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

Filip Mollerup*, Emma Master

*Tämän työn vastaava kirjoittaja

Tutkimustuotos: LehtiartikkeliArticleScientificvertaisarvioitu

3 Sitaatiot (Scopus)
329 Lataukset (Pure)

Abstrakti

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].

AlkuperäiskieliEnglanti
Sivut176-183
Sivumäärä8
JulkaisuData in Brief
Vuosikerta6
DOI - pysyväislinkit
TilaJulkaistu - 1 maaliskuuta 2016
OKM-julkaisutyyppiA1 Julkaistu artikkeli, soviteltu

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  • Projektit

    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

    Projekti: EU: ERC grants

    Laitteet

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