Some Characteristics of β-D-Xylopyranosidases, α-L-Arabinofuranosidases and an Arabinoxylan α-L-arabinofuranohydrolase from Wheat Bran and Germinated Wheat

Enzymes from wheat bran and germinated wheat involved in the degradation of arabinoxylan and arabinoxylooligosaccharides were investigated. Fourp-nitrophenyl-α-l-arabinofuranoside hydrolysing activities (ArafI-IV) and threep-nitrophenyl-β-d-xylopyranoside hydrolysing activities (XylpI-III) were identified in wheat kernels and germinating wheat. Two of these activities, ArafI and XylpII, were purified about 10 000-fold from wheat bran. Both enzymes were inactive towards polymeric arabinoxylan. ArafI produced no arabinose but some xylose from arabinoxylooligosaccharides, while XylpII gave only xylose upon incubation with this substrate. An arabinoxylan arabinofuranohydrolase (AXH) was found in wheat bran and germinated wheat. This enzyme was active towards the polymeric substrate, but was unable to hydrolysep-nitrophenyl-α-l-arabinofuranoside. TheM_rs of these enzymes were determined by size exclusion chromatography and were in the range of 40–50 000, except for ArafIII, for which aM_rof 104 000 was determined. During germination, the levels of these enzymes increased markedly between the third and fifth day, after which some of them decreased again by the seventh day. ArafI-IV were inhibited strongly by arabinonic acid-γ-lactone, while xylonic acid-γ-lactone was a good inhibitor of XylpI-III. The latter lactone also inhibited ArafI. Neither of these lactones inhibited AXH. Endoxylanase activity was demonstrated but not quantified.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH₂), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 3₁₀-helix from residues Pro⁷ to His¹⁰ and a type I β-turn from residues Pro¹⁴ to Cys¹⁷. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC₅₀ of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu² by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser¹² with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.     

Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Two fraction extraction of α-zein from DDGS and its characterization

Zein was recovered from corn distiller's dried grains with solubles (DDGS) by a modified method using 70% (w/w) aqueous 2-propanol (70-IPA) or 70% (v/v) aqueous ethanol (70-EtOH) solvents, and a commercial method using 88% (w/w) aqueous 2-propanol (88-IPA). Yield, purity, and film properties of the isolated zein were determined. The modified procedure extracted two fractions of zeins: a mostly α-zein fraction, and a mostly γ-zein fraction. The modified method increased α-zein yield from 4% to 14%. Enzyme cellulase pretreatment did not improve zein yield, but grinding did. The α-zein fraction showed electrophoretic bands at 40, 22, 19, and 10 kDa, corresponding to α-zein dimer, α₁-zein, α₂-zein, and δ-zein, respectively. The α-zein of DDGS retained its film forming capability. The α-zein film of unmodified DDGS was cloudy and rough, unlike the clear and smooth films of α-zeins isolated from corn gluten meal and enzyme-treated DDGS.     

Reversed phase HPLC analysis of α- and β-carotene from selected raw and cooked vegetables

Traditional AOAC colorimetric procedures for carotenoid analysis are known to lack specificity and accuracy. Newer HPLC methods provide the investigator with a more precise tool for carotenoid quantification in foods and tissues. In the present studies, reverse phase HPLC was utilized to evaluate the - and -carotene content in raw and cooked leaves of lettuce, spinach and winged bean as well as in the carrot root. The vegetables were boiled or steamed and the true retention of - and -carotene in the cooked products was determined. Boiling for 30 minutes resulted in a 53 and 40% loss of -carotene from lettuce and carrots, respectively. Full retention or even an increase in -carotene content in boiled winged bean leaves and spinach was noted. Steaming resulted in very good retention of - and - carotene in all vegetables (83–139% retention). Thus, although cooking procedures (especially boiling) may result in oxidative loss of carotenoids in some vegetables, heat treatment increases the chemical extractability of - and -carotene in others. The presence of carotenoproteins in some vegetables may effect the heat stability of extractability of - and - carotene.     

Studies on α-amylase and trypsin inhibitors in legume seeds using agar diffusion and isoelectric focusing techniques

Amylolytic and tryptic inhibitors of faba bean extracts were determined by an agar diffusion test. The amylolytic inhibitor had protein characters. Furthermore, water-soluble trypsin inhibitors ofCicer arietinum, Lens esculenta, Lupinus termis, Phaseolus vulgaris, Pisum sativum, Trigonella foenum-graecum andVicia faba which were separated by polyacrylamide gel isoelectric focusing (PAGIF) in thin-layers, showed species specific patterns. Negative staining showed 10 bands for French beans, 9 for fenugreek seeds, 8 for lentils and chickpeas, 7 for peas and 6 for faba beans. Lupin seeds were free from trypsin inhibitors. Treatments (soaking, germination and heat processing) of faba beans reduced the number of trypsin inhibitors in PAGIF patterns, less after soaking and germination, but more after roasting and frying. No inhibitors were detected after cooking.     

Content of starch and sugars and in vitro digestion of starch by α-amylase in five minor millets

Five varieties of minor millets were studied for their amylose, soluble amylose, amylopectin, soluble amylopectin, reducing sugar, total sugar and starch contents. Pure starch was isolated from each variety and the enzymic degradation of starch by porcine pancreatic -amylase were examined with and without gelatinisation. Gelatinised sample ofEchinochloa frumentacea (var. K2) showed minimal hydrolysis and gelatinised sample ofPanicum miliaceum (var. CO3) showed maximum hydrolysis of starch by porcine pancreatic -amylase. Gelatinised starch was highly susceptible to enzymic digestion when compared to ungelatinised starch. The extent of starch degradation varied from 71 to 85 percent in gelatinised samples and starch degradation in ungelatinised sample varied from 10 to 18 percent.     

Antibodies to soluble liver antigen and α-enolase in patients with autoimmune hepatitis

BACKGROUND: Antibodies to a cytosolic soluble liver antigen (SLA) are specifically detected in patients with autoimmune hepatitis (AIH). The target of anti-SLA has been identified as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP(Ser)Sec), through the screening of cDNA libraries. A recent report questioned the identity of tRNP(Ser)Sec as the real SLA antigen. The latter study identified alpha-enolase as a major anti-SLA target, through proteomic analysis. METHODS: In an attempt to explain the observed discrepancy we have investigated reactivity of SLA positive sera against alpha-enolase and tRNP(Ser)Sec using rat and primate liver homogenate and the recombinant antigens. Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA antibodies were detected by an inhibition ELISA and confirmed by immunoblot using human liver homogenate. Autoantibody reactivity was further evaluated using preparations of primate and rat liver homogenates. Anti-alpha-enolase antibody reactivity has been tested by immunoblot using recombinant alpha-enolase. An affinity purified goat polyclonal anti-alpha-enolase IgG antibody was used as reference serum sample. Anti-tRNP(Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP(Ser)Sec antigen. RESULTS AND DISCUSSION: The affinity purified IgG antibody directed to human alpha-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP(Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations. All but one anti-SLA antibody positive sera reacted with a ~50 kDa but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP(Ser)Sec protein. None of the anti-SLA negative sera reacted with tRNP(Ser)Sec. Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant alpha-enolase by immunoblot. Pre-incubation of anti-SLA positive sera with tRNP(Ser)Sec completely abolished the 50 kDa band. The findings of the present study indicate that alpha-enolase and tRNP(Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP(Ser)Sec - but not anti-alpha-enolase - correlates with anti-SLA antibody reactivity. CONCLUSION: Our findings indicate that tRNP(Ser)Sec is the most likely target of anti-SLA.     

Pre-maturity α-amylase in wheat: The role of abscisic acid and gibberellins

The occurrence of pre-maturity α-amylase (PMA) is a major cause of poor bread-making quality (low Hagberg Falling Number) in wheat grain. In susceptible genotypes, it involves the excessive accumulation of high isoelectric point (pI) α-amylase in mature grain prior to germination and in the absence of pre-harvest sprouting. Several factors regulate PMA formation in developing grain, including genotype, agronomy, and environmental conditions. In particular, a cold period during mid-grain development has been found to be a major stimulus for PMA induction. Although the factors affecting the PMA occurrence are well known, little is known about the molecular mechanism governing its induction. The plant hormones abscisic acid (ABA) and gibberellins (GAs) influence various aspects of grain development, and it has been suggested that PMA involves changes in the amount of these hormones or the sensitivity of the grain to these hormones. This review summarizes recent studies investigating the role of ABA and GAs in PMA induction and PMA occurrence.     

Elongation and insolubilisation of α-glucans by the action of Neisseria polysaccharea amylosucrase

Amylosucrase (E.C. 2.4.1.4) from Neisseria polysaccharea catalyses a transglycosylation reaction using sucrose as a glucose donor to synthesise an insoluble (1→4)-α-glucan, releasing fructose in the solution. If glycogen is used as a glucosyl unit acceptor, amylosucrase elongates polymer branches from their non-reducing ends. Testing of a large series of acceptors with various molar masses, glycosidic linkages, branching degrees and solubilities showed that chain elongation occurred only on polymers with (1→4)-α- or (1→4)-α- and (1→6)-α- linkages. Synthesis yields were better for completely soluble polymers (1.45 glucosyl units grafted per glucosyl unit for polymers with (1→4)-α- and (1→6)-α- linkages), than for partially soluble polymers (0.27–0.97 glucosyl units grafted per glucosyl unit for polymers with either (1→4)-α- linkages or (1→4)-α- and (1→6)-α- linkages). Elongation occurred randomly at the non-reducing ends of some external chains. Elongation of soluble branched molecules led to rapid gel formation favourable to gelation control inside suspensions and solutions. These polymers showed resistant starch contents (22% (w/w) to 57%) after elongation with amylosucrase.     

Nocapyrones: α- and γ-Pyrones from a Marine-Derived Nocardiopsis sp.

One new α-pyrone (nocapyrone R (1)), and three known γ-pyrones (nocapyrones B, H and L (2–4)) were isolated from the culture extract of a Nocardiopsis strain collected from marine sediment. Structures of these compounds were determined on the basis of spectroscopic data including NMR and MS. γ-Pyrones 2–4 were found to induce adiponectin production in murine ST-13 preadipocyte cells but the α-pyrone 1 had no activity. The absolute configuration of the anteiso-methyl branching in 4 was determined by HPLC comparison of a degraded product of 4 with standard samples as a 2:3 enantiomeric mixture of (R)- and (S)-isomers.     

Biological and chemical evaluation of chick pea seed proteins as affected by germination,extraction and α-amylase treatment

The effects of germination, extraction (double extraction with 70% ethanol and water at isoelectric point) and -amylase treatments of chick pea seed flours on crude protein, total carbohydrate, protein efficiency ratio (PER), biological value (BV), true digestibility (TD), net protein utilization (NPU), essential amino acid composition, in-vitro protein digestibility (IVPD) and actual amino acid indices (essential amino acid index or amino acid score) were evaluated. Crude protein content was increased (8–149%), while total carbohydrate was decreased (11–62%) by germination, extraction and -amylase treatments. Alpha-amylase treatment was more efficient in reducing total carbohydrate and increasing the protein content than that of extraction treatment. The protein quality of chick pea flours as measured by PER, BV, TD, NPU, IVPD and corrected amino acid indices (actual amino acid indices×IVPD) was significantly improved by these treatments. The protein quality of germinated--amylase treatment was comparble with casein, while germinated--amylase treaded seeds appeared nutritionally superior to casein. The results indicate that the germinated--amylase and germinated--amylase-extracted treatments could be used successfully as a source of concentrated high quality protein for baby food production. The corrected amino acid indices gave better prediction of PER, BV, TD and NPU (r=93 to 97) than actual amino acid indices (r=45 to 71). PER was highly correlated with corrected amino acid score (r=0.93). The PER could be predicted from the following simple regression equation: PER=–1.827+0.0561×corrected amino acid score.     

Synthesis and α-glucosidase inhibitory mechanisms of bis(2,3-dibromo-4,5-dihydroxybenzyl) ether, a potential marine bromophenol α-glucosidase inhibitor

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE), derived from the marine algae, is a potential α-glucosidase inhibitor for type 2 diabetes treatment. In the present study, a synthetic route was established as a valid approach to obtain BDDE. Fluorescence spectra, circular dichroism spectra and molecular docking methods were employed to elucidate the inhibitory mechanisms of BDDE against α-glucosidase. The results showed that BDDE could be prepared effectively and efficiently with the established synthetic methods. Synthetic BDDE bound with α-glucosidase and induced minor conformational changes of the enzyme. The docking results indicated the interaction between BDDE and α-glucosidase was driven by both hydrophobic forces and hydrogen bonds. The docked BDDE molecule was completely buried in the α-glucosidase binding pocket with part of the molecule reaching the catalytic center and overlapping with the position of glucose, and the rest of the molecule extending towards protein surface. This study provides useful information for the understanding of the BDDE-α-glucosidase interaction and for the development of novel α-glucosidase inhibitors.     

Associations between caryopsis dormancy, α-amylase activity,and pre-harvest sprouting in barley

Pre-harvest sprouting (PHS) is a concern for barley (Hordeum vulgare L.) producers, grain processors, and researchers worldwide. Pre-harvest sprouting has been mainly attributed to low dormancy, which is determined by genotype, stage of plant maturation, and environmental conditions during caryopsis development. Fourteen barley genotypes were sown in field experiments at two sites in North Dakota in 2004 and 2005. Spikes were harvested at four different stages: ≈500 g kg⁻¹ moisture content, physiological maturity, harvest maturity, and post-harvest maturity. Results indicated that barley genotypes were released from dormancy at different rates. The 14 barley genotypes were divided into three classes based on their dormancy loss rate during caryopsis development. C93-3230-24 was highly dormant, and ‘Stander’ and ‘Legacy’ were highly susceptible to PHS due to lack of dormancy from as early as 20 d after heading date. All other genotypes fell into the third group that had intermediate dormancy loss rate. No significant correlation was detected between barley α-amylase activity and germination percentage. A moderate association between malt α-amylase activity and caryopsis dormancy suggested that cultivars with increased malt α-amylase activity tend to have low dormancy and may be more prone to PHS.     

Studies on bioactivities of tea (Camellia sinensis L.) fruit peel extracts: Antioxidant activity and inhibitory potential against α-glucosidase and α-amylase in vitro

Tea fruit peel is the main byproduct during manufacture of tea seed oil. The great increase in tea seed oil production in recent years brings the challenge of finding application for tea fruit peel. The aims of this study were to obtain tea fruit peel extracts enriched with bioactive compounds by several solvent extraction methods and to evaluate their antioxidant activity and inhibitory potential against α-glucosidase and α-amylase in vitro. Flavonoids and phenolics were accumulated in ethyl acetate, butanol, and chloroform fractions, and these fractions possessed much better antioxidant activities, including scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) cation radicals (ABTS⁺), and reducing activity, compared with those of the 75% ethanol extract and water fraction. Moreover, the ethyl acetate, butanol, and chloroform fractions exhibited excellent inhibitory activity against α-glucosidase, much stronger than that of the positive control (acarbose). These fractions also showed mild inhibition on α-amylase activity.     

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3β2β3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers’ yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH₄HCO₃ or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.     

GA3 and Proline Promote Germination of Wheat Seeds by Stimulating α-Amylase at Unfavorable Temperatures

abstract

The effects of various constant temperatures (4, 9, 14, 19, 24, 29, 34, and 38°C) on the germination of winter wheat seed ( Triticum aestivum L. cv. ‘Koyuki’) in a dark condition were studied. The maximum germination percentage was 98% at 24°C. The speed of germination was fastest at 29°G. These results indicate that the most suitable temperature for germination was in the range of 24 to 29°G. α-Amylase expression during germination was also high at higher temperature, and maximum expression occurred at 29°C, although a high temperature of 38°C prevented the synthesis of α-amylase. The close correlation between germination and α-amylase activity at various temperatures indicates that α-amylase is an essential factor for the temperature-dependent germination of wheat seed. In contrast, accumulation of proline increased at a lower temperature, and was the highest at 4°G. We also studied the effects of gibberellin (GA₃) and proline, a compatible osmotica in alleviating the effect of low and high temperature stresses. Pre-soaking treatment with GA3 and proline was effective in promoting germination and increasing α-amylase expression at a low (4°C) and high (38°C) temperature. These results suggest that GA₃ and proline exhibit positive effects on stress alleviation through the stimulation of α-amylase expression.