Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study
Identifieur interne : 000029 ( Pmc/Corpus ); précédent : 000028; suivant : 000030Eukaryotic Expression System Pichia pastoris Affects the Lipase Catalytic Properties: A Monolayer Study
Auteurs : Madiha Bou Ali ; Yassine Ben Ali ; Imen Aissa ; Youssef GargouriSource :
- PLoS ONE [ 1932-6203 ] ; 2014.
Abstract
Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.
Url:
DOI: 10.1371/journal.pone.0104221
PubMed: 25133585
PubMed Central: 4136768
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Eukaryotic Expression System <italic>Pichia pastoris</italic> Affects the Lipase Catalytic Properties: A Monolayer Study</title><author><name sortKey="Bou Ali, Madiha" sort="Bou Ali, Madiha" uniqKey="Bou Ali M" first="Madiha" last="Bou Ali">Madiha Bou Ali</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ben Ali, Yassine" sort="Ben Ali, Yassine" uniqKey="Ben Ali Y" first="Yassine" last="Ben Ali">Yassine Ben Ali</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Aissa, Imen" sort="Aissa, Imen" uniqKey="Aissa I" first="Imen" last="Aissa">Imen Aissa</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Gargouri, Youssef" sort="Gargouri, Youssef" uniqKey="Gargouri Y" first="Youssef" last="Gargouri">Youssef Gargouri</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25133585</idno><idno type="pmc">4136768</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136768</idno><idno type="RBID">PMC:4136768</idno><idno type="doi">10.1371/journal.pone.0104221</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000029</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000029</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Eukaryotic Expression System <italic>Pichia pastoris</italic> Affects the Lipase Catalytic Properties: A Monolayer Study</title><author><name sortKey="Bou Ali, Madiha" sort="Bou Ali, Madiha" uniqKey="Bou Ali M" first="Madiha" last="Bou Ali">Madiha Bou Ali</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ben Ali, Yassine" sort="Ben Ali, Yassine" uniqKey="Ben Ali Y" first="Yassine" last="Ben Ali">Yassine Ben Ali</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Aissa, Imen" sort="Aissa, Imen" uniqKey="Aissa I" first="Imen" last="Aissa">Imen Aissa</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Gargouri, Youssef" sort="Gargouri, Youssef" uniqKey="Gargouri Y" first="Youssef" last="Gargouri">Youssef Gargouri</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Martinelle, M" uniqKey="Martinelle M">M Martinelle</name></author><author><name sortKey="Holmquist, M" uniqKey="Holmquist M">M Holmquist</name></author><author><name sortKey="Hult, K" uniqKey="Hult K">K Hult</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jaeger, Ke" uniqKey="Jaeger K">KE Jaeger</name></author><author><name sortKey="Eggert, T" uniqKey="Eggert T">T Eggert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sassenfeld, Hm" uniqKey="Sassenfeld H">HM 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25133585</article-id><article-id pub-id-type="pmc">4136768</article-id><article-id pub-id-type="publisher-id">PONE-D-14-14607</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0104221</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Biochemistry</subject><subj-group><subject>Enzymology</subject><subject>Lipids</subject></subj-group></subj-group></subj-group></article-categories><title-group><article-title>Eukaryotic Expression System <italic>Pichia pastoris</italic> Affects the Lipase Catalytic Properties: A Monolayer Study</article-title><alt-title alt-title-type="running-head">Yeast Expression System Affects the Lipase Catalytic Properties</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Bou Ali</surname><given-names>Madiha</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Ben Ali</surname><given-names>Yassine</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Aissa</surname><given-names>Imen</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Gargouri</surname><given-names>Youssef</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><addr-line>Laboratory of Biochemistry, National Engineering School of Sfax (ENIS), University of Sfax, Sfax, Tunisia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Deschenes</surname><given-names>Robert J.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>College of Medicine, University of South Florida, United States of America</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>ytgargouri@yahoo.fr</email></corresp><fn fn-type="conflict"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: YG YBA. Performed the experiments: MB IA. Analyzed the data: YBA YG MB IA. Contributed reagents/materials/analysis tools: YBA YG MB IA. Contributed to the writing of the manuscript: MB YBA YG.</p></fn></author-notes><pub-date pub-type="collection"><year>2014</year></pub-date><pub-date pub-type="epub"><day>18</day><month>8</month><year>2014</year></pub-date><volume>9</volume><issue>8</issue><elocation-id>e104221</elocation-id><history><date date-type="received"><day>1</day><month>4</month><year>2014</year></date><date date-type="accepted"><day>10</day><month>7</month><year>2014</year></date></history><permissions><copyright-year>2014</copyright-year><copyright-holder>Bou Ali et al</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension.</p></abstract><funding-group><funding-statement>The authors have no support or funding to report.</funding-statement></funding-group><counts><page-count count="8"></page-count></counts><custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name><meta-value>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.</meta-value></custom-meta></custom-meta-group></article-meta><notes><title>Data Availability</title><p>The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.</p></notes></front><body><sec id="s1"><title>Introduction</title><p>Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis of triacylglycerols at the interface between the insoluble substrate and water <xref rid="pone.0104221-Martinelle1" ref-type="bibr">[1]</xref> and have been widely used in industrial fields including detergents, dairy products, diagnostics, oil processing and biotransformation due to substrate specificity, regiospecificity, enantiomeric selectivity, thermostability and alkaline stability <xref rid="pone.0104221-Jaeger1" ref-type="bibr">[2]</xref>.</p><p>Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells in order to obtain high levels of expression for both fundamental and applied research purposes. For large scale purification of recombinant proteins, affinity chromatography is usually preferred due to the specificity of the process and the relatively easy purification schemes. Chimeric proteins have been made with affinity tags like L-galactosidase, Escherichia colimaltose binding protein, FLAG peptide, glutathione-S-transferase and hexahistidine tag (His-tag) <xref rid="pone.0104221-Sassenfeld1" ref-type="bibr">[3]</xref>. The His-tag, consisting of four to six consecutive histidine residues, functions as a predominant ligand in immobilized metal affinity chromatography (IMAC) <xref rid="pone.0104221-Porath1" ref-type="bibr">[4]</xref>. The ability of the histidine residue complex to bind to metal ions with high affinity even in the presence of denaturing agents and the requirement of mild elution conditions has made the His-tag a versatile tool for protein purification and characterization <xref rid="pone.0104221-Paborsky1" ref-type="bibr">[5]</xref>, <xref rid="pone.0104221-Nieba1" ref-type="bibr">[6]</xref>. However, few comparative kinetic studies have been performed between tagged and un-tagged eukaryotic enzymes.</p><p>Recent reports have shown that His-tag can alter the binding characteristics of recombinant proteins when compared to the wild-type native protein <xref rid="pone.0104221-GabercPorekar1" ref-type="bibr">[7]</xref>, <xref rid="pone.0104221-Hang1" ref-type="bibr">[8]</xref>. Hang et al. <xref rid="pone.0104221-Hang1" ref-type="bibr">[8]</xref> showed that although the His-tagged subunits of the terminase enzyme from bacteriophage (lamda) formed holoenzymes with wild-type catalytic activity, one of the subunits showed altered interaction with DNA. The length, composition and location of His-tag can require further optimization depending upon the sequences of the native protein <xref rid="pone.0104221-GabercPorekar1" ref-type="bibr">[7]</xref>, <xref rid="pone.0104221-Chaga1" ref-type="bibr">[9]</xref>. Nevertheless, there are several studies where ‘affinity tags’ have no adverse effect on the activity of the native proteins <xref rid="pone.0104221-Casey1" ref-type="bibr">[10]</xref>, <xref rid="pone.0104221-Sharma1" ref-type="bibr">[11]</xref>, <xref rid="pone.0104221-Passafiume1" ref-type="bibr">[12]</xref>.</p><p>Recently, Horchani et al <xref rid="pone.0104221-Horchani1" ref-type="bibr">[13]</xref> studied the interfacial and kinetic properties of the wild type, untagged recombinant and tagged recombinant forms of three staphylococcal lipases (SSL, SXL and SAL3) which were compared using the monomolecular film technique. They showed that independently from the negative effects of the recombinant expression process per se (REC), the presence of an N-terminal tag extension would decrease the catalytic activities (TEC) of staphylococcal lipases by creating a steric hindrance during the interfacial binding step. The effects of the heterologous expression process on the catalytic properties of the staphylococcal lipases are three times more deleterious than the presence of an N-terminal tag extension <xref rid="pone.0104221-Horchani1" ref-type="bibr">[13]</xref>.</p><p>No studies have been conducted to compare the kinetic properties of the wild type form of eukaryotic lipases with those of the tagged and untagged recombinant forms.</p><p>The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. This avian pancreatic lipase contains 450 amino acids and presents an experimental mass of 49665.31 Da <xref rid="pone.0104221-Fendri1" ref-type="bibr">[14]</xref>, <xref rid="pone.0104221-Sayari1" ref-type="bibr">[15]</xref>. To investigate the structure-function relationships of this lipase, TPL was expressed in <italic>Pichia pastoris</italic>. The biochemical characterization of the rTPL, using an emulsified system (pH-Stat), and its comparison to the native TPL show that the rTPL seems to be identical to the native enzyme <xref rid="pone.0104221-BouAli1" ref-type="bibr">[16]</xref>.</p><p>In this study, we report the expression of the tagged TPL (6His-rTPL) in <italic>Pichia pastoris</italic>. The recombinant tagged enzyme was purified and its kinetic properties are compared to the recombinant and the native forms using the monomolecular film technique in order to study the Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC).</p></sec><sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title><sec id="s2a"><title>1. Lipases</title><p>The native turkey pancreatic lipase (nTPL) was purified from delipidated pancreases as described by Sayari et al (2000) <xref rid="pone.0104221-Sayari1" ref-type="bibr">[15]</xref> and the recombinant TPL was expressed in <italic>Pichia pastoris (X33)</italic> and purified using adapted chromatographic methods, as previously described <xref rid="pone.0104221-BouAli1" ref-type="bibr">[16]</xref>. Starting with TPL full-length DNA (1353 pb) cloned into the pGAPZαA, which served as the template, the tagged TPL gene was obtained by PCR amplification using the following forward and reverse primers. Forward primer includes 6 Histidine codons (grey) and both primers including an EcoRI restriction site (underlined):</p><p>Primer Fw: <named-content content-type="gene">5′-GATC<underline>GAATTC</underline>CATCATCATCATCATCATTCTGAAGTTTGCTATGAC-3′</named-content> Primer Rv: <named-content content-type="gene">5′-GATC<underline>GAATTC</underline>TTAGCAAGCAGTAAGGGT-3′</named-content></p><p>The tagged recombinant TPL was expressed in <italic>Pichia pastoris (X33)</italic> and purified to homogeneity in a single step, using immobilized metal affinity chromatographic method (<xref ref-type="fig" rid="pone-0104221-g001">Figure 1</xref>).</p><fig id="pone-0104221-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.g001</object-id><label>Figure 1</label><caption><title>SDS-PAGE of the three TPL forms performed on 12% acrylamide gel.</title><p>Lane 1: Low molecular weight marker; Lane 2, 3 and 4: purified 6His-rTPL, rTPL and nTPL.</p></caption><graphic xlink:href="pone.0104221.g001"></graphic></fig></sec><sec id="s2b"><title>2. Lipids</title><p>1,2-<italic>sn</italic>- and 1,3-<italic>sn</italic>-dicaprin were from Sigma. 2,3-<italic>sn</italic>-dicaprin was prepared from tricaprin (Sigma) by performing stereospecific enzymatic hydrolysis on the <italic>sn</italic>-1 ester bond <xref rid="pone.0104221-Rogalska1" ref-type="bibr">[17]</xref>. As expected, the surface pressure-molecular area curves obtained with 1,2-<italic>sn</italic>-and 2,3-<italic>sn</italic>-dicaprin were found to be superimposable and characteristic of the 1iquid expanded state, and showed no sign of discontinuity throughout the range of surface pressures investigated. The collapse pressure of 1,2-<italic>sn</italic>- and 2,3-<italic>sn</italic>-dicaprin was 40 mN.m<sup>−1</sup>, and that of 1,3-<italic>sn</italic>-dicaprin was 32 mN.m <sup>−1</sup><xref rid="pone.0104221-Rogalska2" ref-type="bibr">[18]</xref>.</p></sec><sec id="s2c"><title>3. Protein concentration determinations</title><p>Protein concentration was determined as described by Bradford et al. <xref rid="pone.0104221-Bradford1" ref-type="bibr">[19]</xref>, using BSA as standard. The lipase extinction coefficients were determined using the absorption UV spectroscopy <xref rid="pone.0104221-Gill1" ref-type="bibr">[20]</xref>. The protein assays on the three lipases were performed simultaneously and in parallel, using the same chemical reagents.</p></sec><sec id="s2d"><title>4. Lipase activity and kinetic properties determination</title><p>The lipase activity was measured titrimetically at pH 8.5 and 37°C with a pH-Stat using olive oil emulsion or Tributyrin (TC<sub>4</sub>) in presence of 4 mM NaDC and Turkey colipase. One lipase unit corresponds to 1 µmol of fatty acid liberated per min.</p><p>The kinetic constants (K<sub>m</sub>app, k<sub>cat</sub>, V<sub>max</sub>, and k<sub>cat</sub>/K<sub>m</sub>app.) calculated from the initial rate activity of native, recombinant and recombinant tagged TPL forms were determined using tributyrine. The effect of TC4 concentration on the hydrolysis rate was measured at various concentrations ranging from 0.68 to 68 mM.</p></sec><sec id="s2e"><title>5. SDS-PAGE</title><p>Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed by the method of Laemmli <xref rid="pone.0104221-Laemmli1" ref-type="bibr">[21]</xref>. The proteins were stained with Coomassie brilliant blue.</p></sec><sec id="s2f"><title>6. Monomolecular film technique for kinetic measurements on lipase</title><p>Measurements were performed with KSV-2000 barostat equipment (KSV-Helsinki, Finland). The principle of the method was described previously by Verger and De Haas <xref rid="pone.0104221-Verger1" ref-type="bibr">[22]</xref>. It involves the use of a “zero-order” trough with a reaction compartment and a reservoir compartment, which are connected to each other by a small surface channel. The enzyme solution was injected into the subphase of the reaction compartment when the lipid film covered both compartments. A mobile barrier, driven automatically by the barostat, moved back and forth over the reservoir to keep the surface pressure (π) constant, thus compensating for the substrate molecules removed from the film by enzyme hydrolysis. The surface pressure was measured in the reservoir compartment with a Wilhelmy plate (perimeter 3.94 cm) attached to an electromicrobalance, which was connected in turn to a microprocessor programmed to regulate the movement of the mobile barrier. As previously established <xref rid="pone.0104221-Verger1" ref-type="bibr">[22]</xref>, the sensitivity of the Wilhelmy plate was estimated at 0.1 mN.m<sup>−1</sup>. Two 2-cm magnetic stirrers running at 250 rpm were placed in the reaction compartment. Kinetic measurements were performed at room temperature (25°C). The reaction compartment had an area of 130 cm<sup>2</sup> and a volume of 130 ml. The reservoir compartment was 148 mm wide and 249 mm long. The monolayers were formed by spreading a chloroform solution at the air/water interface, using a Hamilton microsyringe. Five to ten minutes were then allowed for the chloroform to evaporate and the film to form. Prior to each experiment, the Teflon trough used to form the monomolecular film was cleaned with water and then gently brushed in the presence of distilled ethanol, washed again with tap water, and finally rinsed with double-distilled water.</p><p>The aqueous subphase was composed of buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 21 mM CaCl<sub>2</sub>, and 1 mM EDTA) prepared with double-distilled water and filtered through a 0.45 µm Milli-pore filter. Any residual surface-active impurities were removed before each assay by sweeping the surface and applying suction. The kinetic recordings were usually linear. Activities were expressed as the number of moles of substrate hydrolyzed per unit time and unit reaction compartment area at an arbitrary lipase concentration of 1 M in the “zero-order” trough.</p></sec><sec id="s2g"><title>7. Statistical analysis</title><p>Experimental results were given as mean value±SD of three parallel measurements. Comparisons between values were analyzed by Student's test for unpaired data, and p<0.05 was considered significant. All statistical analysis were conducted using Microsoft Excel software.</p></sec></sec><sec id="s3"><title>Results and Discussion</title><sec id="s3a"><title>1. Variations with surface pressure in the catalytic activities of nTPL, rTPL and 6His-rTPL using the three dicaprin isomers</title><p>Kinetic experiments were performed at various surface pressures using three dicaprin isomers as substrates and three TPL forms. As can be seen in <xref ref-type="fig" rid="pone-0104221-g002">figure 2</xref>, the three dicaprin isomers were clearly differentiated by all the lipases studied, and this differentiation was more pronounced at high surface pressures.</p><fig id="pone-0104221-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.g002</object-id><label>Figure 2</label><caption><title>Surface pressure-activity profiles of nTPL, rTPL and 6His-rTPL measured on 1,2-<italic>sn</italic>-dicaprin (A), 2,3-<italic>sn</italic>-dicaprin (B), and 1,3-<italic>sn</italic>-dicaprin (C).</title><p>Assays were carried out at room temperature in a “zero-order” trough (volume, 130 ml; surface area, 111 cm<sup>2</sup>). Buffer: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 21 mM CaCl2, and 1 mM EDTA. Activities are expressed as the number of moles of substrate hydrolyzed by unit time and unit surface of the reaction compartment of the “zero-order” trough for an arbitrary lipase concentration of 1 M. Bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.001; nTPL versus rTPL. <bold><sup>§</sup></bold>p<0.05, <bold><sup>§§</sup></bold>p<0.01, <bold><sup>§§§</sup></bold>p<0.001; nTPL versus 6His-rTPL. <bold><sup>+</sup></bold>p<0.05, <bold><sup>++</sup></bold>p<0.01, <bold><sup>+++</sup></bold>p<0.001; rTPL versus 6His-rTPL.</p></caption><graphic xlink:href="pone.0104221.g002"></graphic></fig><p>When using the 1,2-<italic>sn</italic>-dicaprin, the nTPL and rTPL present their maximal activities at a surface pressure of 20 mN.m<sup>−1</sup>, but the 6His-rTPL presents its optimal activity at 25 mN.m<sup>−1</sup>. Up to those pressures, the three lipase form activities decreased (<xref ref-type="fig" rid="pone-0104221-g002">Figure 2A</xref>).</p><p>Using 2,3-<italic>sn</italic>-dicaprin and 1,3-<italic>sn</italic>-dicaprin, the nTPL shows a maximum activity with bell-shaped curves at an optimal surface pressure of 20 mN.m<sup>−1</sup> and 15 mN.m<sup>−1</sup> respectively, contrary to the rTPL and 6His-rTPL which exhibit increased activity according to pressure showing high pressure preference (<xref ref-type="fig" rid="pone-0104221-g002">Figures 2B and 2C</xref>). When analyzing the maximum catalytic activities of the three TPL forms (<xref ref-type="fig" rid="pone-0104221-g003">Figure 3A</xref>), one can easily state that nTPL maximum catalytic activities were always higher than those of the rTPL and 6His- rTPL (<xref ref-type="fig" rid="pone-0104221-g003">Figure 3A</xref>).</p><fig id="pone-0104221-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.g003</object-id><label>Figure 3</label><caption><title>Histogram of the maximum activities of the three TPL forms.</title><p>(<bold>A</bold>) maximum activities on monomolecular films of 1,2-<italic>sn</italic>-dicaprin, 2,3-<italic>sn</italic>-dicaprin, and 1,3-<italic>sn</italic>-dicaprin, Data based on <xref ref-type="fig" rid="pone-0104221-g001">figure 1</xref>. Activities are expressed as the number of moles of substrate hydrolysed per unit time (min) and unit surface (cm<sup>2</sup>) at an arbitrary lipase concentration of 1 M in the aqueous subphase of the reaction compartment of a “zero-order” trough (volume, 130 ml; surface area, 111 cm<sup>2</sup>). (<bold>B</bold>) Histogram of kinetic parameters of TPL forms. nTPL (black bars), rTPL (gray bars) and 6His-rTPL (white bars).</p></caption><graphic xlink:href="pone.0104221.g003"></graphic></fig><p>The specific activities of the rTPL and 6His-rTPL are similar when 2,3-<italic>sn</italic>-dicaprin and 1,3-<italic>sn</italic>-dicaprin are used as substrates, but with the 1,2-<italic>sn</italic>-dicaprin, the maximum catalytic activity of the 6His-rTPL is comparable to that of nTPL (<xref ref-type="fig" rid="pone-0104221-g003">Figure 3A</xref>).</p><p>Our results also show that nTPL hydrolyzes the three dicaprin isomers more efficiently than rTPL and 6His-rTPL do. So, the heterologous expression and N-tag extension change the pressure preference and decrease the specific activity of the native enzyme.</p></sec><sec id="s3b"><title>2. Effects of recombinant expression and N-tag extension on catalysis (REC, TEC and TREC)</title><p>To quantify the effects of the recombinant expression process and/or the presence of an N-terminal extension on the catalytic hydrolysis of the three dicaprin isomers, we coined the terms “Recombinant expression Effects on Catalysis” (REC), “N-Tag Effects on Catalysis” (TEC) and “N-Tag and Recombinant expression Effects on Catalysis” (TREC), which were defined as follows:<disp-formula id="pone.0104221.e001"><graphic xlink:href="pone.0104221.e001.jpg" position="anchor" orientation="portrait"></graphic></disp-formula><disp-formula id="pone.0104221.e002"><graphic xlink:href="pone.0104221.e002.jpg" position="anchor" orientation="portrait"></graphic></disp-formula><disp-formula id="pone.0104221.e003"><graphic xlink:href="pone.0104221.e003.jpg" position="anchor" orientation="portrait"></graphic></disp-formula>A (nTPL), A (rTPL) and A (6His-rTPL) are the lipase activities of the wild type, untagged recombinant and tagged recombinant TPL, respectively. The TEC, REC and TREC values were calculated from data presented in <xref ref-type="fig" rid="pone-0104221-g002">figure 2</xref> in the case of the three lipases forms. The TEC, REC and TREC panels showed mostly negative values, with all dicaprin isomers and surface pressures tested. The mean REC (−0,7) value showed a deleterious effect of the heterologous expression process on the catalytic properties of TPL in contrast to the presence of an N-terminal tag extension (TEC = +0,03) which did not have negative effects. The combination of the heterologous expression and the N-terminal tag extension (TREC = −0,56) showed that the expression of tagget TPL reduced the negative effect caused by the heterologous expression (<xref ref-type="table" rid="pone-0104221-t001">Table 1</xref>). The positive effect of the N-terminal tag extension was found when 1,2-<italic>sn</italic>-dicaprin was used as substrat, but when 2,3-<italic>sn</italic>-dicaprin and 1,3-<italic>sn</italic>-dicaprin were used, negative effects on catalytic properties of TPL were recorded.</p><table-wrap id="pone-0104221-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.t001</object-id><label>Table 1</label><caption><title>REC, TEC and TREC values of TPL obtained using 1,2-<italic>sn</italic> dicaprin, 2,3-<italic>sn</italic>-dicaprin and 1,3-<italic>sn</italic>-dicaprin substrates, spread at the air/water interface forming monomolecular films at 20, 25 and 30 mN.m<sup>−1</sup>.</title></caption><alternatives><graphic id="pone-0104221-t001-1" xlink:href="pone.0104221.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td colspan="4" align="left" rowspan="1">REC</td><td colspan="4" align="left" rowspan="1">TEC</td><td colspan="4" align="left" rowspan="1">TREC</td></tr><tr><td align="left" rowspan="1" colspan="1">Pressure (mN.m−1)</td><td align="left" rowspan="1" colspan="1">1,2-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">2,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">1,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">Mean<xref ref-type="table-fn" rid="nt101">a</xref></td><td align="left" rowspan="1" colspan="1">1,2-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">2,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">1,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">Mean<xref ref-type="table-fn" rid="nt101">a</xref></td><td align="left" rowspan="1" colspan="1">1,2-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">2,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">1,3-<italic>sn</italic>-dicaprin</td><td align="left" rowspan="1" colspan="1">Mean<xref ref-type="table-fn" rid="nt101">a</xref></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">−0,55</td><td align="left" rowspan="1" colspan="1">−0,83</td><td align="left" rowspan="1" colspan="1">−0,72</td><td align="left" rowspan="1" colspan="1">−0,7</td><td align="left" rowspan="1" colspan="1">+0,36</td><td align="left" rowspan="1" colspan="1">−0,38</td><td align="left" rowspan="1" colspan="1">−0,57</td><td align="left" rowspan="1" colspan="1">−0,2</td><td align="left" rowspan="1" colspan="1">−0,24</td><td align="left" rowspan="1" colspan="1">−0,92</td><td align="left" rowspan="1" colspan="1">−0,91</td><td align="left" rowspan="1" colspan="1">−0,69</td></tr><tr><td align="left" rowspan="1" colspan="1">25</td><td align="left" rowspan="1" colspan="1">−0,94</td><td align="left" rowspan="1" colspan="1">−0,66</td><td align="left" rowspan="1" colspan="1">−0,61</td><td align="left" rowspan="1" colspan="1">−0,74</td><td align="left" rowspan="1" colspan="1">+0,93</td><td align="left" rowspan="1" colspan="1">−0,42</td><td align="left" rowspan="1" colspan="1">−0,29</td><td align="left" rowspan="1" colspan="1">+0,07</td><td align="left" rowspan="1" colspan="1">−0,08</td><td align="left" rowspan="1" colspan="1">−0,85</td><td align="left" rowspan="1" colspan="1">−0,76</td><td align="left" rowspan="1" colspan="1">−0,56</td></tr><tr><td align="left" rowspan="1" colspan="1">30</td><td align="left" rowspan="1" colspan="1">−0,95</td><td align="left" rowspan="1" colspan="1">−0,56</td><td align="left" rowspan="1" colspan="1">−0,43</td><td align="left" rowspan="1" colspan="1">−0,65</td><td align="left" rowspan="1" colspan="1">+0,94</td><td align="left" rowspan="1" colspan="1">−0,19</td><td align="left" rowspan="1" colspan="1">−0,1</td><td align="left" rowspan="1" colspan="1">+0,22</td><td align="left" rowspan="1" colspan="1">−0,09</td><td align="left" rowspan="1" colspan="1">−0,68</td><td align="left" rowspan="1" colspan="1">−0,51</td><td align="left" rowspan="1" colspan="1">−0,43</td></tr><tr><td align="left" rowspan="1" colspan="1">Overall mean<xref ref-type="table-fn" rid="nt102">b</xref></td><td colspan="4" align="left" rowspan="1">−0,7</td><td colspan="4" align="left" rowspan="1">+0,03</td><td colspan="4" align="left" rowspan="1">−0,56</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label>a</label><p>Mean values of either TEC or REC or TREC for the three dicaprin isomers at a given surface pressure.</p></fn><fn id="nt102"><label>b</label><p>Overall mean TEC, REC and TREC values obtained with the three dicaprin isomers, tested at three surface pressures.</p></fn></table-wrap-foot></table-wrap></sec><sec id="s3c"><title>3. Regioselectivity and Stereospecificity of TPL</title><p>In order to assess the lipase preference for the distal (acyl chains present at sn-1 and sn-3 positions) versus adjacent (acyl chains present at sn-1 and sn-2 or sn-3 and sn-2 positions) ester groups of diglyceride isomers, we have calculated the vicinity index (VI) of native, recombinant and recombinant Tagged TPL as defined by Rogalska et al. <inline-formula><inline-graphic xlink:href="pone.0104221.e004.jpg"></inline-graphic></inline-formula>, where A1,3, A2,3 and A1,2 are lipases activities measured with 1,3-sn-dicaprin, 2,3-sn-dicaprin and 1,2-sn-dicaprin, respectively. The vicinity index value was calculated at two arbitrary surface pressure values, 15 and 25 mN m<sup>−1</sup>, respectively <xref rid="pone.0104221-Rogalska2" ref-type="bibr">[18]</xref>.</p><p>As shown in <xref ref-type="fig" rid="pone-0104221-g004">figure 4A</xref> and <xref ref-type="table" rid="pone-0104221-t002">table 2</xref>, nTPL, as well as 6His-rTPL prefer distal ester groups of the diacylglycerols isomers (1,3-dicaprin) at low surface pressure (15 mN.m<sup>−1</sup>), but at high surface pressure (25 mN.m<sup>−1</sup>) these enzymes prefer adjacent ester of the diacylglycerols isomers (1,2-<italic>sn</italic>-dicaprin and 2,3-<italic>sn</italic>-dicaprin), in contrast to the rTPL which prefers adjacent ester groups at low surface pressure (15 mN.m<sup>−1</sup>) and distal ester groups of the diglyceride isomers (1,3-dicaprin) at high surface pressure (25 mN.m<sup>−1</sup>).</p><fig id="pone-0104221-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.g004</object-id><label>Figure 4</label><caption><title>The vicinity and the stereoselectivity index of the three forms of TPL.</title><p>(<bold>A</bold>) The vicinity index at 25 versus 15 mN m<sup>−1</sup> is calculated from the specific activities measured at 25 and 15 mN m<sup>−1</sup>, respectively. Data for nTPL, rTPL and 6His-rTPL are derived from <xref ref-type="fig" rid="pone-0104221-g001">figure 1</xref>. Data for HPL and PPL are reproduced from previous work <xref rid="pone.0104221-Rogalska1" ref-type="bibr">[17]</xref>. The vicinity index (V.I.) is calculated with the following formula: <inline-formula><inline-graphic xlink:href="pone.0104221.e005.jpg"></inline-graphic></inline-formula>, where A1,3, A2,3 and A1,2 are lipase specific activities measured with l,3-<italic>sn</italic>-dicaprin, 2,3-<italic>sn</italic>-dicaprin and 1,2-<italic>sn</italic>-dicaprin, respectively. (<bold>B</bold>) The stereoselectivity index at 25 versus 15 mN m<sup>−1</sup> is calculated from the specific activities measured at 25 and 15 mN m<sup>−1</sup>, respectively. Data are derived from <xref ref-type="fig" rid="pone-0104221-g001">figure 1</xref>. The stereoselectivity index (SI) is calculated with the following formula: <inline-formula><inline-graphic xlink:href="pone.0104221.e006.jpg"></inline-graphic></inline-formula>, where A1,2 and A2,3 correspond to lipase specific activities measured with 1,2-<italic>sn</italic>-dicaprin and 2,3-<italic>sn</italic>-dicaprin, respectively.</p></caption><graphic xlink:href="pone.0104221.g004"></graphic></fig><table-wrap id="pone-0104221-t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.t002</object-id><label>Table 2</label><caption><title>Regioselectivity and Stereospecificity of nTPL, rTPL and 6His-rTPL.</title></caption><alternatives><graphic id="pone-0104221-t002-2" xlink:href="pone.0104221.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td colspan="3" align="left" rowspan="1">V.I.</td><td colspan="3" align="left" rowspan="1">S.I.</td></tr><tr><td align="left" rowspan="1" colspan="1">surface Pressure (mNm<sup>−1</sup>)</td><td align="left" rowspan="1" colspan="1">nTPL</td><td align="left" rowspan="1" colspan="1">rTPL</td><td align="left" rowspan="1" colspan="1">6His-rTPL</td><td align="left" rowspan="1" colspan="1">nTPL</td><td align="left" rowspan="1" colspan="1">rTPL</td><td align="left" rowspan="1" colspan="1">6His-rTPL</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold>15</bold></td><td align="left" rowspan="1" colspan="1">0,248</td><td align="left" rowspan="1" colspan="1">−0,172</td><td align="left" rowspan="1" colspan="1">0,202</td><td align="left" rowspan="1" colspan="1">−0,13</td><td align="left" rowspan="1" colspan="1">−0,728</td><td align="left" rowspan="1" colspan="1">−0,97</td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>25</bold></td><td align="left" rowspan="1" colspan="1">−0,033</td><td align="left" rowspan="1" colspan="1">0,387</td><td align="left" rowspan="1" colspan="1">−0,98</td><td align="left" rowspan="1" colspan="1">0,05</td><td align="left" rowspan="1" colspan="1">0,711</td><td align="left" rowspan="1" colspan="1">−0,84</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt103"><label></label><p>The vicinity index (V.I.) and the stereospecificity index (S.I.) of the three TPL forms were calculated using the following definitions: V.I. = [A<sub>1,3</sub>−½ (A<sub>2,3</sub>+A<sub>1,2</sub>)]/[A<sub>1,3</sub>+½ (A<sub>2,3</sub>+A<sub>1,2</sub>)] and S.I. = (A<sub>2,3</sub>−A<sub>1,2</sub>)/(A<sub>2,3</sub>+A<sub>1,2</sub>) at two surface pressure values (15 and 25 mN.m<sup>−1</sup>), where A<sub>1,3</sub>, A<sub>1,2</sub> and A<sub>2,3</sub> are lipase activities measured with 1,3-<italic>sn</italic>-dicaprin; 1,2-<italic>sn</italic>-dicaprin and 2,3-<italic>sn</italic>-dicaprin, respectively.</p></fn></table-wrap-foot></table-wrap><p>To study the three TPL forms stereopreferences, catalytic activities were measured using 1,2-sn- and 2,3-<italic>sn</italic>- dicaprin at two surface pressure values (15 and 25 mN m<sup>−1</sup>). The stereoselectivity index (SI) was calculated as defined previously by Rogalska et al <xref rid="pone.0104221-Rogalska2" ref-type="bibr">[18]</xref> using the following definition: <inline-formula><inline-graphic xlink:href="pone.0104221.e007.jpg"></inline-graphic></inline-formula> where A2,3 and A1,2 are lipase activity with 2,3-sn and 1,2-sn-dicaprin, respectively.</p><p>S.I. values presented in <xref ref-type="fig" rid="pone-0104221-g004">figure 4B</xref> and <xref ref-type="table" rid="pone-0104221-t002">table 2</xref> show that the three TPL forms are stereospecific for the <italic>sn</italic>-1 position of the 1,2-<italic>sn</italic>-enantiomer of dicaprin at low surface pressures. But at high surface pressures, the 6His-rTPL remains stereospecific for the <italic>sn</italic>-1 position in contrast to the nTPL and rTPL which become stereospecific for the <italic>sn</italic>-3 position of the 2,3-<italic>sn</italic>-dicaprin (<xref ref-type="fig" rid="pone-0104221-g004">Figure 4B</xref>; <xref ref-type="table" rid="pone-0104221-t002">Table 2</xref>).</p><p>However, the heterologous expression changes the TPL regioselectivity without affecting the stereospecificity contrary to the N-tag extension which retains the TPL regioselectivity and changes the stereospecificity at high surface pressures, so the 6His-rTPL remains <italic>sn</italic>1 specific regardless of the surface pressure. This result can be explained by the large difference between rTPL and 6His-rTPL activities at high surface pressure when 1,2-<italic>sn</italic>-dicaprin was used as substrat (<xref ref-type="fig" rid="pone-0104221-g002">Figure 2A</xref>) and the difference between rTPL and 6His-rTPL regioselectivity at high surface pressures where the 6His-rTPL would be specific to the adjacent ester of the diglyceride isomers (<xref ref-type="fig" rid="pone-0104221-g004">Figure 4A</xref>).</p></sec></sec><sec id="s4"><title>General Discussion</title><p>Nowadays, <italic>Pichia pastoris</italic> is the most frequently used yeast system for heterologous protein production. Over the last few years, several products based on this platform have gained approval as biopharmaceutical drugs. Protein folding and secretion have been identified as significant bottlenecks in yeast expression systems, pinpointing a major target for strain optimization. However, few studies have investigated the heterologous expression and the N-terminal tag extension effects on the expressed proteins properties.</p><p>To our knowledge, this is the first attempt to discriminate between the negative effects of the heterologous expression process and those of the N-terminal tag extension process on pancreatic lipase catalysis. One of the main features of lipolytic enzymes is the fact that, although they are water-soluble, they catalyse substrate hydrolysis at lipid-water interfaces. In the simplest kinetic model developed by Verger and co-workers <xref rid="pone.0104221-Verger1" ref-type="bibr">[22]</xref>, the coupling between the classical Michaelis-Menten step and various interfacial processes such as the penetration and activation of the lipase and the solubilization of the reaction products were described. It is worth noting that the negative effects of the heterologous expression and N-terminal tag extension processes are not very marked when a partly water-soluble (TC4) and an insoluble (Olive oil) substrates are used (<xref ref-type="table" rid="pone-0104221-t003">Table 3</xref>). When k<sub>cat</sub> and catalytic efficiency (k<sub>cat</sub>/k<sub>m app</sub>) were calculated, we can see than there are not differences between native, recombinant and recombinant tagged TPL confirming that there are not significant differences between the three TPL forms when classical analysis techniques were used (<xref ref-type="fig" rid="pone-0104221-g003">Figure 3B</xref>).</p><table-wrap id="pone-0104221-t003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0104221.t003</object-id><label>Table 3</label><caption><title>Specific activity of nTPL, rTPL and 6His-rTPL using olive oil emulsion and TC4 as substrate.</title></caption><alternatives><graphic id="pone-0104221-t003-3" xlink:href="pone.0104221.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">nTPL</td><td align="left" rowspan="1" colspan="1">rTPL</td><td align="left" rowspan="1" colspan="1">6His-rTPL</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Specific activity on Olive oil (U/mg)</td><td align="left" rowspan="1" colspan="1">4300±400</td><td align="left" rowspan="1" colspan="1">5400±300</td><td align="left" rowspan="1" colspan="1">6000±300</td></tr><tr><td align="left" rowspan="1" colspan="1">Specific activity on TC4 (U/mg)</td><td align="left" rowspan="1" colspan="1">9500±500</td><td align="left" rowspan="1" colspan="1">10000±200</td><td align="left" rowspan="1" colspan="1">11000±400</td></tr></tbody></table></alternatives></table-wrap><p>The rTPL and 6His-rTPL seem to share the same biochemical and kinetic properties with the native enzyme under the same experimental conditions (pH stat technique). But when sensitive method such as the mono-molecular film technique was used, we found significant differences in the interfacial properties of the nTPL, rTPL and 6His-rTPL showing the negative effects of the heterologous expression and the N-terminal tag extension on the TPL properties. The most pronounced difference was the significant decrease in the specific activity of the recombinant and the tagged recombinant enzyme in comparison to the native TPL (<xref ref-type="fig" rid="pone-0104221-g003">Figure 3A</xref>). We also found that heterologous expression changed the regioselectivity of the TPL without affecting the stereospecificity contrary to the N-tag extension which retained the regioselectivity of the TPL and changed the stereospecificity at high surface pressures. 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