NickelMaghrebV1, France, Analysis, bibRecord, 000146

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

Identifieur interne : 000146 ( France/Analysis ); précédent : 000145; suivant : 000147

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

Auteurs : RIADH BEN SALAH [Tunisie] ; Ali Gargouri [Tunisie] ; Robert Verger [France] ; Youssef Gargouri [Tunisie] ; Hafedh Mejdoub [Tunisie]

Source :

World journal of microbiology & biotechnology [ 0959-3993 ] ; 2009.

RBID : Pascal:09-0342194

Descripteurs français

Pascal (Inist)
- Triacylglycerol lipase, Rhizopus oryzae, Mutation, Purification, Caractérisation, Pichia pastoris, Mutagenèse dirigée, Surexpression génique.

English descriptors

KwdEn :
- Characterization, Gene overexpression, Mutation, Pichia pastoris, Purification, Rhizopus oryzae, Site directed mutagenesis, Triacylglycerol lipase.

Abstract

The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX₁ promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X₃₃ and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X₃₃ cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His₆-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His₆-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His₆-ROLw) and the mutant (His₆-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.

Affiliations:

Links toward previous steps (curation, corpus...)

to stream PascalFrancis, to step Corpus: 000183
to stream PascalFrancis, to step Curation: 000258
to stream PascalFrancis, to step Checkpoint: 000190
to stream Main, to step Merge: 000527
to stream Main, to step Curation: 000513
to stream Main, to step Exploration: 000513
to stream France, to step Extraction: 000146

Links to Exploration step

Pascal:09-0342194

Le document en format XML

<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en" level="a">Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization</title>
<author><name sortKey="Riadh Ben Salah" sort="Riadh Ben Salah" uniqKey="Riadh Ben Salah" last="Riadh Ben Salah">RIADH BEN SALAH</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Gargouri, Ali" sort="Gargouri, Ali" uniqKey="Gargouri A" first="Ali" last="Gargouri">Ali Gargouri</name>
<affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour, Km 6, P.O. Box « K »</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>2 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Verger, Robert" sort="Verger, Robert" uniqKey="Verger R" first="Robert" last="Verger">Robert Verger</name>
<affiliation wicri:level="3"><inist:fA14 i1="03"><s1>Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS</s1>
<s2>Marseille</s2>
<s3>FRA</s3>
<sZ>3 aut.</sZ>
</inist:fA14>
<country>France</country>
<placeName><region type="region">Provence-Alpes-Côte d'Azur</region>
<region type="old region">Provence-Alpes-Côte d'Azur</region>
<settlement type="city">Marseille</settlement>
</placeName>
</affiliation>
</author>
<author><name sortKey="Gargouri, Youssef" sort="Gargouri, Youssef" uniqKey="Gargouri Y" first="Youssef" last="Gargouri">Youssef Gargouri</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Mejdoub, Hafedh" sort="Mejdoub, Hafedh" uniqKey="Mejdoub H" first="Hafedh" last="Mejdoub">Hafedh Mejdoub</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">INIST</idno>
<idno type="inist">09-0342194</idno>
<date when="2009">2009</date>
<idno type="stanalyst">PASCAL 09-0342194 INIST</idno>
<idno type="RBID">Pascal:09-0342194</idno>
<idno type="wicri:Area/PascalFrancis/Corpus">000183</idno>
<idno type="wicri:Area/PascalFrancis/Curation">000258</idno>
<idno type="wicri:Area/PascalFrancis/Checkpoint">000190</idno>
<idno type="wicri:explorRef" wicri:stream="PascalFrancis" wicri:step="Checkpoint">000190</idno>
<idno type="wicri:doubleKey">0959-3993:2009:Riadh Ben Salah:expression:in:pichia</idno>
<idno type="wicri:Area/Main/Merge">000527</idno>
<idno type="wicri:Area/Main/Curation">000513</idno>
<idno type="wicri:Area/Main/Exploration">000513</idno>
<idno type="wicri:Area/France/Extraction">000146</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a">Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization</title>
<author><name sortKey="Riadh Ben Salah" sort="Riadh Ben Salah" uniqKey="Riadh Ben Salah" last="Riadh Ben Salah">RIADH BEN SALAH</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Gargouri, Ali" sort="Gargouri, Ali" uniqKey="Gargouri A" first="Ali" last="Gargouri">Ali Gargouri</name>
<affiliation wicri:level="1"><inist:fA14 i1="02"><s1>Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, Route de Sidi Mansour, Km 6, P.O. Box « K »</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>2 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Verger, Robert" sort="Verger, Robert" uniqKey="Verger R" first="Robert" last="Verger">Robert Verger</name>
<affiliation wicri:level="3"><inist:fA14 i1="03"><s1>Laboratoire de Lipolyse Enzymatique, UPR 9025, CNRS</s1>
<s2>Marseille</s2>
<s3>FRA</s3>
<sZ>3 aut.</sZ>
</inist:fA14>
<country>France</country>
<placeName><region type="region">Provence-Alpes-Côte d'Azur</region>
<region type="old region">Provence-Alpes-Côte d'Azur</region>
<settlement type="city">Marseille</settlement>
</placeName>
</affiliation>
</author>
<author><name sortKey="Gargouri, Youssef" sort="Gargouri, Youssef" uniqKey="Gargouri Y" first="Youssef" last="Gargouri">Youssef Gargouri</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
<author><name sortKey="Mejdoub, Hafedh" sort="Mejdoub, Hafedh" uniqKey="Mejdoub H" first="Hafedh" last="Mejdoub">Hafedh Mejdoub</name>
<affiliation wicri:level="1"><inist:fA14 i1="01"><s1>Laboratoire de biochimie et de génie enzymatique des lipases, ENIS, Route de Soukra, "BPW"</s1>
<s2>3038 Sfax</s2>
<s3>TUN</s3>
<sZ>1 aut.</sZ>
<sZ>4 aut.</sZ>
<sZ>5 aut.</sZ>
</inist:fA14>
<country>Tunisie</country>
<wicri:noRegion>3038 Sfax</wicri:noRegion>
</affiliation>
</author>
</analytic>
<series><title level="j" type="main">World journal of microbiology & biotechnology</title>
<title level="j" type="abbreviated">World j. microbiol. biotechnol.</title>
<idno type="ISSN">0959-3993</idno>
<imprint><date when="2009">2009</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
<seriesStmt><title level="j" type="main">World journal of microbiology & biotechnology</title>
<title level="j" type="abbreviated">World j. microbiol. biotechnol.</title>
<idno type="ISSN">0959-3993</idno>
</seriesStmt>
</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Characterization</term>
<term>Gene overexpression</term>
<term>Mutation</term>
<term>Pichia pastoris</term>
<term>Purification</term>
<term>Rhizopus oryzae</term>
<term>Site directed mutagenesis</term>
<term>Triacylglycerol lipase</term>
</keywords>
<keywords scheme="Pascal" xml:lang="fr"><term>Triacylglycerol lipase</term>
<term>Rhizopus oryzae</term>
<term>Mutation</term>
<term>Purification</term>
<term>Caractérisation</term>
<term>Pichia pastoris</term>
<term>Mutagenèse dirigée</term>
<term>Surexpression génique</term>
</keywords>
</textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en">The sequence corresponding to the mature lipase of Rhizopus oryzae WPG (ROLw) was subcloned in the pPIC9K expression vector, with a strong AOX<sub>1</sub>
 promoter, to construct a recombinant lipase protein containing six histidine residues at the N-terminal. The His-tagged lipase was expressed in Pichia Pastoris X<sub>33</sub>
 and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography (Ni-NTA resin). High level expression of the lipase by Pichia Pastoris X<sub>33</sub>
 cells harbouring the lipase gene containing expression vector was observed upon induction with 2.5 g/l methanol at 28°C; the specific activity of the purified His<sub>6</sub>
-ROLw was 1,500 or 760 U/mg using olive oil emulsion or tributyrin as substrates, respectively. To check the importance of Asn 134 His substitution in the affinity and substrate selectivity of ROLw, the mutant His<sub>6</sub>
-ROLw-N134H was overexpressed in Pichia Pastoris X33 and purified with the same nickel metal affinity column. The specific activity of the purified His-tagged ROLw-N134H was 5,900 and 35 U/mg using olive oil emulsion or tributyrin as substrate. A comparative study of the wild type (His<sub>6</sub>
-ROLw) and the mutant (His<sub>6</sub>
-ROLw-N134H) proteins was carried out. A 3D structure model of ROLw was built using the RNL structure as template. We have concluded that a slight increase in the exposed hydrophilic residues on the surface of ROLw as compared to RNL (ROLwN134H) could be responsible for a higher selectivity of ROlw for long and short chain triacylglycerols at the lipid/water interface and then explaining the importance of Asn 134 for the chain length specificity of ROLw. This property is quite rare among Rhizopus lipases and gives this new lipase great potential for use in the field of biocatalysis.</div>
</front>
</TEI>
<affiliations><list><country><li>France</li>
<li>Tunisie</li>
</country>
<region><li>Provence-Alpes-Côte d'Azur</li>
</region>
<settlement><li>Marseille</li>
</settlement>
</list>
<tree><country name="Tunisie"><noRegion><name sortKey="Riadh Ben Salah" sort="Riadh Ben Salah" uniqKey="Riadh Ben Salah" last="Riadh Ben Salah">RIADH BEN SALAH</name>
</noRegion>
<name sortKey="Gargouri, Ali" sort="Gargouri, Ali" uniqKey="Gargouri A" first="Ali" last="Gargouri">Ali Gargouri</name>
<name sortKey="Gargouri, Youssef" sort="Gargouri, Youssef" uniqKey="Gargouri Y" first="Youssef" last="Gargouri">Youssef Gargouri</name>
<name sortKey="Mejdoub, Hafedh" sort="Mejdoub, Hafedh" uniqKey="Mejdoub H" first="Hafedh" last="Mejdoub">Hafedh Mejdoub</name>
</country>
<country name="France"><region name="Provence-Alpes-Côte d'Azur"><name sortKey="Verger, Robert" sort="Verger, Robert" uniqKey="Verger R" first="Robert" last="Verger">Robert Verger</name>
</region>
</country>
</tree>
</affiliations>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Wicri/Terre/explor/NickelMaghrebV1/Data/France/Analysis

HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000146 | SxmlIndent | more

HfdSelect -h $EXPLOR_AREA/Data/France/Analysis/biblio.hfd -nk 000146 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Wicri/Terre
   |area=    NickelMaghrebV1
   |flux=    France
   |étape=   Analysis
   |type=    RBID
   |clé=     Pascal:09-0342194
   |texte=   Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization
}}

This area was generated with Dilib version V0.6.27.
Data generation: Fri Mar 24 23:14:20 2017. Site generation: Tue Mar 5 17:03:47 2024

	Serveur d'exploration sur le nickel au Maghreb
	Attention, ce site est en cours de développement ! Attention, site généré par des moyens informatiques à partir de corpus bruts. Les informations ne sont donc pas validées.

Serveur d'exploration sur le nickel au Maghreb

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

Expression in Pichia pastoris X33 of His-tagged lipase from a novel strain of Rhizopus oryzae and its mutant Asn 134 His: purification and characterization

Source :

Descripteurs français

English descriptors

Abstract

Links toward previous steps (curation, corpus...)

Links to Exploration step

Le document en format XML

Pour manipuler ce document sous Unix (Dilib)

Pour mettre un lien sur cette page dans le réseau Wicri