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Fibronectin type III (FNIII) domains were first identified in the eukaryotic plasma protein fibronectin, where they act as structural spacers or enable protein-protein interactions. Recently we characterized two large and multi-domain amylases in Microbacterium aurum B8.A that both carry multiple FNIII and carbohydrate binding modules (CBMs). The role of (multiple) FNIII domains in such carbohydrate acting enzymes is currently unclear. Four hypothetical functions are considered here: a substrate surface disruption domain, a carbohydrate binding module, as a stable linker, or enabling protein-protein interactions. We performed a phylogenetic analysis of all FNIII domains identified in proteins listed in the CAZy database. These data clearly show that the FNIII domains in eukaryotic and archaeal CAZy proteins are of bacterial origin and also provides examples of interkingdom gene transfer from Bacteria to Archaea and Eucarya. FNIII domains occur in a wide variety of CAZy enzymes acting on many different substrates, suggesting that they have a non-specific role in these proteins. While CBM domains are mostly found at protein termini, FNIII domains are commonly located between other protein domains. FNIII domains in carbohydrate acting enzymes thus may function mainly as stable linkers to allow optimal positioning and/or flexibility of the catalytic domain and other domains, such as CBM.

Starch is an important food ingredient and a substrate for the production of many industrial products. Biological and industrial processes involve hydrolysis of raw starch, such as digestion by humans and animals, starch metabolism in plants, and industrial starch conversion for obtaining glucose, fructose and maltose syrup or bioethanol. Raw starch degrading α-amylases (RSDA) can directly degrade raw starch below the gelatinization temperature of starch. Knowledge of the structures and properties of starch and RSDA has increased significantly in recent years. Understanding the relationships between structural peculiarities and properties of RSDA is a prerequisite for efficient application in different aspects of human benefit from health to the industry. This review summarizes recent advances on RSDA research with emphasizes on representatives of glycoside hydrolase family GH13. Definite understanding of raw starch digesting ability is yet to come with accumulating structural and functional studies of RSDA.

The modular structure and organization of most proteins is a fascinating aspect of their origin and evolution. α-Amylases are known to be formed of at least three domains. In a number of bacterial α-amylases, one or several additional domains may exist, which are carbohydrate binding modules, interacting with raw substrates. In animal α-amylases, however, no additional domain has been described. Here we report the presence of a C-terminal domain, previously described only in the bacterium Pseudoalteromonas haloplanktis. This domain is widely distributed in invertebrate α-amylases and must be ancestral, although it has been lost in important phyla or groups, such as vertebrates and insects. Its function is still unknown. In a single genome, enzymes with and without the terminal domain may coexist. In a few instances, this domain has been recruited by other proteins in both bacteria and animals through domain shuffling.

Grain α-amylase presents an apparent paradox for the wheat community. Despite the necessity of α-amylase for the seed germination process, high levels of amylase activity in the grain are considered detrimental for grain quality. Wheat α-amylases (EC 3.2.1.1) are endohydrolases belonging to the GH13_6 subfamily, one of the most studied subclasses of glycoside hydrolase (GH) family GH13. However, no comprehensive study had been done so far to describe and catalogue all the wheat α-amylase isoforms, despite compelling information on the involvement of two α-amylases on economically important issues for the international cereal community, namely pre-harvest sprouting and late maturity α-amylase. This study describes for the first time the genomic localization, nucleotide and amino acid sequences, phylogeny and expression profile of all known α-amylases in wheat, including a hitherto unknown fourth isoform here designated as TaAMY4. Isoform profiling strongly suggested α-amylases to be working in partnership to achieve complete degradation of a starch granule, whereas expression profiling revealed a potential involvement of TaAMY4 in the late maturity α-amylase problem.

Starch hydrolyzing enzymes, amylases, are important commercial enzymes used in several productive areas. A current tendency is to find amylases with high catalytic activity at 20-40°C, to generate products that work well at low temperatures, such as detergents, and for energy saving resources in industrial processes. In this work, an α-glucosidase secreted by the cold-adapted yeast Dioszegia fristingensis was purified and biochemically characterized. The effect of physicochemical parameters on the enzyme activity was evaluated. According to our results, the amylolytic enzyme secreted by D. fristingensis is a monomeric α-glucosidase of about 30 kDa that displayed the highest activity at 37-40°C and at pH 5.5-6.5,in the presence of 10 mM CaCl 2 . The enzyme exhibited a Michaelis-Menten kinetics with K m and k cat of 1.3 g/L and15 min -1 , respectively.

Starch from cereal endosperm is a major energy source for many mammals. The synthesis of this starch involves a number of different enzymes whose mode of action is still not completely understood. ADPglucose pyrophosphorylase is involved in the synthesis of starch monomer (ADP-glucose), a process, which almost exclusively takes place in the cytosol. ADPglucose is then transported into the amyloplast and incorporated into starch granules by starch synthase, starch-branching enzyme and debranching enzyme. Additional enzymes, including starch phosphorylase and disproportionating enzyme, may be also involved in the formation of starch granules, although their exact functions are still obscure. Interactions between these enzymes in the form of functional complexes have been proposed and investigated, resulting more complicated starch biosynthetic pathways. An overall picture and recent advances in understanding of the functions of these enzymes is summarized in this review to provide insights into how starch granules are synthesized in cereal endosperm.

In order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.

Certain lactic acid bacteria produce glycosyltransferases known as glucansucrases, which synthesize α-D-glucans via glucosyl transfer from sucrose. We recently reported on the formation of the unusual trisaccharide isomelezitose in low yields by a variety of glucansucrases. Isomelezitose is a rare non-reducing trisaccharide, with the structure α-d-glucopyranosyl- (1→6)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside. In this work, we describe the synthesis of isomelezitose in high yields by variants of glucansucrases engineered to contain a single point mutation at a key leucine residue involved in acceptor substrate binding. Some variants produce isomelezitose in yields up to 57%. This method is amenable to large-scale production of isomelezitose for food, industrial and biomedical applications.