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Evaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesis

2015-04Journal article Restricted access
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dc.contributor.authorPedro, Augusto
dc.contributor.authorOppolzer, David
dc.contributor.authorBonifácio, M J
dc.contributor.authorMaia, C J
dc.contributor.authorQueiroz, João
dc.contributor.authorPassarinha, L A
dc.date.accessioned2019-12-04T17:11:18Z
dc.date.available2019-12-04T17:11:18Z
dc.date.issued2015-04
dc.description.abstractCatechol-O-methyltransferase (COMT, EC 2.1.1.6) is an enzyme that catalyzes the methylation of catechol substrates, and while structural and functional studies of its membrane-bound isoform (MBCOMT) are still hampered by low recombinant production, Pichia pastoris has been described as an attractive host for the production of correctly folded and inserted membrane proteins. Hence, in this work, MBCOMT biosynthesis was developed using P. pastoris X33 and KM71H cells in shake flasks containing a semidefined medium with different methanol concentrations. Moreover, after P. pastoris glass beads lysis, biologically and immunologically active hMBCOMT was found mainly in the solubilized membrane fraction whose kinetic parameters were identical to its correspondent native enzyme. In addition, mixed feeds of methanol and glycerol or sorbitol were also employed, and its levels quantified using liquid chromatography coupled to refractive index detection. Overall, for the first time, two P. pastoris strains with opposite phenotypes were applied for MBCOMT biosynthesis under the control of the strongly methanol-inducible alcohol oxidase (AOX) promoter. Moreover, this eukaryotic system seems to be a promising approach to deliver MBCOMT in high quantities from fermentor cultures with a lower cost-benefit due to the cheaper cultivation media coupled with the higher titers tipically achieved in biorreactors, when compared with previously reported mammallian cell cultures.pt_PT
dc.description.sponsorshipA.Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from Fundação para a Ciência e Tecnologia and D. Oppolzer acknowledges a fellowship (CENTRO-07-ST24_FEDER-002014- TPCR-2-004) from Programa BMais Centro^ within the scope of QREN–POPH–Advanced Formation programs cofunded by Fundo Social Europeu and MEC. This work was partially funded by Fundação para a Ciência e Tecnologia I.P. (PIDDAC) and Fundo Europeu de Desenvolvimento Regional-FEDER funds through Programa Operacional Factores de Competitividade (POFC)–COMPETE: FCOMP-01-0124-FEDER-027563 and by National Funds through FCT–Fundação para a Ciência e Tecnologia within the scope of Project BEXPL/BBB478/ BQB/0960/2012.
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.doi10.1007/s12010-015-1551-0pt_PT
dc.identifier.urihttp://hdl.handle.net/10400.6/7664
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.subjectPichia pastorispt_PT
dc.subjectMembrane proteinpt_PT
dc.subjectMethanol feedingpt_PT
dc.subjectAlcohol oxidase promoterpt_PT
dc.subjectFermentationpt_PT
dc.titleEvaluation of Mut(S) and Mut⁺ Pichia pastoris strains for membrane-bound catechol-O-methyltransferase biosynthesispt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F81222%2F2011/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/5876-PPCDTI/EXPL%2FBBB-BQB%2F0960%2F2012/PT
oaire.citation.endPage3855pt_PT
oaire.citation.issue8pt_PT
oaire.citation.startPage3840pt_PT
oaire.citation.titleApplied Biochemistry and Biotechnologypt_PT
oaire.citation.volume175pt_PT
oaire.fundingStreamSFRH
oaire.fundingStream5876-PPCDTI
person.familyNameQuaresma Henriques Pedro
person.familyNameOppolzer
person.familyNameMaia Baptista
person.familyNameQueiroz
person.familyNamePaulino Passarinha
person.givenNameAugusto
person.givenNameDavid
person.givenNameCláudio Jorge
person.givenNameJoão
person.givenNameLuís António
person.identifierhttps://scholar.google.com/citations?user=oer44UoAAAAJ&hl=pt-PT&oi=ao
person.identifierhttps://scholar.google.pt/citations?user=hFKOd_QAAAAJ&hl=pt-PT&oi=ao
person.identifierhttps://www.researchgate.net/profile/Luis_Passarinha
person.identifier.ciencia-id7111-B2BE-336D
person.identifier.ciencia-id5115-FCBD-A31F
person.identifier.ciencia-id931E-B66D-E341
person.identifier.ciencia-id621B-74BF-F9A7
person.identifier.orcid0000-0001-8194-6022
person.identifier.orcid0000-0002-8888-4976
person.identifier.orcid0000-0002-5658-5445
person.identifier.orcid0000-0002-3096-8325
person.identifier.orcid0000-0001-6910-7576
person.identifier.ridL-3104-2014
person.identifier.scopus-author-id54080426100
person.identifier.scopus-author-id55623902200
person.identifier.scopus-author-id23097426600
person.identifier.scopus-author-id7003705645
person.identifier.scopus-author-id12445439600
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.embargofctCopyright cedido à editora no momento da publicaçãopt_PT
rcaap.rightsclosedAccesspt_PT
rcaap.typearticlept_PT
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