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.

Biochemical, Thermodynamic and Kinetic Characterization of Glucose Oxidase Purified from Pseudomonas and Actinomyces spp. from Natural Sources

“…There was excellent stability, substrate affinity and catalytic activity perceived in the two enzymes purified has shown that these enzymes can be used in industrial applications as well as in biosensors. The GODs extracted and purified from naturally isolated strains displayed optimum activity at lower temperature than GOD from Aspergillus tubingensis and a recombinant GOD from Penicillium amagasakiense 16,17 .The Km of the GOD from both the isolated strain was similar to the Km and Vmax of GOD from A. niger, and Penicillium species a recombinant one expressed in Pichia pastoris 16,18,19 . Inhibition of the enzyme activity by chemical agents such as chelating and denaturing agents along with substrates were similar to Aspergillus niger.…”

A New Cold-Active Glucose Oxidase From Penicillium: High-Level Expression and Application in Fish Preservation

“…After that, the total enzyme yield and wet cell mass tended to decrease, and the methanol consumption rate apparently reduced, which implied the deadline of fermentation. The maximal enzymatic activity of GOxP 5 in shake flask was much higher than many heterologously expressed enzymes in P. pastoris, such as GOx form P. variable (0.33 U/mL), A. niger (1.23 U/mL), and A. niger NRLL-3 (17.5 U/mL) (Kalisz et al, 1997;Yamaguchi et al, 2007;Kovačević et al, 2014). Although the highest expression level of GOxP 5 in P. pastoris after high-cell-density fermentation was lower than that of GOD-m from Penicillium notatum (615 U/mL) (Gao et al, 2012), it was still of great value due to its excellent characteristics in food preservation area.…”

Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

“…Crognale et al (2006) described a genetically modified P. pastoris X 33 with the gene encoding the GOX from P. variabile P16, and the activity only reached at 50 U/mL. Kovačević et al (2014) cloned several mutated glucose oxidase genes from A. niger M12 and expressed them in P. pastoris KM71H. The highest activity of the GOD came up to 17.5 U/mL of fermentation media.…”