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Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Authorized Users Only
2014
Authors
Kovačević, Gordana
Blažić, Marija
Draganic, Bojana
Ostafe, Raluca
Gavrović-Jankulović, Marija
Fischer, Rainer
Prodanović, Radivoje
Article (Published version)
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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 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 simila...r 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.

Keywords:
Pichia pastoris / Glucose oxidase / Directed evolution / Mutant
Source:
Molecular Biotechnology, 2014, 56, 4, 305-311
Publisher:
  • Humana Press Inc, Totowa
Projects:
  • Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-172049)
  • Study of structure-function relationships in the plant cell wall and modifications of the wall structure by enzyme engineering (RS-173017)

DOI: 10.1007/s12033-013-9709-x

ISSN: 1073-6085

PubMed: 24122283

WoS: 000332972300003

Scopus: 2-s2.0-84896935103
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URI
http://cer.ihtm.bg.ac.rs/handle/123456789/1506
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IHTM

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