Deficiency of maize starch-branching enzyme i results in altered starch fine structure, decreased digestibility and reduced coleoptile growth during germination.
Xia, H., Yandeau-Nelson, M., Thompson, D. B. & Guiltinan, M. J. (2011). BMC Plant Biology, 11(1), 95-107.
Background: Two distinct starch branching enzyme (SBE) isoforms predate the divergence of monocots and dicots and have been conserved in plants since then. This strongly suggests that both SBEI and SBEII provide unique selective advantages to plants. However, no phenotype for the SBEI mutation, sbe1a, had been previously observed. To explore this incongruity the objective of the present work was to characterize functional and molecular phenotypes of both sbe1a and wild-type (Wt) in the W64A maize inbred line. Results: Endosperm starch granules from the sbe1a mutant were more resistant to digestion by pancreatic α-amylase, and the sbe1a mutant starch had an altered branching pattern for amylopectin and amylose. When kernels were germinated, the sbe1a mutant was associated with shorter coleoptile length and higher residual starch content, suggesting that less efficient starch utilization may have impaired growth during germination. Conclusions: The present report documents for the first time a molecular phenotype due to the absence of SBEI, and suggests strongly that it is associated with altered physiological function of the starch in vivo. We believe that these results provide a plausible rationale for the conservation of SBEI in plants in both monocots and dicots, as greater seedling vigor would provide an important survival advantage when resources are limited.
Rapid determination of enzyme purity by a microdialysis-based assay.
Richardson, S., Nilsson, G. S., Torto, N., Gorton, L. & Laurell, T. (1999). Analytical Communications, 36(5), 189-193.
Microdialysis was shown to be useful as a fast on-line sampling method for determining the purity of starch hydrolysing enzymes. The enzymes were characterised using their hydrolytic properties. β-Amylases and pullulanases from different sources and/or manufacturers were investigated, with maltose, maltoheptaose, pullulan, and potato amylopectin starch (PAP) as substrates. The hydrolysis products were sampled via an on-line microdialysis probe and determined in a high-performance anion-exchange chromatographic (HPAEC) system. Comparison between the expected (theoretical) hydrolysis products with those obtained in the experiments made it possible to determine impurities in the enzymes. Two of the β-amylases and one pullulanase released unwanted hydrolysis products, indicating trace impurities in the enzyme preparation. Microdialysis sampling allows on-line sampling and eliminates separate sample preparation and clean-up steps. On-line microdialysis coupled to a HPAEC system is therefore a fast and simple technique for analysing enzyme hydrolysates.
Enzyme-aided investigation of the substituent distribution in cationic potato amylopectin starch.
Richardson, S., Nilsson, G., Cohen, A., Momcilovic, D., Brinkmalm, G. & Gorton, L. (2003). Analytical Chemistry, 75(23), 6499-6508.
The distribution of substituents along the polymer chain in cationic potato amylopectin starch, modified in solution, granular slurry, or dry state, was investigated. The starch derivatives were successively hydrolyzed by different enzymes, followed by characterization of the hydrolysis products obtained by means of electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). ESI-MS and MALDI-MS were proved to be appropriate techniques for identification of the substituted hydrolysis products, for which there are no standard compounds available. No highly substituted oligomers were found in the hydrolysates, which was taken as an indication of a more or less homogeneous distribution of cationic groups in the amylopectin molecules. Furthermore, from the results obtained it was suggested that the enzymes cleave glucosidic linkages only between unsubstituted glucose units and, preferentially, linkages in sequences containing more than two adjacent unsubstituted units. The determination of the amount of unsubstituted glucose produced from every successive hydrolysis step revealed slight differences between the different starch samples with respect to the homogeneity of the substitution pattern. Among the three samples under investigation, starch cationized in solution was found to have the most and dry-cationized starch the least homogeneous distribution of substituents.
Residual amylopectin structures of amylase-treated wheat starch slurries reflect amylase mode of action.
Leman, P., Goesaert, H. & Delcour, J. A. (2009). Food Hydrocolloids, 23(1), 153-164.
Some amylases can delay bread staling and/or starch (amylopectin) retrogradation, but the molecular basis of this effect remains little understood. In order to increase our insight in these aspects of amylase functionality, several amylases were added in a pure wheat-starch-containing model system and subjected to a heating step corresponding to that in the baking phase in bread making. Next, the effects of the limited amylolytic degradation on the rapid visco analyser (RVA) rheological properties of starch were studied and the accompanying changes in the amylopectin molecular properties (such as chain length distribution) investigated. The different amylases clearly affected the molecular structure of amylopectin to a different extent, which could be related to their mode of action and the enzyme activity levels added. Bacillus subtilis and Aspergillus oryzae α-amylases had only a limited impact on the side chain distribution of the amylopectin molecules, presumably due to their preferential hydrolysis of internal chain segments and the low enzyme activity added in the RVA. In contrast, porcine pancreatic α-amylase and Bacillus stearothermophilus maltogenic α-amylase, both with higher degree of multiple attack and used at higher enzyme activity levels, had a marked influence on the amylopectin molecular structure. More in particular, under the test conditions, the maltogenic α-amylase reduced the average chain length of the outer chains by 50%. Presumably, this will affect amylopectin retrogradation to a large extent. The results contribute to a better understanding of amylase functionality in starchy foods.
Differences in structures of starch hydrolysates using saliva from different individuals.
Nantanga, K. K. M., Chan, E., Suleman, S., Bertoft, E. & Seetharaman, K. (2013). Starch‐Stärke, 65(7‐8), 709-713.
High salivary amylase activity is associated with improved glycemic homeostasis in humans. Therefore, high salivary amylase activity is associated with greater digestion of starch. However, it is unclear if the structures of the hydrolysates from different individuals with different salivary amylase activity are the same. To test this, cooked starch (1:2 starch/water ratio) was treated with saliva from six participants at equal activity and conditions mimicking oral digestion. Salivary amylase activities ranged from 470 × 103 to 118 × 103 U/mL among the participants. The composition of the hydrolysates was characterised by gel-permeation chromatography. All samples gave rise to different and complex mixtures of hydrolysates with different breakdown structures. While saliva from participant 2 (high amylase activity) greatly reduced the high MW fraction, the saliva from participant 6 (low amylase activity) more extensively hydrolysed the starch to small MW fractions of oligosaccharides. These results show that different starch hydrolysates are produced during oral digestion by saliva from different individuals. Further research is therefore needed to understand if hydrolysate structure, rather than level of amylase activity, impacts glucose homeostasis.
Structures of human salivary amylase hydrolysates from starch processed at two water concentrations.
Nantanga, K. K. M., Bertoft, E. & Seetharaman, K. (2013). Starch‐Stärke, 65(7‐8), 637-644.
Digestion of starch in humans starts in the mouth and progresses to the small intestine. A thorough understanding of the progression of digestion, of consequence to glycemic and possibly insulinemic responses, requires a better characterization of the digestion products along the gut – products that are the substrates in the subsequent hydrolysis by sucrase-isomaltase and maltase-glucoamylase. This submission focuses on the first step of digestion, i.e., impact of human salivary amylase on the structure of hydrolysis products obtained from cooked starch. Starch was cooked at 1:0.7 (T0.7) or 1:2 (T2) starch:water ratios. To remove the effect of granular structure, starch was also dispersed using DMSO (TD) prior to amylase treatment. Cooked and dispersed starches were subjected to salivary amylase at conditions mimicking oral digestion. All samples gave rise to different and complex mixtures of hydrolysates with broad size-distributions as measured by gel-permeation chromatography (GPC). Following hydrolysis, the smallest dextrins (DP <30) constituted 35% in TD and only ∼20% in both T0.7 and T2. Cooking appeared to protect amylose molecules from hydrolysis with less hydrolysis in T0.7. These results show that the amount of water present during processing of starch affects structures of salivary amylase hydrolysates, which potentially impact on glucose homeostasis.
Debranching of β-dextrins to explore branching patterns of amylopectins from three maize genotypes.
Xia, H. & Thompson, D. B. (2006). Cereal Chemistry, 83(6), 668-676.
The amylopectin (AP) branching pattern is a fundamental feature of AP fine structure but a little-studied one. In this work, we followed enzyme digestion over time for AP from three maize genotypes (wx, du wx, and AP of ae VII). The objective was to describe differences in the progress of β-amylolysis and in subsequent debranching of β-limit dextrins (β-LD). During the progress of β-amylolysis, changes in the distribution of short residual chains show that the enzyme favors hydrolysis farthest from branch points. On treating β-LD with isoamylase (IA) alone, debranching was incomplete. Using IA and pullulanase (PUL) sequentially, a similar increase in the DP 5–7 region and the peak at DP 6 were observed for all samples, indicating a common element in the branching pattern. This similarity suggests that, despite differences in the proportion of short to long B chains, the most closely associated branch points may be arranged in a similar way for these AP. We suggest that the increase in DP 6 after PUL digestion would result from debranching of linear DP 6 residual B chains that originally had two branch points, consistent with interior segment length (ISL) of 1 or 2.
Effects of Chemical and Enzymatic Modifications on Starch–Oleic Acid Complex Formation.
Arijaje, E. O. & Wang, Y. J. (2015). Journal of Agricultural and Food Chemistry, 63(16), 4202-4210.
The solubility of starch-inclusion complexes affects the digestibility and bioavailability of the included molecules. Acetylation with two degrees of substitution, 0.041 (low) and 0.091 (high), combined without or with a β-amylase treatment was employed to improve the yield and solubility of the inclusion complex between debranched potato starch and oleic acid. Both soluble and insoluble complexes were recovered and analyzed for their degree of acetylation, complexation yields, molecular size distributions, X-ray diffraction patterns, and thermal properties. Acetylation significantly increased the amount of recovered soluble complexes as well as the complexed oleic acid in both soluble and insoluble complexes. High-acetylated debranched-only starch complexed the highest amount of oleic acid (38.0 mg/g) in the soluble complexes; low-acetylated starch with or without the β-amylase treatment resulted in the highest complexed oleic acid in the insoluble complexes (37.6–42.9 mg/g). All acetylated starches displayed the V-type X-ray pattern, and the melting temperature generally decreased with acetylation. The results indicate that starch acetylation with or without the β-amylase treatment can improve the formation and solubility of the starch–oleic acid complex.
Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase.
Zhang, B., Zhang, L., Li, F., Zhang, D., Liu, X., Wang, H., Xu, Z., Chu, C. & Zhou, Y. (2017). Nature Plants, 3, 17017.
O-acetylation, a ubiquitous modification of cell wall polymers, has striking impacts on plant growth and biomass utilization and needs to be tightly controlled. However, the mechanisms that underpin the control of cell wall acetylation remain elusive. Here, we show a rice brittle leaf sheath1 (bs1) mutant, which contains a lesion in a Golgi-localized GDSL esterase that deacetylates the prominent hemicellulose xylan. Cell wall composition, detailed xylan structure characterization and enzyme kinetics and activity assays on acetylated sugars and xylooligosaccharides demonstrate that BS1 is an esterase that cleaves acetyl moieties from the xylan backbone at O-2 and O--3 positions of xylopyranosyl residues. BS1 thus plays an important role in the maintenance of proper acetylation level on the xylan backbone, which is crucial for secondary wall formation and patterning. Our findings outline a mechanism for how plants modulate wall acetylation and endow a plethora of uncharacterized GDSL esterases with surmisable activities.
Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways.
Vavitsas, K., Rue, E. Ø., Stefánsdóttir, L. K., Gnanasekaran, T., Blennow, A., Crocoll, C., Gudmundsson, S. & Jensen, P. E. (2017). Microbial cell factories, 16(1), 140.
Background: There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. Results: In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. Conclusions: There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to ‘traditional’ metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.