Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

Localization of α-Glucosidases I,II, and III in Organs of European Honeybees,Apis mellifera L., and the Origin of α-Glucosidase in Honey

Three kinds of α-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an α-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey α-glucosidase, were almost the same as those of α-glucosidase III. Specific antisera against the α-glucosidases were prepared to examine the localization of α-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that α-glucosidase I was present in ventriculus, and α-glucosidase II, in ventriculus and haemolymph. α-Glucosidase III, which became apparent to be honey α-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted α-glucosidase III.     

Purification and Substrate Specificity of Honeybee,Apis mellifera L., α-Glucosidase III

α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high K_m for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k₀ values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K_m values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (A_i’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A_i value at subsite 3 was larger than that of subsite 1.     

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis)

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG’s substrate specificity. PYG exhibited the highest V_max/K_m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.     

A series of cinnamic acid derivatives and their inhibitory activity on intestinal α-glucosidase

Inhibition of α-glucosidase and α-amylase delays the digestion of starch and disaccharides to absorbable monosaccharides, resulting in a reduction of postprandial hyperglycemia. Finding effective mammalian α-glucosidase inhibitors from natural sources can be beneficial in the prevention and treatment of diabetes mellitus. We investigated the inhibitory activity of cinnamic acid derivatives against rat intestinal α-glucosidase and porcine pancreatic α-amylase in vitro. Among 11 cinnamic acid derivatives, caffeic acid, ferulic acid, and isoferulic acid were the most potent inhibitors against intestinal maltase with IC₅₀ values of 0.74?±?0.01, 0.79?±?0.04, and 0.76?±?0.03?mM, respectively, whereas ferulic acid (IC₅₀?=?0.45?±?0.01?mM) and isoferulic acid (IC₅₀?=?0.45?±?0.01?mM) were effective intestinal sucrase inhibitors. However, all cinnamic acid derivatives were found to be inactive in pancreatic α-amylase inhibition. Kinetic analysis revealed that intestinal maltase was inhibited by caffeic acid, ferulic acid, and isoferulic acid in a mixed-inhibition manner. In addition, ferulic acid and isoferulic acid inhibited intestinal sucrase in a mixed type manner, whereas caffeic acid was a non-competitive inhibitor. The combination of isoferulic acid and acarbose showed an additive inhibition on intestinal sucrase. This study could provide a new insight into naturally occurring intestinal α-glucosidase inhibitors that could be useful for treatment of diabetes and its complications.     

Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability

Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized α-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) α-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by α-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the α-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.     

Partial biochemical characterization of α- and β-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae)

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of α- and β-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of α- and β-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Activity of α- and β-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of α- and β-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Results showed that the optimal pH for α- and β-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for α- and β-glucosidase activity in the midgut was obtained as 45 °C. The addition of CaCl₂ (40 mM) decreased midgut β-glucosidase activity whereas α-glucosidase activity was significantly increased at this concentration. The α-glucosidase activity, in contrast to β-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive β-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.     

Studies of glucosidase activities from surface sediments in mangrove swamp

Four transects including sixteen stations were established in the Fugong mangrove (117°54′-117°55′E, 24°22′-24°24′N) of the Jiulong River Estuary, Fujian, China. Besides geochemical characterization and estimation of bacterial abundances, the distribution of α- and β-glucosidase activity was studied to explore the degradation of carbohydrates which can be expected to occur in high quantities in mangrove systems. The distribution pattern of microbial α-glucosidase and β-glucosidase activities was investigated using a fluorogenic model substrate (FMS) technique in order to allow better understanding of in situ enzyme activities, as well as their relation to bacterial biomass, metabolic activity and environmental factors in mangrove sediments. The results showed that the enzyme activities of α-glucosidase (10.83~100.86 µmol g^- 1 h^- 1) and β-glucosidase (39.60~222.75 µmol g^- 1 h^- 1) varied among the different stations, and the enzyme activities of β-glucosidase were higher than those of α-glucosidase at all stations. The extracellular enzyme activities were positively related to organic C, organic matter and bacterial abundance. In addition, the use of the FMS technique to measure extracellular enzyme activities of mangrove sediments could help us to evaluate their catabolic behavior in situ and so lead to a better understanding of the bacterial role in material cycle of mangrove swamp ecosystems.     

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K⁺ and Ca²⁺ significantly increased enzymatic activity, Na⁺ had no effect, and Cu²⁺, Fe²⁺ and Mg²⁺ had the significant negative effects on the enzyme activity. Ca²⁺ and Fe²⁺ increased β-glucosidase activity in the midgut of A. amygdali, Na⁺ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.     

The superiority of bumblebees to honeybees as pollinators: insect visits to raspberry flowers

Abstract.

• 1 The behaviour and activity patterns of Apis mellifera and of five species of Bombus were analysed in relation to climatic variables and nectar quality on three varieties of unsprayed cultivated raspberry (Rubus idaeus) in eastern Scotland.
• 2 Stages of floral morphology and reward were similar for the three varieties: young flowers offered both nectar and pollen, but medium and old flowers offered nectar only, in diminishing quantities.
• 3 A wide range of insects visited raspberry flowers, but bees were dominant, bumblebees being responsible for about 60% of all visits and honeybees making up most of the remaining percentage. All bees had substantial pollen deposited on their bodies during visits, though few specifically collected it.
• 4 Bombus spp. were found to favour young (receptive) flowers strongly, especially early in the morning when pollen was most abundant: whilst Apis visited unselectively. Bumblebees also foraged over substantially longer periods of the day, and in poorer weather, some being present at most times of observation; and they foraged more quickly in terms of flower visits per minute.
• 5 Bombus carried more pollen on their bodies than Apis, and also deposited more pollen on raspberry stigmas, with B.lapidarius and B.terrestris being particularly effective and also being the most abundant species. All bumblebees also foraged over a longer range, moving between canes and rows more frequently than did honeybees.
• 6 Bumblebees are therefore likely to be substantially more important as pollinators of raspberries than are honeybees, especially as raspberries though moderately self-fertile may exhibit metaxenia. Reasons why Bombus may be the preferred pollinator in most sites of raspberry cultivation are discussed, together with implications for present and future growers.

     

Enzymatic transglycosylation of ginsenoside Rg1 by rice seed α-glucosidase

Six α-monoglucosyl derivatives of ginsenoside Rg1 (G-Rg1) were synthesized by transglycosylation reaction of rice seed α-glucosidase in the reaction mixture containing maltose as a glucosyl donor and G-Rg1 as an acceptor. Their chemical structures were identified by spectroscopic analysis, and the effects of reaction time, pH, and glycosyl donors on transglycosylation reaction were investigated. The results showed that rice seed α-glucosidase transfers α-glucosyl group from maltose to G-Rg1 by forming either α-1,3 (α-nigerosyl)-, α-1,4 (α-maltosyl)-, or α-1,6 (α-isomaltosyl)-glucosidic linkages in β-glucose moieties linked at the C6- and C20-position of protopanaxatriol (PPT)-type aglycone. The optimum pH range for the transglycosylation reaction was between 5.0 and 6.0. Rice seed α-glucosidase acted on maltose, soluble starch, and PNP α-D-glucopyranoside as glycosyl donors, but not on glucose, sucrose, or trehalose. These α-monoglucosyl derivatives of G-Rg1 were easily hydrolyzed to G-Rg1 by rat small intestinal and liver α-glucosidase in vitro.     

Structure and Biological Activity of Plant Growth Regulators Produced by Penicillium sp. No. 31f

A phenolic amide, N-p-coumaroyltyramine (1), was isolated as an α-glucosidase inhibitor from methanol extracts of Welsh onion (Allium fistulosum). The inhibitory activity of 1 against a yeast enzyme was as high as K_i 8.4 × 10^?7 m. From a structure-activity relationship study of 1 and its related compounds, the occurrence of α-glucosidase inhibitory activity required a p-coumaramide structure, with an amide hydrogen and alkyl or aralkyl substituent on the amide part.     

CLONING OF MELITTIN cDNA FROM HONEYBEE INTO THE HIGH EXPREHION PLASMID OF ESCHERICHIA COLI*

Abstract Total mRNA from venom glands of newly emerged queen bees was reversely transcribed into cDNA and cloned into the EcoRI site of plasmid λgt11; cDNA library for bee venom was thus constructed. PCR technique was used to produce the melittin coding sequence from the cDNA library. A 87 bp product was produced and inserted into the EcoRI and PstI sites of the high level expression vector pBV220. Recombinant plasmid pBM95 was transformed into the competent cells of E.coli JM101. After screening transformants on LB medium with ampicilin, structure of the recombinant plasmid pBM95 from transformants was analyzed and melittin gene in pBM95 was sequenced. The cloned cDNA coding for honey bee melittin was obtained.     

Nectar and Pollen Sources for Honeybee （Apis cerana cerana Fabr.） in Qinglan Mangrove Area, Hainan Island, China

In the present study, nectar and pollen sources for honeybee （Apls cerana cerana Fabr.） were studied in Qlnglan mangrove area, Hainan Island, China, based on microscopic analysis of honey and pollen load （corblcular and gut contents） from honeybees collected In October and November 2004. Qualitative and quantitative melittopalynologlcal analysis of the natural honey sample showed that the honey is of unlfloral type with Mimosa pudlca L. （Mlmosaceae） as the predominant （89.14%） source of nectar and pollen for A. cerana cerana In October. Members of Araceae are an Important minor （3%-15%） pollen type, whereas those of Arecaceae are a minor （〈3%） pollen type. Pollen grains of Nypa fruticans Wurmb., Rhlzophora spp., Excoecarla agallocha L., Lumnitzera spp., Brugulera spp., Kandella candel Druce, and Ceriops tagal （Perr.） C. B. Rob. are among the notable mangrove texa growing In Qinglan mangrove area recorded as minor taxa In the honey. The absolute pollen count （I.e. the number of pollen grains/10 g honey sample） suggests that the honey belongs to Group V （〉1 000 000）. Pollen analysis from the corblcular and gut contents of A. cerana cerana revealed the highest representation （95.60%） of members of Sonneratia spp. （Sonneratlaceae）, followed by Bruguiera spp. （Rhizophoraceae）, Euphorblaceae, Poaceae, Fabaceae, Arecaceae, Araceae, Anacardlaceae, and Rublaceae. Of these plants, those belonging to Sonneratla plants are the most Important nectar and pollen sources for A. cerana cerana and are frequently foraged and pollinated by these bees in November.     

Validation of reference genes for quantitative real-time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

In quantitative real-time polymerase chain reaction (qRT-PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up- or down-regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT-PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT-PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.     

SNPs selected by information content outperform randomly selected microsatellite loci for delineating genetic identification and introgression in the endangered dark European honeybee (Apis mellifera mellifera)

The honeybee (Apis mellifera) has been threatened by multiple factors including pests and pathogens, pesticides and loss of locally adapted gene complexes due to replacement and introgression. In western Europe, the genetic integrity of the native A. m. mellifera (M‐lineage) is endangered due to trading and intensive queen breeding with commercial subspecies of eastern European ancestry (C‐lineage). Effective conservation actions require reliable molecular tools to identify pure‐bred A. m. mellifera colonies. Microsatellites have been preferred for identification of A. m. mellifera stocks across conservation centres. However, owing to high throughput, easy transferability between laboratories and low genotyping error, SNPs promise to become popular. Here, we compared the resolving power of a widely utilized microsatellite set to detect structure and introgression with that of different sets that combine a variable number of SNPs selected for their information content and genomic proximity to the microsatellite loci. Contrary to every SNP data set, microsatellites did not discriminate between the two lineages in the PCA space. Mean introgression proportions were identical across the two marker types, although at the individual level, microsatellites' performance was relatively poor at the upper range of Q‐values, a result reflected by their lower precision. Our results suggest that SNPs are more accurate and powerful than microsatellites for identification of A. m. mellifera colonies, especially when they are selected by information content.     

Anomer Selective Formation of l-Menthyl α-D-Glucopyranoside by α-Glucosidase-catalyzed Reaction

l-Menthol was glucosylated by the α-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as glucosyl donor. When 50 mg of l-menthol and 1M maltose in 10 mM citrate–phosphate buffer (pH 7.0) were incubated for 24 h at 30°C, a menthylglucoside was selectively obtained as a product. The molar conversion yield based on supplied menthol was 4.5%. The product was identified as l-menthyl α-D-glucopyranoside (α-MenG) by ¹³C-NMR analysis.     

GM2 gangliosidoses in Spain: Analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R²⁰⁰ and A²⁰⁴ inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA^H201L but not rhGAA^WT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA^H201L but not rhGAA^WT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.     

EXPRESSION OF PNA,WGA, SBA,AND Con A LECTIN RECEPTORS IN THE ADULT AND DEVELOPING HONEYBEE ANTENNAL LOBE

Glycoproteins are thought to play a crucial role in cell—cell interactions during nervous system ontogeny both in vertebrates and invertebrates. In order to investigate the putative involvement of such molecules during bee brain ontogeny we used lectins for their ability to bind specifically carbohydrate moieties. The expression of four lectin receptors, i.e. Arachis hypogea (PNA), Triticum vulgaris (WGA), Glycine max(SBA), and Concanavalin A (Con A) has been studied during pupal development and in the adult. The antennal lobe shows a complementary pattern of expression of Con A which stains both neuron somata and glomerular contours, and PNA, which stains the glomerular neuropile. SBA strongly stained the perineurium, trachea and mushroom body neuropile, while other neuropiles were not stained. WGA stained neuronal somata and the core of the glomeruli.