Biochemical and mutational analyses of a Trametes pyranose oxidase and comparison of its mutants in breadmaking

Mengzhu Li,Huiying Luo,Xiaoyun Su,Hong Deng,Rui Ma,Bin Yao

doi:10.1186/s13568-018-0570-y

Abstract

Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking.

Highlights

Pyranose oxidase (POx) (EC 1.1.3.10) is a large and homotetrameric protein that contains covalently bound flavin adenine dinucleotide (FAD) as a co-factor (Hallberg et al 2004)
Expression and purification of recombinant TsPOx The cDNA fragment coding for the mature TsPOx was ligated into pET-28a(+) to obtain the recombinant plasmid pET-28a-Tspox, which was transformed into E. coli competent cells
It catalyzes the oxidation of pyranoses to corresponding keto-sugars and provides hydrogen peroxide to class II peroxidases

Summary

Introduction

Pyranose oxidase (POx) (EC 1.1.3.10) is a large and homotetrameric protein that contains covalently bound flavin adenine dinucleotide (FAD) as a co-factor (Hallberg et al 2004). It is widely distributed in wood-decaying basidiomycetes but less frequently found in ascomycetes like Aspergillus (Pisanelli et al 2012a; Takakura and Kuwata 2003; Volc et al 1985). POx catalyzes the oxidation of aldopyranoses at position C-2 to generate corresponding 2-ketoaldoses and releases H2O2 (Giffhorn et al 2000) In some cases, it can catalyze the oxidation of C-3 of a few sugars to form 3-keto and 2,3-diketo sugars (Freimund et al 1998; Volc et al 2003).

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Results

Conclusion