Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Malene Bech Vester-Christensen, Maher Abou Hachem, Henrik Næsted, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase I promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LID (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K-m,K-app = 0.16 +/- 0.02 mg/mL and k(cat,app) = 79 +/- 10 s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively.
    Original languageEnglish
    JournalProtein Expression and Purification
    Volume69
    Issue number1
    Pages (from-to)112-119
    ISSN1046-5928
    DOIs
    Publication statusPublished - 2010

    Keywords

    • Pullulan hydrolysis
    • High cell-density fermentation
    • Cyclodextrin affinity
    • Pichia pastoris
    • Barley limit dextrinase

    Cite this

    @article{ccbd5625604f4278a7897db7760eeec8,
    title = "Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation",
    abstract = "Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase I promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LID (84{\%} recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K-m,K-app = 0.16 +/- 0.02 mg/mL and k(cat,app) = 79 +/- 10 s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively.",
    keywords = "Pullulan hydrolysis, High cell-density fermentation, Cyclodextrin affinity, Pichia pastoris, Barley limit dextrinase",
    author = "Vester-Christensen, {Malene Bech} and {Abou Hachem}, Maher and Henrik N{\ae}sted and Birte Svensson",
    year = "2010",
    doi = "10.1016/j.pep.2009.08.016",
    language = "English",
    volume = "69",
    pages = "112--119",
    journal = "Protein Expression and Purification",
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    }

    Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation. / Vester-Christensen, Malene Bech; Abou Hachem, Maher; Næsted, Henrik; Svensson, Birte.

    In: Protein Expression and Purification, Vol. 69, No. 1, 2010, p. 112-119.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

    AU - Vester-Christensen, Malene Bech

    AU - Abou Hachem, Maher

    AU - Næsted, Henrik

    AU - Svensson, Birte

    PY - 2010

    Y1 - 2010

    N2 - Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase I promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LID (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K-m,K-app = 0.16 +/- 0.02 mg/mL and k(cat,app) = 79 +/- 10 s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively.

    AB - Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and alpha-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched alpha-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase I promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on beta-cyclodextrin-Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LID (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS-PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K-m,K-app = 0.16 +/- 0.02 mg/mL and k(cat,app) = 79 +/- 10 s(-1) by fitting the uncompetitive substrate inhibition Michaelis-Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k(cat,app)/K-m,K-app = 488 +/- 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed alpha-, beta-, and gamma-cyclodextrin binding to LD with K-d of 27.2, 0.70, and 34.7 mu M, respectively.

    KW - Pullulan hydrolysis

    KW - High cell-density fermentation

    KW - Cyclodextrin affinity

    KW - Pichia pastoris

    KW - Barley limit dextrinase

    U2 - 10.1016/j.pep.2009.08.016

    DO - 10.1016/j.pep.2009.08.016

    M3 - Journal article

    VL - 69

    SP - 112

    EP - 119

    JO - Protein Expression and Purification

    JF - Protein Expression and Purification

    SN - 1046-5928

    IS - 1

    ER -