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Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond

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Abstract

Objective

To improve the stability of E. coli-produced non-glycosylated fungal FAd-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases.

Results

The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys (k cat = 760 s−1, Km = 35 mM, and catalytic efficiency (k cat/Km) = 22 s−1 mM−1) were almost identical to those of the wild-type enzyme (k cat = 780 s−1, Km = 35 mM, k cat/Km = 22 s−1 mM−1). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type.

Conclusion

The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity.

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Acknowledgments

The authors thank Dr. Stefano Ferri for kindly proofreading and revising the manuscript.

Supporting information

Supplementary Table 1 – Specific activities and thermal stabilities of crude enzyme preparations. Supplementary Figure 1 – The nucleotide sequence of the wild-type FAD-GDH gene. Supplementary Figure 2 – SDS-PAGE analysis of expressed wild-type and mutant FAD-GDHs. Supplementary Figure 3 – Amino acid sequence alignment of glucose oxidases and putative glucose dehydrogenases. Supplementary Figure 4 – Tertiary structure comparison of FAD-GDH with AAOx and flavin domain of CDH.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Nakamachi, Koganei, Tokyo, Japan

    Genki Sakai, Yosuke Oonishi & Koji Sode

  2. Ultizyme International Ltd., Minami, Meguro, Tokyo, Japan

    Katsuhiro Kojima, Kazushige Mori & Koji Sode

Authors
  1. Genki Sakai
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  2. Katsuhiro Kojima
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  3. Kazushige Mori
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  4. Yosuke Oonishi
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  5. Koji Sode
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Corresponding author

Correspondence to Koji Sode.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10529_2015_1774_MOESM1_ESM.pptx

Supplementary Figure 1. The nucleotide sequence of the wild-type FAD-GDH gene. The native gene sequence encoding the preprotein (native) is aligned with the codon-optimized gene used in this study, after deletion of the signal sequence and addition of NdeI and HindIII restriction sites (modified). (PPTX 120 kb)

10529_2015_1774_MOESM2_ESM.pptx

Supplementary Figure 2. SDS-PAGE analysis of expressed wild-type and mutant FAD-GDHs. Molecular weight marker (lane M), soluble fractions (odd lanes) and insoluble fractions (even lanes) of wild type (lanes 1 and 2), Val149Cys/Gly190Cys (lanes 3 and 4) were separated on a 10-20% polyacrylamide gel. (PPTX 74 kb)

10529_2015_1774_MOESM3_ESM.pptx

Supplementary Figure 3. Amino acid sequence alignment of glucose oxidases and putative glucose dehydrogenases. The sequences were aligned using ClustalW. Arrows indicate cysteine residues conserved among glucose oxidases. The putative proteins were previously annotated [8]. Glucose oxidases: A. niger GOx (1CF3 and CAC12802), A. oryzae RIB40 GOx (XP_001727544), P. amagasakiense GOx (1GPE), and A. terreus NIH2624 GOx precursor (XP_001216461). Putative glucose dehydrogenases: A. flavus NRRL3357 putative GOx (AFL599), A. niger CBS 513.88 GOx (XP_001391138 and XP_001394544), A. flavus NRRL3357 putative choline dehydrogenase (XP_002385256), and A. carbonarius ITEM 5010 jgi|Aspca1|33771|fgenesh1_pg.00771_#_19 (Aspca1 33771). (PPTX 272 kb)

10529_2015_1774_MOESM4_ESM.pptx

Supplementary Figure 4. Tertiary structure comparison of FAD-GDH with AAOx and flavin domain of CDH. Overall structures of (a) flavin domain of CDH (PDB ID: 1KDG), (b) AAOx (PDB ID: 3FIM), and (c) FAD-GDH (model structure). The regions around the disulfide bonds in AAOx and flavin domain of CDH, and the corresponding regions in the FAD-GDH structural model, are indicated with red frames. Figures were generated using UCSF Chimera. (PPTX 2311 kb)

Supplementary material 5 (DOCX 12 kb)

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Sakai, G., Kojima, K., Mori, K. et al. Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond. Biotechnol Lett 37, 1091–1099 (2015). https://doi.org/10.1007/s10529-015-1774-8

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  • DOI: https://doi.org/10.1007/s10529-015-1774-8

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