Temperature impacts the multiple attack action of amylases

The action pattern of several amylases was studied at 35, 50, and 70 degrees C using potato amylose, a soluble (Red Starch) and insoluble (cross-linked amylose) chromophoric substrate. With potato amylose as substrate, Bacillus stearothermophilus alpha-amylase (BStA) and porcine pancreatic alpha-amylase displayed a high degree of multiple attack (DMA, i.e., the number of bonds broken during the lifetime of an enzyme-substrate complex minus one), the fungal alpha-amylase from Aspergillus oryzae a low DMA, and the alpha-amylases from B. licheniformis, Thermoactinomyces vulgaris, B. amyloliquifaciens, and B. subtilis an intermediate DMA. These data are discussed in relation to structural properties of the enzymes. The level of multiple attack (LMA), based on the relation between the drop in iodine binding of amylose and the increase in total reducing value, proved to be a good alternative for DMA measurements. The LMA of the endo-amylases increased with temperature to a degree depending on the amylase. In contrast, BStA showed a decreased LMA when temperature was raised. Furthermore, different enzymes had different activities on Red Starch and cross-linked amylose. Hence, next to the temperature, the action pattern of alpha-amylases is influenced by structural parameters of the starch substrate.     

Hydrolysis of amylopectin by amylolytic enzymes: structural analysis of the residual amylopectin population

Amylopectin fine structures were studied following limited hydrolysis of gelatinised waxy maize starch by amylases with a different level of inner chain attack (LICA). This was done by size exclusion chromatography as well as by debranching the (partially hydrolysed) amylopectin samples and studying the size distributions of the released chains. α-Amylases from Bacillus amyloliquefaciens and Aspergillus oryzae, with a relatively high LICA, drastically altered amylopectin chain length distribution and reduced the amylopectin molecular size (MS) significantly even at a low to moderate degree of hydrolysis (DH). Porcine pancreatic α-amylase (PPA), with a rather low LICA but a high multiple attack action on amylose, reduced the amylopectin MS much slower. Following hydrolysis by PPA to a DH of 10% and enzymic debranching of the amylopectin residue, several subpopulations of chains consisting of 2-12 glucose units were detected, indicating a multiple attack action on the amylopectin side chains. During the early stages of hydrolysis, the maltogenic Bacillus stearothermophilus α-amylase (BStA) preferentially hydrolysed the exterior chains of amylopectin. However, during the later phases, BStA also hydrolysed inner chains, presumably with a high multiple attack action. The present results clearly show that different enzymes can be used for (limited) conversion of amylopectin into structures differing in molecular weight and chain length distributions.     

Amylase action pattern on starch polymers

Several decades ago, the first reports on differences in action pattern between amylases from different sources indicated that the starch polymers are not degraded in a completely random manner. We here give an overview of different action patterns of amylases on amylose and amylopectin, focusing on the so-called multiple attack action of the enzymes. Nowadays, the multiple attack action is generally an accepted concept to explain the differences in amylase action pattern. However, the pancreatic α-amylase remains one of the few enzymes known with a considerable level of multiple attack action. Despite some recent studies, the molecular mechanism of the multiple attack action is still largely unclear. Probably, the degree to which the active site architecture and binding properties allow both the reorganization (sliding) of the substrate in the active site and the stabilisation of the productive enzyme/substrate complex mainly determine the multiple attack action of amylases.     

Functional groups in the social behavior of a cichlid fish,the Oscar,Astronotus ocellatus

Dummy conspecifics were presented to isolated adults of the cichlid fish Astronotus ocellatus to investigate the functional organization of cichlid social behavior. Body size and 15 dummy-elicited activities were recorded during 15 min sessions and analyzed by principal components analysis (PCA) to reveal their temporal organization. Five principal components explained almost 80% of the variation in dummy-elicited behavior, and these five factors define functional groups for

• 1.(a) investigation,
• 2.(b) attack,
• 3.(c) nesting,
• 4.(d) boldness,
• 5.(e)distress.

Nest-oriented and attack modal action patterns are not mutually inhibitory during this time frame, and biting does not appear to function exclusively during an attack on a conspecific. Comparison with previous studies of New and Old World cichlids suggests evolutionary conservation of the functional organization of social behavior.     

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.     

Amylose–dye complexes in cationic micelles: an optical spectroscopy study

The formation of amylose complexes with rose bengal (RB), erythrosine B (ER), and phenolphthalein (PP) in the presence of the cationic detergent tetradecyltrimethylammonium bromide (TTABr) was studied using optical spectroscopy methods. Absorption spectroscopy, steady-state fluorescence spectroscopy and picosecond time-resolved fluorescence spectroscopy were used to derive association constants k_s of the dyes, critical micelle concentration (CMC) values and structural information on the complexes formed. It seems that PP fits very well into amylose sites, where it forms an efficient inclusion complex with k_s=44,500 M⁻¹. The molecular diameter of RB is too big to fit the amylose cavity. Only part of the xanthene unit may be adopted in the helical cavity of amylose, whereas most of the interaction occurs through electrostatic and/or dipole–dipole interactions with the amylose chain. The ER molecule is an intermediate case, because it may fit the amylose cavity or adsorb on the amylose surface to form a complex. The presence of a surfactant in the amylose–ligand system increases the association constant for all dyes. In the presence of amylose, a decrease of the detergent CMC value of about one order of magnitude is observed. It is probable that the increased number of micelles incorporate more dyes into the amylose vicinity, which finally changes the structure of the amylose chain. On a macro scale, it was noted that the samples with dyes and detergent have a lower tendency to precipitate and the gelation process is delayed compared to that in water.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.     

Degradation of pruning wastes by Phanerochaete sordida growing in SSF: Ultrastructural,chemical, and enzymatic studies

A solid standard fermentation (SSF) with the fungus Phanerochaete sordida in a medium with Nephrolepis cordifolia (entire pinnae separated from the rachis) and Laurus nobilis (fragmented leaves) was performed over 92 days to study the degradation of leaves with histological, chemical, and enzymatic methods. The fungus entered the leaves early, through the stomata in N. cordifolia and L. nobilis, and also through mechanical cuts that had been made in the latter. The initial attack affected the mesophyll in both plant species, and the phloem in L. nobilis. The vascular bundle of N. cordifolia was protected by a sheath of cells with thick lignified walls. The collenchyma cell walls situated near the principal vein in L. nobilis swelled during the initial stages of enzymatic action, but reduced their thickness afterwards, mainly in regions of contact with the hyphae. At the end of the experiment, no species had leaves with mesophyll. In L. nobilis, phloem was also lacking, and a partial and heterogeneous attack on the xylem became evident. The histological changes are compared with the enzymatic activities and the chemical composition of the culture media, describing the stages of fungal colonization.     

Barley alpha-amylase Met53 situated at the high-affinity subsite -2 belongs to a substrate binding motif in the beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and is critical for activity and substrate specificity.

Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amylases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta-->alpha loop 2 of the catalytic (beta/alpha)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild-type, respectively. Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-d-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5. Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation - promoted probably by slow substrate hydrolysis - to produce up to maltoundecaose from PNPG6.     

Enhanced emissions of floral volatiles by Diplotaxis erucoides (L.) in response to folivory and florivory by Pieris brassicae (L.)

The main function of floral emissions of volatile organic compounds (VOCs) in entomophilous plants is to attract pollinators. Floral blends, however, can also contain volatile compounds with defensive functions. These defensive volatiles are specifically emitted when plants are attacked by pathogens or herbivores. We characterized the changes in the floral emissions of Diplotaxis erucoides induced by folivory and florivory by Pieris brassicae. Plants were continually subjected to folivory, florivory and folivory + florivory treatments for two days. We measured floral emissions with proton transfer reaction/mass spectroscopy (PTR-MS) at different times during the application of the treatments. The emissions of methanol, ethyl acetate and another compound, likely 3-butenenitrile, increased significantly in response to florivory. Methanol and 3-butenenitrile increased 2.4- and 26-fold, respectively, in response to the florivory treatment. Methanol, 3-butenenitrile and ethyl acetate increased 3-, 100- and 9-fold, respectively, in response to the folivory + florivory treatment. Folivory alone had no detectable effect on floral emissions. All VOC emissions began immediately after attack, with no evidence of delayed induction in any of the treatments. Folivory and florivory had a synergistic effect when applied together, which strengthened the defensive response when the attack was extended to the entire plant.     

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.     

Monte Carlo simulation of multiple attack mechanism of α-amylase

Porcine pancreatic α-amylase (EC 3.2.1.1) produces short maltooligosaccharides from a single enzyme-substrate complex without dissociation by multiple or repetitive attack. Multiple attack is caused by relative sliding of the enzyme along the product chain of the enzyme-product complex without dissociation to form another productive complex. The Monte Carlo method was applied to the multiple attack mechanism to predict product distribution from amylose and amylopectin molecules of arbitrary sizes. The position of the initial attack to make the enzyme-substrate complex and branched reaction paths from the enzyme-product complex were selected by random numbers and probabilities. A simulated product distribution from 100,000 samples of amylose of chain length greater than 80 agreed completely with experimental data at the early stage of hydrolysis of amylose of mean chain length 90. On the other hand, the simulated product distribution from amylopectin agreed with experimental data of potato amylopectin when the effective chain length of the A chain was 9. Since the mean chain length of the A chain of potato amylopectin is 15, it is possible that amylopectin is partially compact in solution, so that the enzyme can recognize and act only on the outer side of the A chain at the early stage of digestion. © 1996 John Wiley & Sons, Inc.     

Coupling of polysaccharides activated by means of chloroacetaldehyde dimethyl acetal to amines or proteins by reductive amination

A novel method has been developed for the coupling of modified polysaccharides to proteins or other amines. Chloroacetaldehyde dimethyl acetal has been used for the introduction of O-(2,2-dimethoxyethyl) groups into amylose, dextran, and a linear (1→3)-β-d-glucan. In amylose and the (1→3)-β-d-glucan, these groups were attached preponderantly at O-6 and in dextran at O-2. Mild treatment with acid then gave polysaccharide derivatives substituted with aldehyde groups which were coupled in good yields to proteins and other amines by reductive amination with sodium cyanoborohydride in aqueous solution at pH 7. An aminated (1→3)-β-d-glucan derivative that induced antitumor activity in mouse macrophages in vitro is reported.     

Influence of anomeric configuration on mechanochemical degradation of polysaccharides: cellulose versus amylose

Cellulose and amylose are (1-->4)-linked polysaccharides that are used extensively in the textiles, paper, and food and feed industries and are finding increasing use as alternative fuels and so forth. At the molecular level, cellulose and amylose differ only in their anomeric configuration: beta in cellulose, alpha in amylose. During processing and end use, these polymers experience a variety of mechanochemical stresses, many through contact with transient elongational flow fields. Here, we subject solutions of both polysaccharides to extended periods of ultrasonic irradiation, as the cavitational bubble collapse characteristic of ultrasound experiments creates flow fields strictly analogous to those encountered in other transient elongational flow scenarios. With the use of multidetector size-exclusion chromatography, the effects of anomeric configuration on both the limiting molar mass, beyond which polymers do not degrade in transient elongation flow ( M lim), and the rate of degradation have been isolated in these (1-->4)-linked polysaccharides. This effect was found to be pronounced; for example, M lim (cellulose) = 5( M lim (amylose)). Also, while extensive change was observed in molar mass averages, distribution, polydispersity, and size of the analytes during degradation, their structure was found to remain invariant. A modified "path theory" of transient elongational flow degradation was proposed, with the persistence length identified as a parameter which embodies the minimum continuous path length and flexibility requirements of the theory.     

A model for starch breakdown in higher plants

An in vitro system for the breakdown of starch granules by mixtures of α- and β-amylase is developed and discussed with reference to information concerning the degradation of starch in vivo. β-Amylase has no action on starch granules and has very little effect on the rate of starch granule digestion by α-amylase. It does, however, affect the product distribution in an α-amylase digest and is considered to attack dextrin intermediates produced by the action of α-amylase on the starch granules.     

Assessment of quantitative techniques in paleobiogeography

A series of multivariate methods has been compared to assess their effectiveness in extracting essential information out of a complex micropaleontological data-set. The data-set used for this experiment consists of relative frequencies (percentages) of Miocene coccolith taxa or groups of taxa in cores of the Deep Sea Drilling Project (DSDP) from the Atlantic Ocean. All methods tested are varieties of principal components analysis in R- and Q-mode, and “true” factor analysis. Various secondary rotational procedures ancillary to some of these methods are also tested.A test, denoted Δ-Test, is developed, which assesses how well principal components or factors reproduce the data-set. Δ-Test may be used for determining the optimum number of principal components or factors, the most relevant rotational procedure, and thus the most suitable analytical technique. The Δ-Test does not rely on mathematical testing, but on simple inspection of the compositions of the principal components or factors, and their relations to correlations existing in the data-set.Our experiment reveals that the most efficient methods are the maximum-likelihood factor analysis and the R-mode principal components analysis, within which the varimax (orthogonal) rotations best reproduce correlations. Of these methods, maximum-likelihood factor analysis is considered the optimum method, because of the greater simplicity of compositions. In addition to these methods, Kaiser's second generation “Little Jiffy” factor analysis was also found to be efficient. Three methods provide less sensitive reduction of the data: the “true” R-mode principal components analysis (without secondary rotations), the Q-mode principal components analysis, and the correspondence analysis.     

Metabolism of the reserve polysaccharide of Streptococcus mitis: Properties of a transglucosylase

1. A transglucosylase has been separated from cell extracts of Streptococcus mitis, and has been partially purified by chromatography on DEAE-cellulose. 2. The transglucosylase was present in the six strains of Streptococcus mitis that were examined, and the activity of the enzyme was the same whether the cells had grown on glucose or on maltose. Four of the strains could store intracellular iodophilic polysaccharide when grown on high concentrations of glucose or maltose (1%), but none of the strains stored polysaccharide during growth on 0·1% glucose. The activity of transglucosylase in cell extracts was the same whether or not the cells had stored polysaccharide. 3. The transglucosylase degrades amylose in the presence of a suitable acceptor, transferring one or more glucosyl residues from the non-reducing end of the donor to the non-reducing end of the acceptor. With [¹⁴C]glucose as acceptor the maltodextrins produced were labelled in the reducing glucose unit only. 4. The enzyme can synthesize higher maltodextrins from maltose and maltotriose. Maltotetraose is disproportionated to give products of sufficient chain length to give a stain with iodine. 5. The action pattern of S. mitis during the degradation of synthetic amylose was shown to be intermediate between the single-chain and multi-chain mechanism.     

Cryptochrome, Phytochrome, and the Photoregulation of Anthocyanin Production under Blue Light

The principle of equivalent light action predicts that two light treatments (wavelengths ^λ₁ and λ₂) producing the same Pfr/P ratio (_λ1 = _λ2) and the same rate of phytochrome photoconversion (k_λ1 = k_λ2) are perceived by phytochrome as being the same and should produce the same effect. The results of experiments based on the principle of equivalent light action indicate that cryptochrome is involved in the photoregulation of anthocyanin production elicited by blue light in tomato seedlings. This was also the case for one strain of cabbage seedlings. For another strain of cabbage seedlings, the results suggest that cryptochrome is either not involved or that the state of phytochrome is the principal limiting factor.     

The variability of different larch clone provenances on the response to the attack by its main pests and fungal diseases

A wide range of pests and diseases attacks larch, and over 3 years in an orchard in north-western Romania, the most frequently occurring were larch needle disease (Hypodermella laricis), the case-bearer (Coleophora laricella) and woolly adelgid (Adelges laricis) insects. Their attack on seven larch clones obtained through selection from different geographic populations from Romania was studied. To evaluate the attack intensity, four branches per tree were analysed in the upper, middle and lower third of the crowns in all cardinal directions. According to the position in the crown, a high attack intensity by H. laricis was recorded on the branches in the north direction. A high intensity of C. laricella attack was registered on the upper third of the crown, especially in the east, west and south directions. A positive relationship was identified between the damaged sprouts caused by the C. laricella and A. laricis attack. The differences among the reaction of the clones and the heritability coefficients illustrated that the response to the attacks had a strong genetic determinism, with resistance being clearly influenced by genotype. In addition to the identification of gene sources that are useful for larch breeding, this study offers technical information about the potential to develop efficient treatments based on the attacks on the tree crowns.