Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification

Henrik Næsted, Birte Kramhøft, F. Lok, K. Bojsen, S. Yu, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams a-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters K-m, V-max, and k(cat) were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants. (c) 2005 Elsevier Inc. All rights reserved.
    Original languageEnglish
    JournalProtein Expression and Purification
    Volume46
    Issue number1
    Pages (from-to)56-63
    ISSN1046-5928
    DOIs
    Publication statusPublished - 2006

    Keywords

    • Barley high pI α-glucosidase
    • High cell-density fermentation
    • Pichia pastoris
    • Metal-chelating affinity chromatography
    • Maltose

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