1 Department of Systems Biology, Technical University of Denmark2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark3 Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark
Starch is the primary energy storage of higher plants and an import nutrient for mammals, fungi and bacteria. The mobilization of starch is thus essential in both the germination of the barley grain and in human digestion of foods. α-Amylase (1,4-α-D-glucan glucanohydrolase, EC. 220.127.116.11) catalyzes the hydrolysis of internal 1,4-α-D-glucosidic bonds in starch and related polysaccharides. The present thesis concerns studies of two α-amylases: 1) secondary substrate binding sites in barley α-amylase 1 (AMY1), and 2) the involvement of anti-nutrients in in vitro digestion of starch in legumes by porcine pancreatic α-amylase. The degradation of insoluble polysaccharides by glycoside hydrolases is relative inefficient as the polysaccharide chains often are barely inaccessible to the active site of the enzymes. Many raw starch-degrading enzymes contain secondary carbohydrate binding sites on the catalytic domain or on separate starch binding domains. AMY1 is so far the simplest enzyme known to possess two distinct carbohydrate surface binding sites (here called starch binding site 1 and 2, in short: SBS1 and SBS2). The substrate binding cleft of AMY1 is composed of two aglycone and seven glycone binding subsites, of which subsite –6 (key residue: Y105) is characterized by having the highest binding affinity and being situated at the opposite end of the enzyme molecule of SBS2 at a distance of 60 Å. SBS1 (key residues: W278 and W279) and SBS2 (key residues: Y380 and H395) along with the high affinity subsite –6 have been studied individually by site-directed mutagenesis the joint role, however, of SBS1, SBS2 and subsite –6 is still unknown. The combined functional properties of SBS1 and SBS2 were investigated in the present project along with the high affinity subsite –6 using site-directed mutagenesis. Y105A/Y380A and Y105A/Y380M showed decreased activity (kcat) on amylose, reminiscent of single SBS2 mutant proteins, Y380A and Y380M, and KM increased approx. 10-fold for amylose, similarly to Y105A. Y105A/Y380A, Y105A/Y380M, and Y380A/H395A showed a decrease in the degree of multiple attack (DMA) similar to Y380A whereas H395A had the same DMA as wild-type AMY1. Thus hydrophobic interactions between Tyr380 and amylose in SBS2 are involved in multiple attack; presumably SBS2 plays the role as an important, but remote secondary binding site outside the substrate binding cleft. The affinity for β-cyclodextrin (β-CD) was analyzed using surface plasmon resonance and showed the presence of two surface binding sites with a 20-fold difference in affinity. W278A/W279A/Y380A lost the ability to bind β-CD although a trace affinity was still observed, perhaps reflecting a weak interaction in the substrate binding cleft. SBS1 and SBS2 were found to be essential for the adsorption to starch granules. The affinity to barley starch granules at SBS1 was 4-fold stronger than at SBS2, while the dual SBS1 and SBS2 mutants lost the ability to adsorb onto barley starch granules. The decrease in affinity was accompanied by a similar decrease in the efficiency to hydrolyze starch granules. The affinity of AMY1 for starch granules was depending on the starch composition and morphology. A high amylose content, in particular, resulted in significant decrease in affinity, which may be attributed to considerable differences in morphology between high amylose starch granules and normal starch granules. Legumes (beans, peas, and lentils) are characterised by low blood glucose raising potential, which is proportional to the in vitro starch digestion rates. The high amount of anti-nutritional factors (phytate, proteinaceous inhibitors, tannins, and lectins) in legumes has been associated with the slow starch digestion. However, it is still debated in literature to which extent the legume starch digestibility is affected by anti-nutritional factors. The in vitro starch digestion (hydrolytic index, HI) of pea (Pisum sativum) and mixtures of pea starch with different pea protein isolates and fiber has been investigated to learn about the involvement of anti-nutrients for the digestibility of legume starches. All tested samples gave high HI-values, indicating a rapid digestion. In conclusion, the effect of anti-nutrients in legumes is either minor or non-existing compared to the effect due to botanically encapsulation of starch.