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Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

dc.contributor.authorAizemberg, Raquel [UNESP]
dc.contributor.authorTerrazas, Werner D. M. [UNESP]
dc.contributor.authorFerreira-Dias, Suzana
dc.contributor.authorValentini, Sandro Roberto [UNESP]
dc.contributor.authorGattas, Edwil Aparecida de Lucca [UNESP]
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.contributor.institutionUniv Tecn Lisbon
dc.date.accessioned2014-05-20T13:24:16Z
dc.date.available2014-05-20T13:24:16Z
dc.date.issued2011-07-01
dc.description.abstractThe extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.en
dc.description.affiliationSão Paulo State Univ UNESP, Sch Pharmaceut Sci, BR-14801902 Araraquara, SP, Brazil
dc.description.affiliationUniv Tecn Lisbon, CEER Biosyst Engn, Inst Agron, P-1349017 Lisbon, Portugal
dc.description.affiliationUnespSão Paulo State Univ UNESP, Sch Pharmaceut Sci, BR-14801902 Araraquara, SP, Brazil
dc.format.extent329-335
dc.identifierhttp://www.ftb.com.hr/49/FTB_49_329.html
dc.identifier.citationFood Technology and Biotechnology. Zagreb: Faculty Food Technology Biotechnology, v. 49, n. 3, p. 329-335, 2011.
dc.identifier.issn1330-9862
dc.identifier.lattes5333250355049814
dc.identifier.lattes4006598610021833
dc.identifier.urihttp://hdl.handle.net/11449/7481
dc.identifier.wosWOS:000293877500010
dc.language.isoeng
dc.publisherFaculty Food Technology Biotechnology
dc.relation.ispartofFood Technology and Biotechnology
dc.relation.ispartofjcr1.168
dc.relation.ispartofsjr0,365
dc.rights.accessRightsAcesso abertopt
dc.sourceWeb of Science
dc.subjectPichia pastorisen
dc.subjectrecombinant glycerol kinaseen
dc.subjectcarbon sourceen
dc.subjectbiomassen
dc.subjectresponse surface methodologyen
dc.titleOptimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastorisen
dc.typeArtigopt
dcterms.rightsHolderFaculty Food Technology Biotechnology
dspace.entity.typePublication
relation.isDepartmentOfPublication5004bcab-94af-4939-b980-091ae9d0a19e
relation.isDepartmentOfPublication.latestForDiscovery5004bcab-94af-4939-b980-091ae9d0a19e
relation.isOrgUnitOfPublication95697b0b-8977-4af6-88d5-c29c80b5ee92
relation.isOrgUnitOfPublication.latestForDiscovery95697b0b-8977-4af6-88d5-c29c80b5ee92
unesp.author.lattes5333250355049814
unesp.author.lattes4006598610021833[5]
unesp.author.orcid0000-0003-3655-8201[3]
unesp.campusUniversidade Estadual Paulista (UNESP), Faculdade de Ciências Farmacêuticas, Araraquarapt
unesp.departmentAlimentos e Nutrição - FCFpt
unesp.departmentCiências Biológicas - FCFpt

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