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dc.creatorKovačević, Gordana
dc.creatorBlažić, Marija
dc.creatorDraganic, Bojana
dc.creatorOstafe, Raluca
dc.creatorGavrović-Jankulović, Marija
dc.creatorFischer, Rainer
dc.creatorProdanović, Radivoje
dc.date.accessioned2019-01-30T17:41:02Z
dc.date.available2019-01-30T17:41:02Z
dc.date.issued2014
dc.identifier.issn1073-6085
dc.identifier.urihttp://cer.ihtm.bg.ac.rs/handle/123456789/1506
dc.description.abstractAspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZ alpha A vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k (cat) of 189.4 s(-1) and K (m) of 28.26 mM while M12 GOx had k (cat) of 352.0 s(-1) and K (m) of 13.33 mM for glucose at pH 5.5. Specificity constants k (cat)/K (m) of wt (6.70 mM(-1) s(-1)) and M12 GOx (26.7 mM(-1) s(-1)) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.en
dc.publisherHumana Press Inc, Totowa
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS//
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/173017/RS//
dc.rightsrestrictedAccess
dc.sourceMolecular Biotechnology
dc.subjectPichia pastorisen
dc.subjectGlucose oxidaseen
dc.subjectDirected evolutionen
dc.subjectMutanten
dc.titleCloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71Hen
dc.typearticle
dc.rights.licenseARR
dcterms.abstractБлажић, Марија; Фисцхер, Раинер; Остафе, Ралуца; Продановиц, Радивоје; Драганиц, Бојана; Ковацевиц, Гордана; Гавровиц-Јанкуловиц, Марија;
dc.citation.volume56
dc.citation.issue4
dc.citation.spage305
dc.citation.epage311
dc.citation.other56(4): 305-311
dc.citation.rankM22
dc.identifier.pmid24122283
dc.identifier.doi10.1007/s12033-013-9709-x
dc.identifier.rcubConv_3130
dc.identifier.scopus2-s2.0-84896935103
dc.identifier.wos000332972300003
dc.type.versionpublishedVersion


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