Maltose Units Research Articles

Biochemical Characterization of Starch Excess4 from Storage Crops

Starch is a water‐insoluble branched polymer of glucose. Its degradation requires the concerted action of glucan phosphatases and glucan dikinases. Reversible starch phosphorylation allows for the solubilization of the starch surface, allowing for amylase to degrade starch into maltose units. However, our understanding is limited on how these enzymes contribute to starch degradation in storage crops such as potato and cassava. In this study, we biochemically characterized cassava and potato SEX4 enzymes using enzymatic assays and differential scanning fluorometry. We determined the kinetics of SEX4 using both generic and physiologically relevant substrates. We also explored the utilization of these glucan phosphatase orthologs to enhance starch degradation in‐vitro. Our findings reveal that cassava and potato SEX4 show similar allosteric activation mechanisms but varying abilities to degrade starch under in‐vitro conditions.

GtfC Enzyme of Geobacillus sp. 12AMOR1 Represents a Novel Thermostable Type of GH70 4,6-α-Glucanotransferase That Synthesizes a Linear Alternating (α1 → 6)/(α1 → 4) α-Glucan and Delays Bread Staling

Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 → 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 → 6)/(α1 → 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.

Purification and Interaction of β-amylase from Dioscorea alata (Water Yam) with Epicatechin

β-amylase is an enzyme that hydrolyzes the α-1,4-glucan bonds from the non-reducing ends of starch and other carbohydrate polymers reducing it to maltose units. Maltose has much application with food processing and pharmaceutical industries. The enzyme was purified to apparent homogeneity with a monomeric molecular weight of 30.1 kDa based on SDS-PAGE. The binding Constant (Ka), Kd ΔH, ΔS and ΔG values were 1.53´103Lmol-1 ,3.12x10-4Lmol-1, 19.35kJmol-1, 56.67Jmol-1K-1, and -18.17kJmol-1 respectively. The binding profile of β-amylase with epicatechin was spontaneous with a stoichiometric ratio of 2:1. Hydrophobic bonding played a major role in stabilizing the β-amylase-ligand complex. The mode of reaction was by static quenching. It further dictates that the binding reaction is entropy driven. The inhibitory effect of this plant polyphenols on β-Amylase might contribute to the regulation of β-Amylase activity in plants.

Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses

The β-amylase (BAM) gene family, known for their property of catalytic ability to hydrolyze starch to maltose units, has been recognized to play critical roles in metabolism and gene regulation. To date, BAM genes have not been characterized in oil crops. In this study, the genome-wide survey revealed the identification of 30 BnaBAM genes in Brassica napus L. (B. napus L.), 11 BraBAM genes in Brassica rapa L. (B. rapa L.), and 20 BoBAM genes in Brassica oleracea L. (B. oleracea L.), which were divided into four subfamilies according to the sequence similarity and phylogenetic relationships. All the BAM genes identified in the allotetraploid genome of B. napus, as well as two parental-related species (B. rapa and B. oleracea), were analyzed for the gene structures, chromosomal distribution and collinearity. The sequence alignment of the core glucosyl-hydrolase domains was further applied, demonstrating six candidate β-amylase (BnaBAM1, BnaBAM3.1-3.4 and BnaBAM5) and 25 β-amylase-like proteins. The current results also showed that 30 BnaBAMs, 11 BraBAMs and 17 BoBAMs exhibited uneven distribution on chromosomes of Brassica L. crops. The similar structural compositions of BAM genes in the same subfamily suggested that they were relatively conserved. Abiotic stresses pose one of the significant constraints to plant growth and productivity worldwide. Thus, the responsiveness of BnaBAM genes under abiotic stresses was analyzed in B. napus. The expression patterns revealed a stress-responsive behaviour of all members, of which BnaBAM3s were more prominent. These differential expression patterns suggested an intricate regulation of BnaBAMs elicited by environmental stimuli. Altogether, the present study provides first insights into the BAM gene family of Brassica crops, which lays the foundation for investigating the roles of stress-responsive BnaBAM candidates in B. napus.

An amylopullulanase (ApuNP1) from Geobacillus thermoleovorans NP1: biochemical characterization and its potential industrial applications

An amylopullulanase was produced by Geobacillus thermoleovorans NP1. The optimum enzyme production occurred at 45°C and pH 7.0 (12 hr). NP1 amylopullulanase (ApuNP1) exhibited the maximal activity at 50°C and pH 6.0 and was stable between 30–50°C, and pH 3.0–12.0 for 24 hr. The enzyme showed two bands with molecular weights of 112 and 107 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amylopullulanase retained 100% of its activity in the presence of 10 mM of Ca2+, Ba2+, Zn2+, Mg2+, Cu2+, EDTA, and PMSF. While the enzyme showed resistance to 5% of TritonX-100, Tween 20, and Tween 80, the activity was inhibited by 5% β-mercaptoethanol and H2O2. While the hydrolysis products of pullulan were maltose, maltotriose, and maltodextrin, the starch was hydrolyzed to maltose, maltotriose, and maltodextrin units. This shows that NP1 pullulanase is a type II pullulanase (amylopullulanase). After the liquefaction assay, 12% glucose content was measured with a refractometer in the presence of 20% starch. According to the wash performance tests, the mixture of ApuNP1 and 1% detergent removed almost all of the stains. This novel thermo-acidic amylopullulanase has a potency to be used in detergent, starch, food, baking, textile, and cosmetic industries.

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis

Cyclic α-maltosyl-(1→6)-maltose (CMM, cyclo-{→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium Arthrobacter globiformis and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase-related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely -3', -2, -1, and +1'. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.

Fingerprinting processive β-amylases

Many polysaccharide hydrolases are capable of processive action, where they perform repeated attacks without dissociating from the substrate molecule. In this paper, we extend the “Fingerprinting method”, a method that can be used to determine the relative specificities that an enzyme has for the various reactions that it can catalyze in a multi-step reaction scheme, to reaction schemes involving processive action. We do this for three case studies for the hydrolysis of linear malto-oligosaccharides by β-amylase, an exoenzyme that successively removes maltose units from the non-reducing ends of the molecules. In these case studies, we demonstrate that it is possible to use reaction profiles to determine, simultaneously, the relative specificities that the enzyme has for the different species in the reaction mixture and the probabilities of processive action occurring. These parameters can be used in models for designing and optimizing hydrolysis processes and also to guide protein engineering programs.

GH57 amylopullulanase from Desulfurococcus amylolyticus JCM 9188 can make highly branched cyclodextrin via its transglycosylation activity.

Desulfurococcus amylolyticus is an anaerobic and hyperthermophilic crenarchaeon that can use various carbohydrates as energy sources. We found a gene encoding a glycoside hydrolase family 57 amylolytic enzymes (DApu) in a putative carbohydrate utilization gene cluster in the genome of D. amylolyticus. This gene has an open reading frame of 1,878 bp and consists of 626 amino acids with a molecular mass of 71 kDa. Recombinant DApu (rDApu) completely hydrolyzed pullulan to maltotriose by attacking α-1,6-glycosidic linkages, and was able to produce glucose and maltose from soluble starch and amylopectin. Although rDApu showed no activity toward α-cyclodextrin (CD) and β-CD, maltooctaose (G8) was detected from reaction with γ-CD. The highest activity of rDApu was measured at pH 5.0 and 95 °C. The half-life of rDApu was 12.7 h at 95 °C and 27 min at 98 °C. Interestingly, rDApu was able to transfer a maltose unit to 6-O-α-maltosyl-β-CD via transglycosylation. Structure analysis using MALDI-TOF/TOF MS and nuclear magnetic resonance revealed that the new transglycosylated products were 61, 64-di-O-maltosyl-β-CD and 61, 63, 65-tri-O-maltosyl-β-CD.

Incorporation of DNA/PEI polyplexes into gelatin/chitosan hydrogel scaffolds: a μ-DSC study

Polyplexes between a double-stranded Salmon DNA and hyperbranched poly(ethyleneimine) (PEI) as well as a maltosylated PEI-Mal were incorporated into a gelatin/chitosan hydrogel scaffold. Calorimetric experiments of the polyplexes show a decrease of the melting temperature in presence of PEI and a peak splitting in presence of PEI-Mal, which can be interpreted to a partial compaction of the DNA strands in presence of PEI-Mal. When the polyplexes are incorporated into a gelatin/chitosan scaffold in the swollen state, the DNA melting peaks at 90 and 93 °C, respectively, indicate in both cases the release of the DNA at the surface of the hydrogel scaffold in a more compact form. Specific interactions between the PEI-Mal shell and gelatin are responsible for the tuning of the release properties in presence of the maltose units in the hyperbranched PEI.

Interactions of Nitroxide-Conjugated and Non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies.

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Influence of core and maltose surface modification of PEIs on their interaction with plasma proteins—Human serum albumin and lysozyme

Regardless of the route of administration, some or all of a therapeutic agent will appear in the blood stream, where it can act on blood cells and other components of the plasma. Recently we have shown that poly(ethylene imines) (PEIs) which interact with plasma proteins are taken up into erythrocyte membranes. These observations led us to investigate the interactions between maltose functionalized hyperbranched PEIs (PEI-Mal) and plasma proteins. Two model proteins were chosen − human serum albumin (HSA) (albumins constitute ∼60% of all plasma proteins), and lysozyme. HSA is a negatively charged 66kDa protein at neutral pH, whereas lysozyme is a positively charged 14kDa protein. Fluorescence quenching and changes in the conformation of the amino acid tryptophan, diameter and zeta potential of proteins were investigated to evaluate the interaction of PEI-Mal with proteins. PEI-Mal interacts with both types of proteins. The strength of dendritic glycopolymer interactions was generally weak, especially with lysozyme. Greater changes were found with HSA, mainly triggered by hydrogen bonds and the electrostatic interaction properties of dendritic glycopolymers. Moreover, the structure and the size of PEI-Mal macromolecules affected these interactions; larger macromolecules with more sugar groups (95% maltose units) interacted more strongly with proteins than smaller ones with lower sugar modification (33% maltose units). Due to (i) the proven overall low toxicity of sugar-modified PEIs and, (ii) their ability to interact preferentially through hydrogen bonds with proteins of human plasma or possibly with other interesting protein targets, PEI-Mal is a good candidate for creating therapeutic nanoparticles in the fast developing field of nanomedicine.

β-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress.

During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. β-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly β-amylase-3 (BAM3). A second β-amylase isoform, β-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of β-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.

Change in size, morphology and stability of DNA polyplexes with hyperbranched poly(ethyleneimines) containing bulky maltose units

Polyplexes between Salmon DNA and non-modified hyperbranched poly(ethyleneimines) of varying molar mass, i.e., PEI(5k) with 5000g/mol and PEI(25k) with 25,000g/mol, and modified PEI(5k) with maltose units (PEI-Mal) were investigated in dependence on the molar N/P ratio by using dynamic light scattering (DLS), zeta potential measurements, micro differential scanning calorimetry (μ-DSC), scanning-transmission electron microscopy (STEM), and cryo-scanning electron microscopy (cryo-SEM). A reloading of the polyplexes can be observed by adding the unmodified PEI samples of different molar mass. In excess of PEI a morphological transition from core-shell particles (at N/P 8) to loosely packed onion-like polyplexes (at N/P 40) is observed. The shift of the DSC melting peak from 88°C to 76°C indicates a destabilization of the DNA double helix due to the complexation with the unmodified PEI. Experiments with the maltose-modified PEI show a reloading already at a lower N/P ratio. Due to the presence of the sugar units in the periphery of the polycation electrostatic interactions between DNA become weaker, but cooperative H-bonding forces are reinforced. The resulting less-toxic, more compact polyplexes in excess of the PEI-Mal with two melting points and well distributed DNA segments are of special interest for extended gene delivery experiments.

Characterization of oligosaccharide-functionalized hyperbranched poly(ethylene imine) and their complexes with retinol in aqueous solution

Structure, internal density distribution, and size of hyperbranched poly(ethylene imine) (PEI) functionalized with various amounts of maltose (PEI-Mal) in phosphate buffer were studied by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). The value of pH was varied in the range from 3 to 9. Virtually no effect of pH on the nanostructure was found in this interval. The SAXS results revealed a broad segmental radial density distribution, i.e. a “fluffy” globular structure rather than a distinct core–shell structure with a high-density compact core and a low-density corona. This suggests that the maltose units are rather evenly distributed both in the interior and on the surface of the species with a PEI-core of molar mass of 25,000g/mol. The DLS measurements showed that the overall size of the PEI-Mal derivatives increased as the number of maltose units in the PEI-Mal structures rises. The interaction of the hydrophobic model drug retinol with PEI or PEI-Mal derivatives was also investigated. The UV–visible spectroscopy results disclosed that the solubility of retinol in the phosphate buffer is very poor and it takes a very long time to solubilize retinol. Moreover, retinol induces aggregation of dendritic glycopolymers where the growth of aggregates occurs continuously over several days and then remains virtually constant.

Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

Influence of nonionic branched-chain alkyl glycosides on a model nano-emulsion for drug delivery systems

The effect of incorporating new nonionic glycolipid surfactants on the properties of a model water/nonionic surfactant/oil nano-emulsion system was investigated using branched-chain alkyl glycosides: 2-hexyldecyl-β(/α)-d-glucoside (2-HDG) and 2-hexyldecyl-β(/α)-d-maltoside (2-HDM), whose structures are closely related to glycero-glycolipids. Both 2-HDG and 2-HDM have an identical hydrophobic chain (C16), but the former consists a monosaccharide glucose head group, in contrast to the latter which has a disaccharide maltose unit. Consequently, their hydrophilic–lipophilic balance (HLB) is different. The results obtained have shown that these branched-chain alkyl glycosides affect differently the stability of the nano-emulsions. Compared to the model nano-emulsion, the presence of 2-HDG reduces the oil droplet size, whereas 2-HDM modify the properties of the model nano-emulsion system in terms of its droplet size and storage time stability at high temperature. These nano-emulsions have been proven capable of encapsulating ketoprofen, showing a fast release of almost 100% in 24h. Thus, both synthetically prepared branched-chain alkyl glycosides with mono- and disaccharide sugar head groups are suitable as nano-emulsion stabilizing agents and as drug delivery systems in the future.

Weissella confusa Cab3 dextransucrase: Properties and in vitro synthesis of dextran and glucooligosaccharides

Food-derived Weissella spp. have gained attention during recent years as efficient dextran producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR had a molar mass of 178kDa and was activated by Co2+ and Ca2+ ions. Glycerol and Tween 80 enhanced enzyme stability, and its half-life at 30°C increased from 10h to 74h and 59h, respectively. The 1H and 13C NMR spectral analysis of the produced dextran confirmed the presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8×107g/mol. Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with reducing end maltose units. To the best of our knowledge, this is a first report on native W. confusa dextransucrase.

Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13% sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (Pyrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (Km) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The kcat values were 381.0 min(-1) for G3 and 1,545.0 min(-1) for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the Ki constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates

β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme–product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.

Structure of a novel α-amylase AmyB fromThermotoga neapolitanathat produces maltose from the nonreducing end of polysaccharides

An intracellular α-amylase, AmyB, has been cloned from the hyperthermophilic bacterium Thermotoga neapolitana. AmyB belongs to glycoside hydrolase family 13 and liberates maltose from diverse substrates, including starch, amylose, amylopectin and glycogen. The final product of AmyB is similar to that of typical maltogenic amylases, but AmyB cleaves maltose units from the nonreducing end, which is a unique property of this α-amylase. In this study, the crystal structure of AmyB from T. neapolitana has been determined at 2.4 Å resolution, revealing that the monomeric AmyB comprises domains A, B and C like other α-amylases, but with structural variations. In the structure, a wider active site and a putative extra sugar-binding site at the top of the active site were found. Subsequent biochemical results suggest that the extra sugar-binding site is suitable for recognizing the nonreducing end of the substrates, explaining the unique activity of this enzyme. These findings provide a structural basis for the ability of an α-amylase that has the common α-amylase structure to show a diverse substrate specificity.