGSAMGSLGANTSIAN 233 I. lacteus SDGIDVWGTNYWIHDIEVTNRDECVTVKSPANHIQVEQIWCNQSGGSAIGSLGANTTIQN 234
A. aculeatus IVYRNVYTWSSNQMYMIKSNGGSGTVSNVLLENFIGHGNAYSLDIDGYWSSMTAVAGDGV 295 A. niger ILYRNVYTWSSNQMYMIKSNGGSGTVNNTLLENFIGRGNRYSLDVDSYWSSMTAVDGDGV 296 P. placenta ILYQNVYTVGGNQAYMIKSNGGSGTVKDVVFQNFISRDTAYGLDVNQYWASESTQPGDGI 293 I. lacteus VLYRNVYTNGGNQIFMIKSNGGSGTVQNVNLENFIARNTAYGLDIDQYWSSQSTAPGNGV 294
A. aculeatus QLNNITVKNWKGTEANGATRPPIRVVCSDTAPCTDLTLEDIAIWTESGSSELYLCRSAYG 355 A. niger QLSNITFKNWKGTEADGAERGPIKVVCSDTAPCTDITIEDFAMWTESGDEQTYTCESAYG 356 P. placenta QLYNITFKNWDGNVANGVQRSPIQILCADGAPCYDINLDDVYMWSLT-DEATWKCESAYG 352 I. lacteus QLKDITFSNWDGFITDGARRAPIQVLCADGAPCTDININNVNLWAAN-NQATNKCRSAYG 353
A. aculeatus SGYCLKDSSSHTSYTTTSTVTAAPSGYS-ATTMAADLATAFGLTASIPIPTIPTSFYPGL 414 A. niger DGFCLEDSDSTTSYTTTQTVTTAPSGYS-ATTMAADLTTDFGTTASIPIPTIPTSFYPGL 415 P. placenta TGACLKSGSSHKSYAVETTTYTQPSGYTTPTTMSGDLTAGFGSTTLIPTPTIPTMFYPEL 412 I. lacteus TGACLKSGS-GGSYSQVTKTISKPPAFTTPATMSGDLSDGFPTNSPIPIPTIPPSFFPGT 412
A. aculeatus TPYSALAG----- 422 A. niger TAISPLASAATTA 428 P. placenta PQISPLMKNK--- 422 I. lacteus QPLKALAGK---- 421
..*
Fig. 3 CLUSTAL-W alignmentof RG-hydrolases sequencesfrom A. aculeatus (UniProtKBaccession no. Q00001), A. niger(UniProtKB accessionno. P87160), P. placenta(UniProtKB accession no.XP_002471859) and I. lacteus.Conserved cysteins are boxed.Aminoacids of theRG-hydrolase active site from A.aculeatus are in bold underlined
Analysis of degradation products of I. lacteusRG-hydrolase
To explain the degradation products from acetylated or non-acetylated substrates, oligomers were referred to as acombination of R for rhamnose, U for uronic acid and G forgalactose, each letter being followed by a figure indicating thenumber of each monomer. They constitute a homogenousseries with a GalA at the reducing end. As an example, thedeveloped formula of R2U2 is Rha-GalA-Rha-GalA.
Time course of the non-acetylated and acetylated RG bythe recombinant rhamnogalacturonan hydrolase was followedby HPAEC pH 13 and response factors determined fromstandard oligomers were used to quantify the products.
When non-acetylated RG was submitted to hydrolysis bythe RG-hydrolase (Fig. 6a), R3U3 appeared as the majorproduct of degradation. Beyond the major product, themajor oligomers were in decreasing order: R2U2, R2U2G2,R2U3 and R1U2. The plateau was reached after 7 h ofdegradation for all the products.
The products obtained from the hydrolysis of acetylatedRG were also followed by HPAEC pH 13 (Fig. 6b). Due tothe high pH of the eluent, all the ester groups were removedduring analysis and separation took place according to acombination of DP and charge density as for the productsobtained from non-acetylated RG hydrolysis.
When acetylated substrate was degraded with the sameamount of enzyme, the major products were R2U2 and thenin decreasing order: R2U2G2, R4U4, R5U5, R1U2 andR2U3. The plateau was not reached for all the productsafter 48 h hydrolysis.
The final sum of identified products was 249.2 μg/mLon non-acetylated RG and 164.5 μg/mL on acetylated RG,when 1 mg/mL of substrate was subjected to degradation.
Discussion
We isolated for the first time a cDNA encoding a rhamnoga-lacturonase from I. lacteus by RACE-PCR experiments. Theisolated 1,437-bp cDNA contains an open reading frame of1,329 bp that corresponded to a 421-amino-acid protein. Thededuced 421-amino-acid sequence showed high similaritieswith glycoside hydrolase from family 28 (Henrissat 1991) inwhich all known rhamnogalacturonan hydrolases are classi-fied. The 15 peptides that have been previously identifiedfrom the purified rhamnogalacturonase from I. lacteus(Normand et al. 2010) were present in the deduced proteinsequence (Fig. 2). Thus, the cDNA has been inserted in a P.
Fig. 5 Influence of pH (a) and temperature (b) on the activity of therecombinant rhamnogalacturonase on non-acetylated rhamnogalactur-onan (closed markers) and acetylated rhamnogalacturonan (openmarkers). Activity is expressed as a percentage of the maximum
Fig. 4 SDS-PAGE and silver staining. A (lane 1) Kaleidoscopeprestained molecular weight markers, (lane 2) culture supernatant; B(lane 1) molecular weight markers, (lane 2) pool issued of first size-exclusion chromatography; C (lane 1) molecular weight markers,(lane 2) Rhamnogalacturonase fraction of second size-exclusionchromatography
pastoris expression vector for the production of a recombi-nant RG-hydrolase. Predicted molecular weight and isoelec-tric point are 44.6 kDa and 6.2, respectively. Compared tothe 55-kDa native protein that show an isoelectric point of7.2 (Normand et al. 2010), the differences could signify thatthe native protein is glycosylated. Indeed, the sequenceexhibits 13 predicted glycosylation sites. The SDS-PAGE ofthe recombinant rhamnogalacturonase allowed estimating itsmolar mass to 56 kDa. This is, as for the native enzyme,about 10 kDa more than the predicted molecular weight andthis shows no hyperglycosylation of the recombinant proteinby P. pastoris. The biochemical characteristics of the nativeand the recombinant enzymes are highly similar. Analysis ofhydrolysis products of the recombinant rhamnogalacturonaseby HPAEC showed oligosaccharides very similar to thoseobtained by the action of the native enzyme. Because thestructures of these products have been elucidated for the
native enzyme (Normand et al. 2010), and because therecombinant enzyme is able to act on acetylated rhamnoga-lacturonan, it is likely that the structures of these products aresimilar. Therefore the recombinant rhamnogalacturonase isable to accept an acetyl group in its active site, similarly tothe native one.
However, the recombinant rhamnogalacturonase activityis only 5.7-fold lower on acetylated RG than on non-acetylated RG, whereas the same ratio was 12.2 for thenative enzyme (Normand et al. 2010). This recombinantrhamnogalacturonan hydrolase is then more tolerant thanthe native enzyme to the substitution of the substrate byacetyl groups.
Alignment analysis of the recombinant rhamnogalacturo-nase with the A. aculeatus rhamnogalacturonase A (Petersenet al. 1997) showed 52% identities and 71% homology. Inthose identities it has to be noted that the catalytic amino
Fig. 6 Kinetics of degradationof non-acetylated rhamnogalac-turonan (a) and acetylatedrhamnogalacturonan (b)by the recombinantrhamnogalacturonase
acids were conserved as well as all the cysteine residuesinvolved in disulfide bridges (Petersen et al. 1997). In astructural point of view, I. lacteus rhamnogalacturonase ismore closely related to A. aculeatus RG-hydrolase and A.niger RG-hydrolase A (Kofod et al. 1994; Suykerbuyk et al.1995) than to A. niger RG-hydrolase B and the B. fuckelianaone, that exhibit an extra C-terminal extension (Suykerbuyket al. 1997; Fu et al. 2001).
No major differences can be observed regarding molecularweight, isoelectric point, optimum parameters, or catalytic andstructurally important amino acids, between this RGase fromI. lacteus and A. aculeatus RGase A. However, A. aculeatusRGase A is not active on acetylated substrate (Schols et al.1990; Kofod et al. 1994). Thus, none of these parameterscould explain why Irpex RGase is tolerant to the acetylationof its substrate. Similarly to what was observed for I. lacteusRGase with acetyl groups, the endopolygalacturonase fromFusarium moniliforme was shown to be tolerant to themethylation of its substrate (Bonnin et al. 2002). Aspolygalacturonases are known to be hindered on methyl-esterified pectin, structures of complexes of F. moniliformeendopolygalacturonase with non-methylated or partly meth-ylated homogalacturonans were modelled to identify theresidues involved in substrate binding and to correlate withthe experimental data (André-Leroux et al. 2005). Theexplanation for the tolerance may be found in catalyticgroove amino acids better than in catalytic amino acidsthemselves. In 2009, complete sequencing of the genome ofthe brown-rot basidiomycete, P. placenta (Martinez et al.2009), revealed the existence of a gene encoding a putativerhamnogalacturonase. The predicted protein shares 66% ofsimilarity with the I. lacteus rhamnogalaturonase (Fig. 3).However, because this protein had not been purified, noinformation is available on its specificity and its eventualtolerance to the acetylation of the substrate. In a recent study,the plant cell-wall-decomposing machinery of anotherbrown-rot basidiomycete, Serpula lacrymans (Eastwood etal. 2011), was also investigated. Although existence of genesencoding enzyme from the GH28 family was expected, nosequence sharing significant similarity with the I. lacteus onewas identified. Next studies have to be focused onimprovement of the recombinant protein expression yield.Recombinant rhamnogalacturonase can thus be used to studyrhamnogalacturonan structure and properties, as well as itsacetylation pattern. Moreover, this new enzyme will berelevant to produce new pectic oligosaccharides for whichnew biological activities can be investigated, as for instanceimmuno-modulative (Wong 2008), prebiotic (Manderson etal. 2005; Mandalari et al. 2007), or antiproliferative activities(Kang et al. 2006).
Acknowledgements Sylviane Daniel and Amandine Leloup areacknowledged for their technical assistance.
References
André-Leroux G, Tessier D, Bonnin E (2005) Endopolygalacturonasesreveal molecular features for processivity pattern and tolerancetowards acetylated pectin. Biochim Biophys Acta 1794:53–64
Axelos MAV, Thibault J-F (1991) Influence of the substituentscarboxyl and of the rhamnose content on the solution propertiesand flexibility of pectins. Int J Biol Macromol 13:77–82
Bonnin E, Brunel M, Gouy Y, Lesage-Meessen L, Asther M, ThibaultJ-F (2001) Aspergillus niger I-1472 and Pycnoporus cinnabar-inus MUCL39533, selected for the biotransformation of ferulicacid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases. Enz Microb Technol28:70–80
Bonnin E, Le Goff A, Körner R, Vigouroux J, Roepstorff P, ThibaultJ-F (2002) Hydrolysis of pectins with different degrees andpatterns of methylation by the endopolygalacturonase of Fusariummoniliforme. Biochim Biophys Acta 1596:83–94
Bonnin E, Clavurier K, Daniel S, Kauppinen S, Mikkelsen JDM,Thibault J-F (2008) Pectin acetylesterases from Aspergillus areable to deacetylate homogalacturonan as well as rhamnogalacturonan.Carbohydr Pol 74:411–418
Bradford MMA (1976) A rapid and sensitive method for thequantitation of protein utilizing principle of protein-dye binding.Anal Biochem 72:248–255
Cereghino GPL, Cregg JM (1999) Applications of yeast in biotech-nology: protein production and genetic analysis. Curr OpinBiotechnol 10:422–427
Eastwood D, Floudas D, Binder M, Majcherczyk A, Schneider P,Aerts A, Asiegbu F, Baker S, Barry K, Bendiksby M, BlumentrittM, Coutinho P, Cullen D, de Vries R, Gathman A, Goodell B,Henrissat B, Ihrmark K, Kauserud H, Kohler A, LaButti K,Lapidus A, Lavin J, Lee YH, Lindquist E, Lilly W, Lucas S,Morin E, Murat C, Oguiza J, Park J, Pisabarro A, Riley R,Rosling A, Salamov A, Schmidt O, Schmutz J, Skrede I, StenlidJ, Wiebenga A, Xie X, Kües U, Hibbett D, Hoffmeister D,Högberg N, Martin F, Grigoriev I, Watkinson S (2011) The plantcell wall-decomposing machinery underlies the functional diversityof forest fungi. Science 333:762–765
Fu J, Prade R, Mort A (2001) Expression and action pattern ofBotryotinia fuckeliana (Botrytis cinerea) rhamnogalacturonanhydrolase in Pichia pastoris. Carbohydr Res 330:73–81
Gottlieb M, Chavko M (1987) Silver staining of native and denaturedeukaryotic DNA in agarose gels. Anal Biochem 165:33–37
Henrissat B (1991) A classification of glycosyl hydrolases based onamino-acid sequence similiraties. Biochem J 280:309–316
Ishii T (1995) Pectic polysaccharides from bamboo shoot cell-walls.Mokuzai Gakkaishi 41:669–676
Kang HJ, Jo C, Kwon JH, Son JH, An BJ, ByunMW (2006) Antioxidantand cancer cell proliferation inhibition effect of citrus pectin-oligosaccharide prepared by irradiation. J Med Food 9:313–320
Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP,Dörreich K, Dalbøge H (1994) Cloning and characterization of twostructurally and functionally divergent rhamnogalacturonases fromAspergillus aculeatus. J Biol Chem 269:29182–29189
Mandalari G, Palop CN, Tuohy K, Gibson GR, Bennett RN, WaldronKW, Bisignano G, Narbad A, Faulds CB (2007) In vitroevaluation of the prebiotic activity of a pectic oligosaccharide-richextract enzymatically derived from bergamot peel. Appl MicrobiolBiotechnol 73:1173–1179
Manderson K, Pinart M, Tuohy KM, Grace WE, Hotchkiss AT,Widmer W, Yadhav MP, Gibson GR, Rastall RA (2005) In vitrodetermination of prebiotic properties of oligosaccharides derivedfrom an orange juice manufacturing by-product stream. ApplEnviron Microbiol 71:8383–8389
Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M,Kubicek CP, Ferreira P, Ruiz-Duenas FJ, Martinez AT, Kersten P,Hammel KE,VandenWymelenberg A, Gaskell J, Lindquist E, SabatG, BonDurant SS, Larrondo LF, Canessa P, Vicuña R, Yadav J,Doddapaneni H, Subramanian V, Pisabarro AG, Lavin JL, OguizaJA, Master E, Henrissat B, Coutinho PM, Harris P, Magnuson JK,Baker SE, Bruno K, Kenealy W, Hoegger PJ, Kües U, Ramaiya P,Lucash S, Salamov A, Shapiro H, Tu H, Chee CL, Misra M, Xie G,Teter S, Yaver D, James T, Mokrejs M, Pospisek M, Grigoriev IV,Brettin T, Rokhsar D, Berka R, Cullen D (2009) Genome,transcriptome, and secretome analysis of wood decay fungus Postiaplacenta supports unique mechanisms of lignocellulose conversion.Proc Natl Acad Sci U S A 106:1954–1959
Nelson N (1944) A photometric adaptation of the Somogoyi methodfor determination of glucose. J Biol Chem 153:375–380
Normand J, Ralet M-C, Thibault J-F, Rogniaux H, Delavault P, Bonnin E(2010) Purification, characterization and mode of action of arhamnogalacturonan-hydrolase from Irpex lacteus, tolerant to anacetylated substrate. Appl Microbiol Biotechnol 86:577–588
Petersen TN, Kauppinen S, Larsen S (1997) The crystal structure ofrhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel β helix. Structure 5:533–544
Schols HA, Voragen AGJ (1994) Occurrence of pectic hairy regions invarious plant cell wall materials and their degradability byrhamnogalacturonase. Carbohydr Res 256:83–95
Schols HA, Voragen AGJ (1996) Complex pectins: structure elucida-tion using enzymes. In: Visser J, Voragen AGJ (eds) Progress inbiotechnology 14. Pectins and pectinases. Elsevier, Amsterdam,pp 3–19
Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, KormelinkFJM, Voragen AGJ (1990) Rhamnogalacturonase: a novelenzyme that degrades the hairy regions of pectins. CarbohydrRes 206:105–115
Sunga AJ, Tolstorukov I, Cregg JM (2008) Posttransformationalvector amplification in the yeast Pichia pastoris. FEMS YeastResearch 8:870–876
Suykerbuyk MEG, Schaap PJ, Stam H, Musters W, Visser J (1995)Cloning, sequence and expression of the gene coding forrhamnogalacturonase of Aspergillus aculeatus; a novel pectinolyticenzyme. Appl Microbiol Biotechnol 43:861–870
Suykerbuyk MEG, Kester HCM, Schaap PJ, Stam H, Musters W,Visser J (1997) Cloning and characterization of two rhamnoga-lacturonases from Aspergillus niger. Appl Environ Microbiol63:2507–2515
Voragen AGJ, Pilnik W, Thibault J-F, Axelos MAV, Renard CMGC(1995) Pectins. In: Stephen AM (ed) Food polysaccharides andtheir applications. Marcel Dekker, New York, pp 287–339
Williamson G (1991) Purification and characterization of pectinacetylesterase from orange peel. Phytochem 30:445–449
Wong D (2008) Enzymatic deconstruction of backbone structures ofthe ramified regions in pectins. Protein J 27:30–42
