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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
Funding / 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
[ Google Scholar ]
22
18
URI
https://cer.ihtm.bg.ac.rs/handle/123456789/1506
Collections
  • Radovi istraživača / Researchers' publications
Institution/Community
IHTM
TY  - JOUR
AU  - Kovačević, Gordana
AU  - Blažić, Marija
AU  - Draganic, Bojana
AU  - Ostafe, Raluca
AU  - Gavrović-Jankulović, Marija
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2014
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1506
AB  - 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 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.
PB  - Humana Press Inc, Totowa
T2  - Molecular Biotechnology
T1  - Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H
VL  - 56
IS  - 4
SP  - 305
EP  - 311
DO  - 10.1007/s12033-013-9709-x
ER  - 
@article{
author = "Kovačević, Gordana and Blažić, Marija and Draganic, Bojana and Ostafe, Raluca and Gavrović-Jankulović, Marija and Fischer, Rainer and Prodanović, Radivoje",
year = "2014",
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 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.",
publisher = "Humana Press Inc, Totowa",
journal = "Molecular Biotechnology",
title = "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H",
volume = "56",
number = "4",
pages = "305-311",
doi = "10.1007/s12033-013-9709-x"
}
Kovačević, G., Blažić, M., Draganic, B., Ostafe, R., Gavrović-Jankulović, M., Fischer, R.,& Prodanović, R.. (2014). Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology
Humana Press Inc, Totowa., 56(4), 305-311.
https://doi.org/10.1007/s12033-013-9709-x
Kovačević G, Blažić M, Draganic B, Ostafe R, Gavrović-Jankulović M, Fischer R, Prodanović R. Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H. in Molecular Biotechnology. 2014;56(4):305-311.
doi:10.1007/s12033-013-9709-x .
Kovačević, Gordana, Blažić, Marija, Draganic, Bojana, Ostafe, Raluca, Gavrović-Jankulović, Marija, Fischer, Rainer, Prodanović, Radivoje, "Cloning, Heterologous Expression, Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H" in Molecular Biotechnology, 56, no. 4 (2014):305-311,
https://doi.org/10.1007/s12033-013-9709-x . .

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