Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the ceralpha assay: Collaborative study.
McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). Journal of AOAC International, 85(5), 1096-1102.
This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.
The effect of carbohydrates on α-amylase activity measurements.
Baks, T., Janssen, A. E. & Boom, R. M. (2006). Enzyme and Microbial Technology, 39(1), 114-119.
The Ceralpha method can be used for α-amylase activity measurements during the hydrolysis of starch at high substrate concentrations (>40 wt.%). However, the results are affected by the carbohydrates present in the samples. The effect of carbohydrates on the Ceralpha α-amylase activity measurements was measured over a broad concentration range. It was found that starch has the largest influence and glucose has the lowest influence on the Ceralpha assay procedure. These results were explained by considering substrate inhibition and substrate competition. A simple kinetic model was used to describe the observed phenomena quantitatively. This model was also used to estimate the Michaelis–Menten constant for a large number of substrates and it requires only a single experiment for each Km determination.
Cross-inhibitory activity of cereal protein inhibitors against α-amylases and xylanases.
Sancho, A. I., Faulds, C. B., Svensson, B., Bartolomé, B., Williamson, G. & Juge, N. (2003). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1650(1), 136-144.
The purification and characterisation of a xylanase inhibitor (XIP-I) from wheat was reported previously. In our current work, XIP-I is also demonstrated to have the capacity to inhibit the two barley α-amylase isozymes (AMY1 and AMY2). XIP-I completely inhibited the activity of AMY1 and AMY2 towards insoluble Blue Starch and a soluble hepta-oligosaccharide derivative. A ternary complex was formed between insoluble starch, a catalytically inactive mutant of AMY1 (D180A), and XIP-I, suggesting that the substrate–XIP-I interaction is necessary for inhibition of barley α-amylases. Ki values for α-amylase inhibition, however, could not be calculated due to the nonlinear nature of the inhibition pattern. Furthermore, surface plasmon resonance and gel electrophoresis did not indicate interaction between XIP-I and the α-amylases. The inhibition was abolished by CaCl2, indicating that the driving force for the interaction is different from that of complexation between the barley α-amylase/subtilisin inhibitor (BASI) and AMY2. This is the first report of a proteinaceous inhibitor of AMY1. BASI, in addition, was demonstrated to partially inhibit the endo-1,4-β-D-xylanase from Aspergillus niger (XylA) of glycoside hydrolase family 11. Taken together, the data demonstrate for the first time the dual target enzyme specificity of BASI and XIP-I inhibitors for xylanase and α-amylase.
Overexpression of the Arabidopsis syntaxin PEP12/SYP21 inhibits transport from the prevacuolar compartment to the lytic vacuole in vivo.
Foresti, O., daSilva, L. L. P. & Denecke, J. (2006). The Plant Cell, 18(9), 2275-2293.
Golgi-mediated transport to the lytic vacuole involves passage through the prevacuolar compartment (PVC), but little is known about how vacuolar proteins exit the PVC. We show that this last step is inhibited by overexpression of Arabidopsis thaliana syntaxin PEP12/SYP21, causing an accumulation of soluble and membrane cargo and the plant vacuolar sorting receptor BP80 in the PVC. Anterograde transport proceeds normally from the endoplasmic reticulum to the Golgi and the PVC, although export from the PVC appears to be compromised, affecting both anterograde membrane flow to the vacuole and the recycling route of BP80 to the Golgi. However, Golgi-mediated transport of soluble and membrane cargo toward the plasma membrane is not affected, but a soluble BP80 ligand is partially mis-sorted to the culture medium. We also observe clustering of individual PVC bodies that move together and possibly fuse with each other, forming enlarged compartments. We conclude that PEP12/SYP21 overexpression specifically inhibits export from the PVC without affecting the Golgi complex or compromising the secretory branch of the endomembrane system. The results provide a functional in vivo assay that confirms PEP12/SYP21 involvement in vacuolar sorting and indicates that excess of this syntaxin in the PVC can be detrimental for further transport from this organelle.
Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo.
Bottanelli, F., Foresti, O., Hanton, S. & Denecke, J. (2011). The Plant Cell, 23(8), 3007-3025.
We tested if different classes of vacuolar cargo reach the vacuole via distinct mechanisms by interference at multiple steps along the transport route. We show that nucleotide-free mutants of low molecular weight GTPases, including Rab11, the Rab5 members Rha1 and Ara6, and the tonoplast-resident Rab7, caused induced secretion of both lytic and storage vacuolar cargo. In situ analysis in leaf epidermis cells indicates a sequential action of Rab11, Rab5, and Rab7 GTPases. Compared with Rab5 members, mutant Rab11 mediates an early transport defect interfering with the arrival of cargo at prevacuoles, while mutant Rab7 inhibits the final delivery to the vacuole and increases cargo levels in prevacuoles. In contrast with soluble cargo, membrane cargo may follow different routes. Tonoplast targeting of an α-TIP chimera was impaired by nucleotide-free Rha1, Ara6, and Rab7 similar to soluble cargo. By contrast, the tail-anchored tonoplast SNARE Vam3 shares only the Rab7-mediated vacuolar deposition step. The most marked difference was observed for the calcineurin binding protein CBL6, which was insensitive to all Rab mutants tested. Unlike soluble cargo, α-TIP and Vam3, CBL6 transport to the vacuole was COPII independent. The results indicate that soluble vacuolar proteins follow a single route to vacuoles, while membrane spanning proteins may use at least three different transport mechanisms.
Measurement of α-amylase activity by Sequential Injection Analysis.
Min, R. W., Carlsen, M., Nielsen, J. & Villadsen, J. (1995). Biotechnology Techniques, 9(10), 763-766.
A Sequential Injection Analysis (SIA) system for monitoring α-amylase activity is described. The SIA analyser is a further development of previously investigated Flow Injection Analysis (FIA) analyser. The analysis of α-amylase activity is based on monitoring the decoloration of an iodine-starch complex. Performances of the SIA analyser have been compared with the FIA analyser. A good agreement has been obtained between the SIA measurements and the FIA measurements.
Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger.
Juge, N., Svensson, B. & Williamson, G. (1998). Applied Microbiology and Biotechnology, 49(4), 385-392.
Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.
Targeting of the plant vacuolar sorting receptor BP80 is dependent on multiple sorting signals in the cytosolic tail.
daSilva, L. L. P., Foresti, O. & Denecke, J. (2006). The Plant Cell, 18(6), 1477-1497.
Although signals for vacuolar sorting of soluble proteins are well described, we have yet to learn how the plant vacuolar sorting receptor BP80 reaches its correct destination and recycles. To shed light on receptor targeting, we used an in vivo competition assay in which a truncated receptor (green fluorescent protein-BP80) specifically competes with sorting machinery and causes hypersecretion of BP80-ligands from tobacco (Nicotiana tabacum) leaf protoplasts. We show that both the transmembrane domain and the cytosolic tail of BP80 contain information necessary for efficient progress to the prevacuolar compartment (PVC). Furthermore, the tail must be exposed on the correct membrane surface to compete with sorting machinery. Mutational analysis of conserved residues revealed that multiple sequence motifs are necessary for competition, one of which is a typical Tyr-based motif (YXXΦ). Substitution of Tyr-612 for Ala causes partial retention in the Golgi apparatus, mistargeting to the plasma membrane (PM), and slower progress to the PVC. A role in Golgi-to-PVC transport was confirmed by generating the corresponding mutation on full-length BP80. The mutant receptor was partially mistargeted to the PM and induced the secretion of a coexpressed BP80-ligand. Further mutants indicate that the cytosolic tail is likely to contain other information besides the YXXΦ motif, possibly for endoplasmic reticulum export, endocytosis from the PM, and PVC-to-Golgi recycling.
Isolation, characterization and inhibition by acarbose of the α-amylase from Lactobacillus fermentum: comparison with Lb. manihotivorans and Lb. plantarum amylases.
Talamond, P., Desseaux, V., Moreau, Y., Santimone, M. & Marchis-Mouren, G. (2002). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 133(3), 351-360.
Extracellular α-amylase from Lactobacillus fermentum (FERMENTA) was purified by glycogen precipitation and ion exchange chromatography. The purification was approximately 28-fold with a 27% yield. The FERMENTA molecular mass (106 000 Da) is in the same range as the ones determined for L. amylovorus (AMYLOA), L. plantarum (PLANTAA) and L. manihotivorans (MANIHOA) α-amylases. The amino acid composition of FERMENTA differs from the other lactobacilli considered here, but however, indicates that the peptidic sequence contains two equal parts: the N-terminal catalytic part; and the C-terminal repeats. The isoelectric point of FERMENTA, PLANTAA, MANIHOA are approximately the same (3.6). The FERMENTA optimum pH (5.0) is slightly more acidic and the optimum temperature is lower (40°C). Raw starch hydrolysis catalyzed by all three amylases liberates maltotriose and maltotretaose. Maltose is also produced by FERMENTA and MANIHOA. Maltohexaose FERMENTA catalyzed hydrolysis produces maltose and maltotriose. Finally, kinetics of FERMENTA, PLANTAA and MANIHOA using amylose as a substrate and acarbose as an inhibitor, were carried out. Statistical analysis of kinetic data, expressed using a general velocity equation and assuming rapid equilibrium, showed that: (1) in the absence of inhibitor kcat/Km are, respectively, 1×109, 12.6×109 and 3.2×109 s-1 M-1; and (2) the inhibition of FERMENTA is of the mixed non-competitive type (K1i=5.27 µM; L1i=1.73 µM) while the inhibition of PLANTAA and MANIHOA is of the uncompetitive type (L1i=1.93 µM and 1.52 µM, respectively). Whatever the inhibition type, acarbose is a strong inhibitor of these Lactobacillus amylases. These results indicate that, as found in porcine and barley amylases, Lactobacillus amylases contain in addition to the active site, a soluble carbohydrate (substrate or product) binding site.
The activity of barley α-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase.
Juge, N., Nøhr, J., Le Gal-Coëffet, M. F., Kramhøft, B., Furniss, C. S., Planchot, V., Archer, D. B., Willianson, G. & Svensson, B. (2006). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1764(2), 275-284.
High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley α-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg × L-1 and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1–5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U × nmol-1 and Kd = 0.13 mg × mL-1, respectively, were both improved with respect to the values 0.015 U × nmol-1 and 0.67 mg × mL-1 for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.
Impact of formulation and technological factors on the acrylamide content of wheat bread and bread rolls.
Claus, A., Mongili, M., Weisz, G., Schieber, A. & Carle, R. (2008). Journal of Cereal Science, 47(3), 546-554.
This study clearly demonstrates that formulation and baking technology have strong influence on the acrylamide content in the baked products. NaCl plays an ambiguous role: Whereas low doses up to 2% lowered acrylamide by inhibition of the enzyme activities, higher addition remarkably increased the contents due to growth inhibition of the yeast. The results of previous model studies concerning the influence of cysteine could be confirmed in pilot plant experiments. Its addition to the dough resulted in significantly lower acrylamide content whereas its application to the crust proved to be ineffective. Furthermore, it was demonstrated that enzyme-bearing bakery improvers had no influence on acrylamide formation. In pilot plant experiments acrylamide was reduced with increasing fermentation time, and minimum acrylamide levels were already reached after 60 min thus avoiding flattened breads due to prolonged amylase activity. Besides formulation and fermentation also process technology is crucial. As shown by our data, reduced baking temperature and prolonged heat treatment is favorable. Furthermore, convection ovens seem to enhance acrylamide formation compared to deck oven.
Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae.
Brain-Isasi, S., Álvarez-Lueje, A. & Higgins, T. J. V. (2017). Microbial Cell Factories, 16(1), 110.
Background: Phaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications. Results: A synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein. Conclusions: This is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.
Analysis of Nanobody–Epitope Interactions in Living Cells via Quantitative Protein Transport Assays.
Früholz, S. & Pimpl, P. (2017). Plant Protein Secretion, Methods in Molecular Biology, 1662, pp. 171-182, Humana Press, New York, NY.
Over the past few decades, quantitative protein transport analyses have been used to elucidate the sorting and transport of proteins in the endomembrane system of plants. Here, we have applied our knowledge about transport routes and the corresponding sorting signals to establish an in vivo system for testing specific interactions between soluble proteins. Here, we describe the use of quantitative protein transport assays in tobacco mesophyll protoplasts to test for interactions occurring between a GFP-binding nanobody and its GFP epitope. For this, we use a secreted GFP-tagged α-amylase as a reporter together with a vacuolar-targeted RFP-tagged nanobody. The interaction between these proteins is then revealed by a transport alteration of the secretory reporter due to the interaction-triggered attachment of the vacuolar sorting signal.