AACC International - 2009 Meeting Abstract - Barley limit dextrinase and its interaction the proteinaceous barley limit dextrinase inhibitor

Meeting Abstract - Oral Presentation

Barley limit dextrinase and its interaction the proteinaceous barley limit dextrinase inhibitor
B. SVENSSON (1), M. B. Vester-Christensen (2), J. M. Jensen (2), P. Hagglund (2), M. Abou Hachem (2)
(1) Enzyme and Protein Chemistry, Kgs Lyngby, DENMARK; (2) Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark
Cereal Foods World 54:A29

The starch debranching enzyme limit dextrinase (LD) hydrolyses α-1,6-glucoside linkages in limit dextrins and amylopectin during seed germination. LD is highly activity towards pullulan with K_m = 0.16 ± 0.02 mg·ml^–1 and k_cat = 79 ± 10 s^–1. Besides a role in germination, LD is important in brewing by hydrolysing a-limit dextrins¹ during mashing and malting. LD occurs in aqueous malt extracts in a free, active and a “bound” inactive form². The inactive form stems partly from binding of the endogenous limit dextrinase inhibitor (LDI). LDI is a CM family member of about 12 kDa, contains 4 disulfide bonds and has a free thiol group present in mixed disulfide with cysteine or glutathione. LDI competitively inhibits LD hydrolysis of pullulan with subnanomolar K_i. Both LD and LDI were produced recombinantly in Pichia pastoris and fitting of a 1:1 binding model to surface plasmon resonance data of LDI and LD confirmed very tight binding of K_D = 40 ± 3 × 10^–12 M. This high affinity stemmed from very slow k_off of ~5 × 10^–5 s^–1, while k_on of 1 × 10⁶ M^–1·s^–1 was in the usual range for proteinaceous inhibitor and amylolytic enzymes. Binding was optimal at slightly acidic to neutral pH and 0.15–0.3 mM NaCl. The interaction was monitored at 15–45°C and analysis of the van’t Hoff thermodynamics showed favourable enthalpic and entropic components of binding free energy (ΔGº = –58 kJ mol^–1) suggesting that the LD-LDI complexation is driven by hydrophobic interactions. The available systems for recombinant production of LD and LDI is utilised in mutational analysis of pivotal structural elements in the very high affinity interaction. This work is supported by a DTU Ph.D. scholarship, (MBVC), Danish Natural Science Research Council, the Carlsberg Foundation. References 1. Kristensen M et al. 1999, Biochim. Biophys. Acta 1431,:538-546. 2. MacGregor AW et al. 1994, Cereal Chemistry 71: 610-617.