Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris

Filip Mollerup*, Emma Master

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

3 Citations (Scopus)
327 Downloads (Pure)

Abstract

Herein, we report the extracellular expression of carbohydrate active fusion enzymes in Pichia pastoris. Particularly, CBM29-1-2 from Piromyces equi was separately fused to the N- and C-terminus of galactose 6-oxidase (GaO, D-galactose: oxygen 6-oxidoreductase, EC 1.1.13.9, CAZy family AA5) from Fusarium graminearum, generating CBM29-GaO and GaO-CBM29, respectively. P. pastoris was transformed with expression vectors encoding GaO, CBM29-GaO and GaO-CBM29, and the fusion proteins were expressed in both shake-flask and 2L bioreactor systems. Volumetric production yields and specific GaO activity increased when expression was performed in a bioreactor system compared to shake-flask cultivation. This was observed for both CBM29-GaO and GaO-CBM29, and is consistent with previous reports of GaO expression in P. pastoris (Spadiut et al., 2010; Anasontzis et al., 2014) [1,2]. Fusion of CBM29 to the C-terminal of GaO (GaO-CBM29) resulted in a stable uniform protein at the expected calculated size (107 kDa) when analyzed with SDS-PAGE. By comparison, the expression of the N-terminal fusion protein (CBM29-GaO) was low, and two truncated versions of CBM29-GaO were coexpressed with the full-sized protein. Despite differences in protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme. A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al., 2015) [3].

Original languageEnglish
Pages (from-to)176-183
Number of pages8
JournalData in Brief
Volume6
DOIs
Publication statusPublished - 1 Mar 2016
MoE publication typeA1 Journal article-refereed

Keywords

  • Carbohydrate binding modules
  • CBM29
  • Enzyme fusion
  • Fermentation
  • Galactose oxidase
  • Protein production
  • Protein purification

Fingerprint Dive into the research topics of 'Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris'. Together they form a unique fingerprint.

  • Projects

    BHIVE: Bio-derived HIgh Value polymers through novel Enzyme function

    Mollerup, F., Karppi, J., Jurak, E., Aumala, V., Master, E., Monschein, M. & Leamon, A. K. M. A. A.

    01/09/201531/10/2020

    Project: EU: ERC grants

    Equipment

  • Cite this