Purification and characterization of recombinant high pI Barley α-Glucosidase

Henrik Næsted, Kirsten Bojsen, Birte Svensson

    Research output: Contribution to conferencePosterResearch

    Abstract

    α-glucosidase activity in barley seeds plays a crucial role in embryonic development. The concerted action of α-glucosidase, α-amylase, α-amylase, and limit dextrinase serves as the machinery responsible for the supply of glucose as energy source for the developing embryo in the germinating seed (MACGREGOR & sissons). recently expression and characterization of the recombinant full length and fully functional barley high pi α-glucosidase in pichia pastoris has been achieved. in order to facilitate protein yield in the mg range, a clone representing an n-terminal hexa histidine tagged recombinant form of the enzyme was grown under high cell-density fermentation conditions. this approach enabled a successful protein expression profile under the highly sensitive methanol utilization phase using a biotatb 5 l reactor for the fermentation procedure. the enzyme was purified from 3.5 liter α-glucosidase containing culture using a four step purification strategy yielding 42 mg of pure enzyme. remarkably, the purified enzyme exhibits a higher molecular mass in sds-page than expected from the primary structure. the apparent molecular mass of 100 kda compared to 92 kda calculated from the amino acid sequence of the native enzyme indicates a possible post-translational glycosylation of the recombinant α-glucosidase. preliminary enzyme kinetic analysis has demonstrated that the purified α-glucosidase displayed high stability during the 5 day long fermenentation and exhibited a specific activity in the range of the native enzyme purified from malt (frandsen et al.). the kinetic values km, vmax and kcat are determined to 1.7 mm, 139 nm s-1 and 85 s-1 using maltose as substrate. the presented data illustrate the first successful production of enzymatically active full length recombinant high pi barley α-glucosidase (tibbot et al.; FRANDSEN et al.). This project is funded under the 5th Framework Programme of the European Commission, Contract Reference QLRT-2000-02400, "New Products from Starch-Derived 1,5-Anhydro-D-Fructose (NEPSA)" References T.P. FRANDSEN, F. LOK, E. MIRGORODSKAYA ET AL. 2000. Plant Physiology. 123, 275-286. B.K. TIBBOT, C.Y. HENSON, R.W. SKADSEN 1998. Plant Molecular Biology. 38, 379-391. A.W MACGREGOR & M.J. SISSONS. 1994. Journal of Cereal Science. 19, 161-169.
    Original languageEnglish
    Publication date2004
    Publication statusPublished - 2004
    EventALAMY - Slovakia
    Duration: 1 Jan 2004 → …

    Conference

    ConferenceALAMY
    CitySlovakia
    Period01/01/2004 → …

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