Volume 2, Issue 1

An α-amylase from Thermoactinomyces vulgaris, TVA I, hydrolyzes both α-1,4- and α-1,6-glucosidic linkages. Two variants of TVA I have been previously constructed, one containing a substitution of three residues, Ala357- Gln359-Tyr360, with Val-Asn-Glu (AQY/VNE), and the other bearing a deletion of 11 residues from Ala363 to Asn373 (Del11). The activities of both AQY/VNE and Del11 for the α-1,4-glucosidic linkage of maltotriose were decreased compared to that of wild-type TVA I, while the activities of the two variants for the α-1,6-glucosidic linkage of a trisaccharide, isopanose, were less significantly altered. Here, we determined the crystal structures of AQY/VNE and Del11. The structure of AQY/VNE was almost isomorphous with that of wild-type TVA I. On the other hand, the structure of Del11 showed that a conformational change in domain B was induced by the 11-residue deletion, causing narrowing of the catalytic cleft. Taken together with the results of kinetic analysis, this narrower catalytic cleft is likely responsible for the preference of the TVA I enzyme for the α-1,6-glucosidic linkage.

The control of postprandial blood glucose level via the inhibition of α‐amylase is a relevant strategy for the treatment of type 2 diabetes. Several antidiabetic plants are known but there is no information about their α-amylase inhibitory activity. This in vitro study tries to reveal the answer. Hot water extracts of 58 medicinal plants and spices were examined. Activity measurements of human salivary α-amylase (HSαA) on 0.14 m/v % starch substrate was carried out by isothermal titration calorimetry in the presence or absence of plant extracts. Water soluble antioxidant capacity of each extract was measured with photo-chemiluminescence method. Results have confirmed the inhibitory activity of several plant extracts against HsαA. The green tea, cinnamon and allspice, furthermore leaves of blackberry, raspberry and strawberry deserve particular mention (IC 50 ≤ 1.2 mg/mL). A few extracts had significant water-soluble antioxidant capacity compared to ascorbic acid and a weak correlation was recognised between the obtained IC 50 and antioxidant capacity values. Inhibition of amylases located in digestive system can be reached via daily intake of most active extracts. These plants could be inserted effectively into a diabetic diet as food supplements.

Isomaltooligosaccharides (IMOS) are sugars with health promoting properties that make them relevant for the pharmaceutical and food industries. IMOS have ample chemical diversity achieved by different α-glucosidic linkages and polymerization degrees, forming linear, branched and cyclic structures. Enzymatic synthesis of these compounds can be carried out by glycoside hydrolases (GHs) with transglycosylating activity. Different substrates are used for the synthesis: combinations of disaccharides and monosaccharides, or polymeric carbohydrates such as starch or dextran, which are converted to IMOS by a combination of hydrolysis and transglucosylation. In this review, the structural features of different enzyme families (GH31, GH13, GH70, GH57 and GH66) involved in IMOS synthesis are analysed. Focus is placed on structural traits that affect substrate and product specificity, and on the relative efficiency of transglucosylation and hydrolysis. Information resulting from site-directed mutagenesis and sequence alignments complements structural data to understand the role of specific residues in the performance of the enzymes. Altogether, these studies provide a frame of knowledge which may be used to design new enzymes with improved properties

α-Amylases have been of interest in diverse fields for many years because of their importance in basic biology, agriculture, and industry. Starch hydrolysis in plants has been studied extensively in germinating cereal seeds. It is generally accepted that α-amylases are secretory enzymes with a pivotal role in the breakdown of starch reserves in the endosperm. Intriguingly, however, recent investigations reveal that some α-amylases degrade starch in the plastids of living cells. The recent solving of the crystal structure of rice AmyI-1 isoform shows that the binding pocket of starch binding site 1 situated outside of the active site cleft interacts with the substances other than oligosaccharides. These findings provided novel insights into structural and cell biological aspects of α-amylase functions in intracellular transport, organelle targeting, and organ-specific actions. Under global warming, abnormal high temperatures during rice grain filling increase grain chalkiness, resulting in yield loss. Intensive “omics” analyses of developing caryopses and mature grains grown under heat stress showed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. Transgenic studies using ectopic overexpression and suppression of α-amylase revealed that α-amylase is a key factor in grain chalkiness. Here we discuss unique new functions of α-amylase in rice cells.

The starchy foods including staled or leftover bread, insect infested or damaged cereal grains and bakery wastes are usually discarded, which is a threat to public health due to extensive fungal growth. In the present study we have utilized them as raw materials for synthesis of maltose and glucose syrups using pullulan hydrolase from Thermococcus kodakarensis (TK-PUL). The novelty of the process was that whole process was completed: (i) in the absence of any liquefying amylase; (ii) without pre-gelatinization of starch; and (iii) using undisrupted E. coli cells, expressing TK-PUL gene, as a source of extremely thermostable TK-PUL protein. Since glucose and maltose can serve as precursors for a variety of biotechnological products, it is therefore anticipated that hydrolysis of starchy food wastes by TK-PUL would be highly beneficial. It will serve as cost effective measure not only to extract valuables from rubbish but also lower the level of environmental pollutants.

Amylopullulanases are endoacting bifunctional enzymes capable of hydrolyzing α-1,4- and α-1,6-glycosidic linkages in starch, amylose, pullulan, amylopectin and related oligosaccharides. These enzymes possess single or dual active site(s) for cleaving α-1,4- and α-1,6-glycosidic bonds; the former are called amylopullulanases, and the latter, α-amylase-pullulanases. These are grouped into GH13 and GH57 families based on the architecture of the catalytic domain and the number of conserved sequence regions. The amylopullulanases/α-amylasepullulanases are produced by bacteria as well as archaea, and among them, thermophilic and hyperthermophilic species are the major producers. The thermostable amylopullulanases find application in one-step starch liquefaction-saccharification to form various sugar syrups and maltooligosaccharides. The starch saccharification process catalysed by amylopullulanases minimizes the use of other amylolytic enzymes, like α-amylase and glucoamylase, thereby reducing the cost of sugar syrups. The enzymes also find applications in bread making as an anti-stale and as a detergent additive.