Amyloglucosidase (Rhizopus sp.)

A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and β-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of α-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing β-glucan. Pure β-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of β-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 β-glucanase and A. niger β-glucosidase. β-glucosidase from almonds does not completely hydrolyze mixed linkage β-glucooligosaccharides from barley or oat β-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than β-glucan, and thus an overestimation of β-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and α- and β-glucosidase.

Dietary fiber is mainly composed of plant cell wall polysaccharides such as cellulose, hemicellulose, and pectic substances, but it also includes lignin and other minor components (1). Basically, it covers the polysaccharides that are not hydrolyzed by the endogenous secretions of the human digestive tract (2,3). This definition has served as the target for those developing analytical procedures for the measurement of dietary fiber for quality control and regulatory considerations (4). Most procedures for the measurement of total dietary fiber (TDF), or specific polysaccharide components, either involve some enzyme treatment steps or are mainly enzyme-based. In the development of TDF procedures such as the Prosky method (AOAC International 985.29, AACC 32—05) (5), the Uppsala method (AACC32-25) (6), and the Englyst method (7), the aim was to remove starch and protein through enzyme treatment, and to measure the residue as dietary fiber (after allowing for residual, undigested protein and ash). Dietary fiber was measured either gravimetrically or by chemical or instrumental procedures. Many of the enzyme treatment steps in each of the methods, particularly the prosky (5) and the Uppsala (6) methods are very similar. As a new range of carbohydrates is being introduced as potential dietary fiber components, the original assay procedures will need to be reexamined, and in some cases slightly modified, to ensure accurate and quantitative measurement of these components and of TDF. These “new” dietary fiber components include resistant nondigestible oligosaccharides; native and chemically modified polysaccharides of plant and algal origin (galactomannan, chemically modified celluloses, and agars and carrageenans); and resistant starch. To measure these components accurately, the purity, activity, and specificity of the enzymes employed will become much more important. A particular example of this is the mesurement of fructan. This carbohydrate consists of a fraction with a high degree of polymerization (DP) that is precipitated in the standard Prosky method (5,8) and a low DP fraction consequently is not measured (9). Resistant starch poses a particular problem. This component is only partially resistant to degradation by α-amylase, so the level of enzyme used and the incubation conditions (time and temperature) are critical.

An enzyme-linked assay for the measurement of amyloglucosidase in commercial enzyme mixtures and crude culture filtrates is described. A method for the synthesis of the substrate employed, p-nitrophenyl β-D-maltoside, is also described. The substrate is used in the presence of saturating levels of β-glucosidase. With a range of Aspergillus sp. culture filtrates, an excellent correlation was found for values obtained with this assay and a conventional assay employing maltose as substrate with measurement of released glucose.

Culture filtrates of Cladosporium resinae ATCC 20495 contain a mixture of enzymes able to convert starch and pullulan efficiently into D-glucose. Culture conditions for optimal production of the pullulan-degrading activity have been established. The amylolytic enzyme preparation was fractionated by ion-exchange and molecular-sieve chromatography, and shown to contain α-D-glucosidase, α-amylase, and two glucoamylases. The glucoamylases have been purified to homogeneity and their substrate specificities investigated. One of the glucoamylases (termed P) readily hydrolyses the (1→6)-α-D linkages in pullulan, amylopectin, isomaltose, panose, and 6³-α-D-glucosylmaltotriose. Each of the glucoamylases cleaves the (1→6)-α-D linkage in panose much more readily than that in isomaltose.

Carbon allocation between source and sink organs determines plant growth and is influenced by environmental conditions. Under water deficit, plant growth is inhibited before photosynthesis and shoot growth tends to be more sensitive than root growth. However, the modulation of source-sink relationship by rootstocks remain unsolved in citrus trees under water deficit. Citrus plants grafted on Rangpur lime are drought tolerant, which may be related to a fine coordination of the source-sink relationship for maintaining root growth. Here, we followed ¹³C allocation and evaluated physiological responses and growth of Valencia orange trees grafted on three citrus rootstocks (Rangpur lime, Swingle citrumelo and Sunki mandarin) under water deficit. As compared to plants on Swingle and Sunki rootstocks, ones grafted on Rangpur lime showed higher stomatal sensitivity to the initial variation of water availability and less accumulation of non-structural carbohydrates in roots under water deficit. High ¹³C allocation found in Rangpur lime roots indicates this rootstock has high sink demand associated with high root growth under water deficit. Our data suggest that Rangpur lime rootstock used photoassimilates as sources of energy and carbon skeletons for growing under drought, which is likely related to increases in root respiration. Taken together, our data revealed that carbon supply by leaves and delivery to roots are critical for maintaining root growth and improving drought tolerance, with citrus rootstocks showing differential sink strength under water deficit.

Potato cell walls (PCW) are a low value by-product from the potato starch industry. Valorisation of PCW is hindered by its high water-binding capacity (WBC). The composition of polysaccharides and interactions between these entities, play important roles in regulating the WBC in the cell wall matrix. Here, we show that in vivo exo-truncation of RG-I β-(1→4)-D-galactan side-chains decreased the WBC by 6-9%. In contrast, exo-truncation of these side-chains increased the WBC by 13% in vitro. We propose that degradation of RG-I galactan side-chains altered the WBC of PCW, due to cell wall remodelling and loosening that affected the porosity. Our findings reinforce the view that RG-I galactan side-chains play a role in modulating WBC, presumably by affecting polysaccharide architecture (spacing) and interactions in the matrix. Better understanding of structure-function relationships of pectin macromolecules is needed before cell wall by-products may be tailored to render added-value in food and biobased products.

Instead of β-cyclodextrin (β-CD), branched β-CDs have been increasingly used in many aspects as they possess better solubility and higher bioadaptability. But most commercialized branched β-CDs were chemically synthesized. Thus, the glucosyl-β-cyclodextrin (G₁-β-CD) prepared via enzymatic approach could be a nice substitute. However, the yield of G₁-β-CD was low. Here, we reported a controlled two-step reaction to efficiently prepare G₁-β-CD from maltodextrins by β-cyclodextrin glucosyltransferase (β-CGTase) and amyloglucosidase (AG). Compared to the single β-CGTase reaction, controlled two-step reaction caused a yield increase of G₁-β-CD by 130%. Additionally, the percentage of G₁-β-CD was enhanced from 2.4% to 24.0% and the side products α-CD and γ-CD were hydrolyzed because of the coupling activity of β-CGTase. Thus, this controlled two-step reaction might be an efficient approach for industrial production of pure G₁-β-CD.

In comparison with natural α- and β-cyclodextrin (CD), γ-CD has attracted much attention due to their large hydrophobic cavities, high water solubility, and bioavailability. However, the production of γ-CD is still rather expensive and time-consuming. To overcome the high cost and long induction time, pUC119 was selected as the gene expression vector, and the recombinant enzyme production time was reduced to 8 h from 72 h. Furthermore, for the first time, we have successfully produced γ-CD using β-CD by cyclodextrin opening reactions through the recombinant CGTase in the presence of maltose. The kinetic mechanism of the coupling reaction was investigated. Moreover, the production of γ-CD could be affected by several key parameters, such as solvent type, reaction time, pH, and temperature. A maximum γ-CD yield of 32.9% was achieved by recombinant CGTase in the presence of 5-cyclohexadecen-1-one. This could be a promising method for the industrial production of γ-CD.

Tree species from the Amazonian floodplains have to cope with low oxygen availability due to annual pulses of inundation that can last up to 7 months. Species capable of adapting to flooding and/or waterlogged conditions usually partition their storage to favor starch and allocate it to roots, where carbohydrates are used to maintain respiration rates during waterlogging. In spite of climate change, virtually nothing is known about how elevated atmospheric CO₂ concentration ([CO₂]) will affect plants when combined with waterlogging. In this work, we used open top chambers to evaluate the effect of elevated [CO₂] during a period of terrestrial phase and in subsequent combination with waterlogged conditions to determine if the surplus carbon provided by elevated [CO₂] may improve the waterlogging tolerance of the fast-growing Amazonian legume tree Senna reticulata. During the terrestrial phase, photosynthesis was ca. 28 % higher after 30, 45 and 120 days of elevated [CO₂], and starch content in the leaves was, on average, 49 % higher than with ambient [CO₂]. Total biomass was inversely correlated to the starch content of leaves, indicating that starch might be the main carbohydrate source for biomass production during the terrestrial phase. This response was more pronounced under elevated [CO₂], resulting in 30 % more biomass in comparison to ambient [CO₂] plants. After 135 days at elevated [CO₂] an inversion has been observed in total biomass accumulation, in which ambient [CO₂] presented a greater increment in total biomass in comparison to elevated [CO₂], indicating negative effects on growth after long-term CO₂ exposure. However, plants with elevated [CO₂]/waterlogged displayed a greater increment in biomass in comparison with ambient [CO₂]/waterlogged that, unlike during the terrestrial phase, was unrelated to starch reserves. We conclude that S. reticulata displays mechanisms that make this species capable of responding positively to elevated [CO₂] during the first pulse of growth. This response capacity is also associated with a “buffering effect” that prevents the plants from decreasing their biomass under waterlogged conditions.

Starch synthesis requires the formation of a primer that can be subsequently elongated and branched. How this primer is produced, however, remains unknown. The control of the number of starch granules produced per chloroplast is also a matter of debate. We previously showed starch synthase 4 (SS4) to be involved in both processes, although the mechanisms involved are yet to be fully characterised. The present work shows that SS4 displays a specific localization different from other starch synthases. Thus, this protein is located in specific areas of the thylakoid membrane and interacts with the proteins fibrillin 1a (FBN1a) and 1b (FBN1b), which are mainly located in plastoglobules. SS4 would seem to be associated with plastoglobules attached to the thylakoids (or to that portion of the thylakoids where plastoglobules have originated), forming a complex that includes the FBN1s and other as-yet unidentified proteins. The present results also indicate that the localization pattern of SS4, and its interactions with the FBN1 proteins, are mediated through its N-terminal region, which contains two long coiled-coil motifs. The localization of SS4 in specific areas of the thylakoid membrane suggests that starch granules are originated at specific regions of the chloroplast.

The dietary fibre (DF) content in wheat grain based food products have been established with both the classical AOAC 985.29 dietary fibre and the new AOAC 2009.01 total dietary fibre protocol. There is a good agreement between the high molecular weight dietary fibre (HMWDF) contents measured with the AOAC 2009.01 method and (DF) content measured with the classical AOAC 985.29 method in wheat grain based food products. With the AOAC 2009.01 method also a significant amount of low molar weight dietary fibre (LMWDF), ranging from 1% to 3% w/w, was measured which is not quantified with the AOAC 985.29 method. With semi-preparative GPC the LMWDF (DP ≥ 3) fractions in the wheat grain based food products were isolated. The monosaccharide composition of the dissolved LMWDF constituents was determined. Glucose was by far the most abundant monosaccharide present with arabinose, galactose, xylose and mannose as minor constituents. It appeared that the LMWDF contains still not fully converted digestible starch/malto-oligosaccharide fragments with DP ≥ 3, which are erroneously quantified as LMWDF. By introducing an extra AMG hydrolysis step in the AOAC 2009.01 protocol after evaporation of the ethanol and dissolving the residue in deionised water, these malto-oligosaccharides are fully hydrolysed resulting in that way in a correct and lower LMWDF content.

Crassulacean acid metabolism (CAM) is a physiological adaptation of plants that live in stress environment conditions. A good model of CAM modulation is the epiphytic bromeliad, Guzmania monostachia, which switches between two photosynthetic pathways (C₃–CAM) in response to different environmental conditions, such as light stress and water availability. Along the leaf length a gradient of acidity can be observed when G. Monostachia plants are kept under water deficiency. Previous studies showed that the apical portions of the leaves present higher expression of CAM, while the basal regions exhibit lower expression of this photosynthetic pathway. The present study has demonstrated that it is possible to induce the CAM pathway in detached leaves of G. monostachia kept under water deficit for 7 d. Also, it was evaluated whether CAM expression can be modulated in detached leaves of Guzmania and whether some spatial separation between NO₃^- reduction and CO₂ fixation occurs in basal and apical portions of the leaf. In addition, we analyzed the involvement of endogenous cytokinins (free and ribosylated forms) as possible signal modulating both NO₃^- reduction and CO₂ fixation along the leaf blade of this bromeliad. Besides demonstrating a clear spatial and functional separation of carbon and nitrogen metabolism along G. monostachia leaves, the results obtained also indicated a probable negative correlation between endogenous free cytokinins – zeatin (Z) and isopentenyladenine (iP) – concentration and PEPC activity in the apical portions of G. monostachia leaves kept under water deficit. On the other hand, a possible positive correlation between endogenous Z and iP levels and NR activity in basal portions of drought-exposed and control leaves was verified. Together with the observations presented above, results obtained with exogenous cytokinins treatments, strongly suggest that free cytokinins might act as a stimulatory signal involved in NR activity regulation and as a negative regulator of PEPC activity in CAM-induced leaves of G. monostachia during a diel cycle.

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO₃^-/NH₄⁺ or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary, GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH₄⁺ assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad's leaves.

An improved method for direct determination of available carbohydrates in low-level products has been developed and validated for a low-carbohydrate soy infant formula. The method involves modification of an existing direct determination method to improve specificity, accuracy, detection levels, and run times through a more extensive enzymatic digestion to capture all available (or potentially available) carbohydrates. The digestion hydrolyzes all common sugars, starch, and starch derivatives down to their monosaccharide components, glucose, fructose, and galactose, which are then quantitated by high-performance anion-exchange chromatography with photodiode array detection. Method validation consisted of specificity testing and 10 days of analyzing various spike levels of mixed sugars, maltodextrin, and corn starch. The overall RSD was 4.0 across all sample types, which contained within-day and day-to-day components of 3.6 and 3.4, respectively. Overall average recovery was 99.4 (n = 10). Average recovery for individual spiked samples ranged from 94.1 to 106 (n = 10). It is expected that the method could be applied to a variety of low-carbohydrate foods and beverages.

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.

The use of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) coupled on-line with electrospray mass spectrometry (ESI-MS) for analysis of the substitution pattern in chemically modified starch, has been investigated. In order to characterise the distribution of substitution groups along the polymer chain, hydroxypropylated potato amylopectin starch (HPPAP) was subjected to enzymic hydrolysis, followed by analysis of the degradation products by HPAEC-PAD-MS. When using conventional chromatographic techniques for characterisation of enzymic hydrolysates, standard compounds are required for identification of the hydrolysis products. However, the on-line coupling with ESI-MS allowed identification of all products obtained, substituted as well as unsubstituted, and also of those compounds that co-eluted, without the need for standards. Further, HPAEC-PAD-MS was shown to be useful for analysis of the substitution pattern in modified starch; from results obtained it was suggested that the hydroxypropyl groups were homogeneously distributed in the amylopectin molecule. It was also shown that the starch hydrolysing enzymes were hindered by the hydroxypropyl groups and preferentially cleaved glucosidic linkages between unsubstituted glucose units.

The distribution of substituents in hydroxypropylated potato amylopectin starch (amylose deficient) modified in a slurry of granular starch (HPPAPg) or in a polymer ‘solution’ of dissolved starch (HPPAPs), was investigated. The molar substitution (MS) was determined by three different methods: proton nuclear magnetic resonance (¹H NMR) spectroscopy, gas-liquid chromatography (GLC) with mass spectrometry, and a colourimetric method. The MS values obtained by ¹H NMR spectroscopy were higher than those obtained by GLC–mass spectrometry analysis and colourimetry. The relative ratio of 2-, 3-, and 6-substitution, as well as un-, mono-, and disubstitution in the anhydroglucose unit (AGU) were determined by GLC–mass spectrometry analysis. Results obtained showed no significant difference in molar distribution of hydroxypropyl groups in the AGU between the two derivatives. For analysis of the distribution pattern along the polymer chain, the starch derivatives were hydrolysed by enzymes with different selectivities. Debranching of the polymers indicated that more substituents were located in close vicinity to branching points in HPPAPg than in HPPAPs. Simultaneous α-amylase and amyloglucosidase hydrolysis of HPPAPg liberated more unsubstituted glucose units than the hydrolysis of HPPAPs, indicating a more heterogeneous distribution of substituents in HPPAPg.