β-Amylase (Barley)

A procedure previously developed for the assay of cereal-flour β-amylase has been improved and standardised. The improved procedure uses the substrate p-nitrophenyl maltopentaose (PNPG5) in the presence of near saturating levels of α-glucosidase. PNPG5 is rapidly hydrolysed by β-amylase but less readily by cereal α-amylases. The substrate is hydrolysed by β-amylase to maltose and p-nitrophenyl maltotriose (PNPG3). With the levels of α-glucosidase used in the substrate mixture, PNPG3 is rapidly cleaved to glucose and p-nitrophenol, whereas PNPG5 is resistant to hydrolysis by the α-glucosidase. The assay procedure has been standardised for several β-amylases and the exact degree of interference by cereal α-amylases determined. The procedure can be readily applied to the selective measurement of β-amylase activity in cereal and malted cereal-flours.

Mass spectrometry (MS) enables detection of different chemical species with a very high specificity; however, it can be limited by its throughput. Integrating MS with microfluidics has a tremendous potential to improve throughput and accelerate biochemical research. In this work, we introduce Drop-NIMS, a combination of a passive droplet loading microfluidic device and a matrix-free MS laser desorption ionization technique called nanostructure-initiator mass spectrometry (NIMS). This platform combines different droplets at random to generate a combinatorial library of enzymatic reactions that are deposited directly on the NIMS surface without requiring additional sample handling. The enzyme reaction products are then detected with MS. Drop-NIMS was used to rapidly screen enzymatic reactions containing low (on the order of nL) volumes of glycoside reactants and glycoside hydrolase enzymes per reaction. MS “barcodes” (small compounds with unique masses) were added to the droplets to identify different combinations of substrates and enzymes created by the device. We assigned xylanase activities to several putative glycoside hydrolases, making them relevant to food and biofuel industrial applications. Overall, Drop-NIMS is simple to fabricate, assemble, and operate and it has potential to be used with many other small molecule metabolites.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBD_St1 and SBD_St2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBD_GA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBD_GA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3–9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBD_GA-TuαGT and-SBD_St2-TuαGT showed K_d of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBD_St1-TuαGT had 3-5-fold lower affinity. SBD_St2 contributed more than SBD_St1 to activity, substrate binding, and the stability of TuαGT fusions.

Six cross-bred barley lines developed by a breeding strategy with the target to enhance the fructan synthesis activity and reduce the fructan hydrolysis activity were analyzed together with their parental lines, and a reference line (Gustav) to determine whether the breeding strategy also affected the content and molecular structure of amylopectin and β-glucan. The highest fructan and β-glucan content achieved in the novel barley lines was 8.6 % and 12 %, respectively (12.3-fold and 3.2-fold higher than in Gustav). The lines with low fructan synthesis activity had higher starch content, smaller building blocks in amylopectin, and smaller structural units of β-glucans than the lines with high-fructan synthesis activity. Correlation analysis confirmed that low starch content was associated with high amylose, fructan, and β-glucan content, and larger building blocks in amylopectin.

In an interlaboratory study we compare different methods to determine the weight-average molecular weight (M_w) and molecular weight distribution of six cereal beta-glucan isolates of nutritional importance. Size-exclusion chromatography (SEC) with multi-angle light scattering (MALS), capillary viscometry, sedimentation velocity analytical ultracentrifugation and one asymmetric flow field-flow fractionation (AF4)-MALS method all yielded similar M_w values for mostly individual chains of dissolved beta-glucan molecules. SEC with post-column calcofluor detection underestimated the M_w of beta-glucan >500 × 10³ g/mol. The beta-glucan molecules analysed by these methods were primarily in a random coil conformation as evidenced from individual Mark-Houwink-Kuhn-Sakurada (MHKS) scaling coefficients between 0.5 and 0.6 and Wales-Van Holde ratios between 1.4 and 1.7. In contrast, a second AF4-MALS method yielded much larger M_w values for these same samples indicating the presence and detection of beta-glucan aggregates. Storage of the six beta-glucan solutions in the dark at 4°C for 4 years revealed them to be stable. This suggests an absence of storage-induced irreversible aggregation phenomena or chain-scission. Shear forces in SEC and the viscometer capillary and hydrostatic pressure in analytical ultracentrifugation probably led to the reversable dissociation of beta-glucan aggregates into molecularly dissolved species. Thus, all these methods yield true weight-average molecular weight values not biased by the presence of aggregates as was the case in one of the AF4 based methods employed.

This study aims to enhance the understanding of the structure of maize phytoglycogen nanoparticles, and the effect of shear scission on their architecture, radius, stiffness, and deformability. Compared to amylopectin, phytoglycogen had a lower A:B chain ratio, a lower number of chains per B chain, and a much higher number of A_fingerprint chains. Phytoglycogen (M_w = 28.0 × 10⁶ g/mol) was subjected to high-shear extrusion with varying Specific Mechanical Energies (SMEs) using different screw speeds, showing a maximum stable molecular weight M_w of ~9.31 × 10⁶ g/mol and a particle radius R reduction of 36 %, with a corresponding 20 % increase in the average mass density. Atomic force microscopy force spectroscopy revealed that nanoparticles extruded at the lowest SME (122 Wh/kg) exhibited a 20 % increase in Young's modulus. Higher SME values (up to 488 Wh/kg) resulted in an overall decrease in stiffness without further significant reductions in radius.

A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and B_fp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and ¹³C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6−12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and B_fp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.

Oca (Oxalis tuberosa) is an underutilized species and represents a novel starch source. Composition and structure of starches from tubers of two commercial oca varieties grown in New Zealand were compared to those of normal maize and potato starches. The phosphorus content of oca starch was ∼60% of that of potato starch. The amylose content of oca starch (∼21%) was lower than that of maize and potato starches (concanavalin A precipitation method). The fine structure of oca amylopectin was much more similar to that of potato amylopectin than to that of maize amylopectin. Oca amylopectin had a shorter internal chain length and less fingerprint B-chains than potato amylopectin. The two oca starches were structurally and compositionally similar. Oca starch granules had a volume moment mean size of 34.5 μm and B-type polymorph. Comparative analysis suggested that oca starch has the potential to be developed as a novel starch source.

New Zealand Maori potatoes (Taewa) represent unique genetic resources for potato quality, though they are much underutilized. In this report, the composition and molecular structure of starches from 5 Maori potato varieties were studied. In particular, the internal unit chain composition of the amylopectins in the form of β-limit dextrins were highlighted. Starches from a commercial modern potato variety and a maize variety with normal amylose contents were employed for comparison. Genetic diversity in the amylose (e.g., 22.6% in Moemoe to 28.6% in Turaekuri) and phosphorus (5.4 mg/100 g in Turaekuri to 7.0 mg/100 g in Kowiniwini) contents as well as the molecule structure of the starches (e.g., external chain length of amylopectin ranged from 13.0 glucosyl residues in Turaekuri to 15.8 glucosyl residues in Karuparera) has been revealed. Maori potato amylopectins have the highest amount of long unit and internal chains and the lowest amount of these chains among amylopectins from different sources. Overall, Maori potato starch appeared to be structurally and compositionally similar to modern potato starch.

Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and β-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify β-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels.