title: Cloning, heterologous expression, purification and characterization of M12 mutant of Aspergillus niger glucose oxidase in yeast Pichia pastoris KM71H.
abstract: Aspergillus 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 pPICZαA 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.
authors.affiliation: Faculty of Chemistry, University of Belgrade, Studentski trg 12, 11000, Belgrade, Serbia.
authors.forename: Gordana
authors.initials: G
authors.lastname: Kovačević
authors.forename: Marija
authors.initials: M
authors.lastname: Blažić
authors.forename: Bojana
authors.initials: B
authors.lastname: Draganić
authors.forename: Raluca
authors.initials: R
authors.lastname: Ostafe
authors.forename: Marija
authors.initials: M
authors.lastname: Gavrović-Jankulović
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanović
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005949:Glucose Oxidase / Q000096:biosynthesis / Q000235:genetics*; D010843:Pichia / Q000235:genetics
chemical_list: D005949:Glucose Oxidase