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Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.

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.

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