Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

BmSUC1 is essential for glycometabolism modulation in the silkworm, Bombyx mori

Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal β-fructofuranosidase (β-FFase, EC 3.2.1.26)-encoding gene that was firstly cloned and identified in silkworm, Bombyx mori. BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from β-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi-BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.     

Consumption of sugars, hemicellulose, starch, pectin and cellulose by the grasshopper Arcris flavolineata

Adults (0.61 g, fresh-weight) of Abracris flavolineata De Geer (Orthoptera: Acrididae) feeding on Brassica oleracea acephala leaves ingest 21 mg dry-weight/day with an approximate digestibility of 42%. Chemical determinations performed on the leaves ingested and on the feces expelled led to the determination of the approximate digestibilities (%) of the major carbohydrates of leaves as follows: soluble carbohydrates, 91; pectin, 32.1; hemicellulose, 0; starch, 66; cellulose, 15. The results are not sufficient to disregard the possibility that digestible hemicellulose polymers contaminate the pectin and the cellulose fraction. Thus, it is possible that the digestibility of hemicellulose is different from zero, and that the digestibility of pectin and cellulose are somewhat lower than reported. The data are used to propose physiological roles of the enzyme activities previously found in the A. flavolineata midgut.     

Plasma membrane-associated amylase and trypsin: Intracellular distribution of digestive enzymes in the midgut of the cassava hornworm,Erinnyis ello

Differential centrifugation of homogenates of midgut cells prepared in isotonic solutions has been carried out and hydrolase and enzyme marker activities have been determined in the isolated fractions. α- and β-Glucosidase and trehalase seem to occur loosely associated with large structures, from which they are set free by homogenizing in water. They are also found in the cytosolic fraction. Aminopeptidase activity follows that of alkaline phosphatase, whereas that of amylase and trypsin occur mainly among particulate fractions. Part of the amylase present in the particulate fractions seems to correspond to soluble amylase bound by membranes. The enrichment factor for alkaline phosphatase and aminopeptidase in microvilli purified from midgut cells is 4 and for amylase 1.5 Amylase and trypsin are only partly released from a membrane fraction after several washings in different media, including ultracentrifugation in a discontinuous glycerol gradient. About 50% of the amylase and trypsin are solubilized from the membranes by treatment with Triton X-100. The results support the proposal that intermediate and final digestion in Erinnyis ello occur under the action of glycocalyx-associated hydrolases (α- and β-glucosidase and trehalase) and of plasma membrane-bound enzymes (aminopeptidase, and perhaps also amylase and trypsin).     

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.     

β-Glucosidase in four phytophagous Lepidoptera

β-Glucosidase in larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), was studied using helicin as the substrate. Enzyme activity was found in various tissues, with the midgut exhibiting the highest activity. Among the midgut subcellular fractions, the soluble fraction was the most active. The apparent K_m value for the enzyme was 0.63 mM. The enzyme was fairly stable for 8 weeks when stored at −10°C. Activity was mainly located in the 60% fraction when the midgut soluble fraction was fractionated with ammonium sulfate. The system also hydrolyzed a variety of glucosides including numerous toxic plant allelochemicals. Substrate specificity of β-glucosidase was different in the fall armyworm, velvetbean caterpillar, cabbage looper and corn earworm, suggesting a qualitative difference in the enzyme among these species. In general, d-amygdalin, helicin, d(+)-cellobiose, p-nitrophenyl β-d-glucoside and 4-methylumbelliferyl β-d-glucoside were the preferred substrates, whereas sinigrin, phloridzin, α-solanine, tomatine and linamarin were poor substrates for the enzyme in these insects. Toxicity tests reveal that, in most instances, the glucosides were less toxic than their corresponding aglycones to the fall armyworm, indicating that β-glucosidase is an activation enzyme.     

Biochemical characterization of a new β-glucosidase (Cel3E) from Penicillium piceum and its application in boosting lignocelluloses bioconversion and forming disaccharide inducers: New insights into the role of β-glucosidase

Fungal genome sequencing has revealed the presence of multiple putative β-glucosidases; however, information regarding these new β-glucosidases is limited. A new β-glucosidase from Penicillium piceum, designated as PpCel3E, was first isolated and characterized. Using p-nitrophenyl-β-d-glucoside as substrate, PpCel3E showed the lowest Km among the β-glucosidases among all fungi studied. Moreover, PpCel3E exhibited a high transglycosylation activity of 1100 mg gentiobiose/mg and 142 mg sophorose/mg using glucose as the donor. PpCel3E is a novel bifunctional glycoside hydrolase with both β-glucosidase and β-xylosidase activity. Our results show that PpCel3E plays an important role in forming soluble cellulose inducer compounds, as well as in amplifying weak cellulase inducer signal and hemicellulase synthesis via its high transglycosylation activity. Supplementing PpCel3E at low concentrations (40 μg/g substrate) increased the saccharification efficiency of different cellulases by 20% to 27% by removing multiple inhibitors.     

Biochemical characterization of digestive α-amylase, α-glucosidase and β-glucosidase in pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

The pistachio green stink bug, Brachynema germari, has 3–5 generations per year and causes severe damages to pistachio crops in Iran. Physiological digestive processes, such as digestive carbohydrases, can be used to design new strategies in IPM programs for controlling this pest. The enzyme α-amylase digests starch during the initial stage of digestion. Complete breakdown of carbohydrates takes place in the midgut where α- and β-glucosidic activities are highest. Alpha-amylase and α- and β-glucosidase activities were found in the midgut and salivary glands of pistachio green stink bug adults. Overall enzyme activities were significantly higher in the midgut than in salivary glands. The highest α-amylase and α- and β-glucosidase activities were in section v3, whereas the lowest activities were in section v4. V_max was higher and K_m was lower in the midgut than in the salivary glands for these enzymes. In the pistachio green stink bug, the optimal pH was pH 5–6.5 and the optimal temperature was 30 °C to 35 °C for these enzymes. Alpha-amylase activity in the midgut and salivary glands decreased as the concentrations of MgCl₂, EDTA and SDS increased. Enzyme activities in both midgut and salivary glands increased in the presence of NaCl, CaCl₂, and KCl. NaCl had a negative effect on alpha-amylase extracted from salivary glands.     

Ultrastructural and biochemical basis of the digestion of nectar and other nutrients by the moth Erinnyis ello

The midgut epithelium of adult Erinnyis ello L. (Lepidoptera: Sphingidae) forms ramified villus-like folds. The epithelial cells exhibit long microvilli and narrow and sinuous basal channels studded with particles. These morphological features are thought to be involved in the absorption of nutrients present in low concentration in food. Enzymatic assays in E. ello adults showed the presence of amylase in salivary glands, and the occurrence of aminopeptidase, -fructosidase, - and -glucosidase, trehalase and trypsin in the midgut. Calcium differential precipitation of midgut homogenates showed that aminopeptidase and -glucosidase are integral proteins of the microvillar cell membranes, whereas the other enzymes are soluble or partly soluble and partly membrane bound. Detergent-solubilized aminopeptidase (pH optimum 7.5) sediments as a protein with Mr 120000, whereas trehalase (pH optimum 6.5), as a protein with Mr 95000. The activities of amylase (pH optimum 5.5), -glucosidase (pH optimum 5.0) and trypsin (pH optimum 9.5) were not sufficiently high to be further characterized, whereas -fructosidase and membrane-bound -glucosidase were characterized elsewhere. The data suggest that nectar-feeding E. ello adults are able to some extent to digest and absorb starch and proteins, in addition to nectar sugars. Initial digestion is considered to occur in midgut lumen and final digestion on the surface of midgut cells under the action of microvillar enzymes.

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Résumé L'épithélium de l'intestin moyen des adultes de E. ello L; (Lépido., Sphingidae) est replié en villosités. Les cellules épithéliales présentent de longs microvilli et d'étroits canaux sinueux basaux garnis de particules. Ces caractères morphologiques peuvent être interprétés comme impliquant l'absorption d'aliments présents en faible concentration dans la nourriture. Des études enzymatiques ont révélé la présence d'amylase dans les glandes salivaires, et la découverte d'aminopeptidase, de -fructosidase, d'- et -glucosidases, de tréhalase et de trypsine dans l'intestin moyen. La précipitation calcique différentielle d'homogénats de l'intestin moyen a montré que l'aminopeptidase et l'-glucosidase sont des protéines appartenant aux membranes microvillaires cellulaires, tandis que les autres enzymes sont entièrement ou partiellement solubles et partiellement liées aux membranes. L'aminopeptidase solubilisée par un détergent (pH optimum: 7,5) sédimente comme une protéine de Mr 120000, tandis que la tréhalase (pH optimum: 6,5) comme une protéine de Mr 95000. Les activités de l'amylase (pH optimum: 5,5), de la -glucosidase (pH optimum 5,0) et de la trypsine (pH optimum 9,5) n'étaient pas suffisantes pour être caractérisées, tandis que la -fructosidase et l'-glucosidase liée à la membrane ont été caractérisées ailleurs. Les résultats suggèrent que les adultes E. ello consommateurs de nectar sont capables de digérer et d'absorber une certaine quantité d'amidon et de protéines, en plus des sucres du nectar. La digestion doit débuter dans la lumière de l'intestin moyen pour s'achever à la surface des cellules intestinales sous l'action des enzymes des microvilli.

     

Purification and partial characterization of a midgut membrane-bound α-glucosidase from Quesada gigas (Hemiptera: Cicadidae)

Adults of Quesada gigas (Hemiptera: Cicadidae) have a major α-glucosidase bound to the perimicrovillar membranes, which are lipoprotein membranes that surround the midgut cell microvilli in Hemiptera and Thysanoptera. Determination of the spatial distribution of α-glucosidases in Q. gigas midgut showed that this activity is not equally distributed between soluble and membrane-bound isoforms. The major membrane-bound enzyme was solubilized in the detergent Triton X-100 and purified to homogeneity by means of gel filtration on Sephacryl S-100, and ion-exchange on High Q and Mono Q columns. The purified α-glucosidase is a protein with a pH optimum of 6.0 against the synthetic substrate p-nitrophenyl α-d-glucoside and M_r of 61,000 (SDS-PAGE). Taking into account V_Max/K_M ratios, the enzyme is more active on maltose than sucrose and prefers oligomaltodextrins up to maltopentaose, with lower efficiency for longer chain maltodextrins. The Q. gigas α-glucosidase was immunolocalized in perimicrovillar membranes by using a monospecific polyclonal antibody raised against the purified enzyme from Dysdercus peruvianus. The role of this enzyme in xylem fluid digestion and its possible involvement in osmoregulation is discussed.     

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A β-glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both β-glucosidase and β-xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.     

Carbohydrates and carbohydrate esters of ferulic acid released from cell walls of Lolium multiflorum by treatment with cellulolytic enzymes

41% of the cell walls from mature leaf blades of Lolium multiflorum were digested by treatment during 14 days with C₁ enzyme (cellulase) which had been purified by gel filtration and ion-exchange chromatography. Cellobiose was the main sugar released from the walls, together with some glucose and higher oligosaccharides. Considerable amounts of carbohydrate esters of ferulic and p-coumaric acids were also released. When the C₁ enzyme was further purified by isoelectric focusing, only 8% of the cell walls were digested. Purified C_x (CM-cellulase) containing β-glucosidase digested 51% of the cell walls in 16 hours: the major component detected in the soluble products was glucose together with some β (1 → 4)-xylobiose, xylose and arabinose. Higher oligosaccharides and carbohydrate esters of ferulic and p-coumaric acids were also present. It was shown that these acids were present in the cell walls mainly in the trans-configuration.     

Isolation and characterization of β-glucosidase from the cytosol of rat kidney cortex

A procedure is described for the preparation of extensively purified β-d-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the β-glucosidase in the high speed supernatant (100 000 × g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. β-Glucosidase activity co-chromatographs with β-d-galactosidase, β-d-fucosidase, α-l-arabinosidase and β-d-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of β-glucosidase, respectively. The specific activity of the apparently homogeneous β-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-β-d-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6–7) and heat lability, and co-migrate on polyacrylamide disc gels at ph 8.9 (R_F, 0.67). β-Glucosidase activity is inhibited competitively by glucono-(1 → 5)-lactone (K_I, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5′-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of β-d-glucose, it will not hydrolyze xylosyl-O-serine, β-d-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000–58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of β-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convulated tubule. The enzyme is also present in relatively large ammounts in the villus cells, but not crypt cells, of the intestine. the physiological subtrates and function of the enzyme are unknown.     

Comparison of the ability of phospholipids from rat liver lysosomes to reconstitute glucocerebrosidase

The in situ lipid activator of rat liver glucocerebrosidase was investigated. Rat liver lysosomes were purified (42.9-fold relative to the crude homogenate) by sequential isopycnic centrifugation in sucrose and metrizamide gradients. Lipids were extracted with chloroform:methanol (2:1) and phospholipids were separated by one-dimensional thinlayer chromatography. The phospholipid content of the lysosome preparation was 0.28 μmol lipid phosphorus/mg protein. Phosphatidylcholine was present as the major non-acidic phospholipid (39.3%). Of the acidic phospholipids, phosphatidylinositol and phosphatidylserine were present in the greatest amounts (12.0 and 19.7%, respectively). The resolved phospholipids were tested separately and in the presence of a heat-stable factor from Gaucher spleen for their ability to reconstitute butanol-delipidated rat liver glucocerebrosidase activity. Alone or in the presence of the heat-stable factor, phosphatidylserine and phosphatidylinositol were the most effective activators of glucocerebrosidase. Bis(monoacylglyceryl) phosphate derived from rat liver tritosomes or rabbit lung macrophages was also effective in reconstituting β-glucosidase activity.     

Influence of internal sugar levels on apoplasmic retrieval of exogenous sucrose in source leaf tissue

Sugar levels in Beta vulgaris leaves were increased by heat-girdling the petiole and returning the plant to the controlled-environment chamber for 10 and 34 hours. After 10 hours, sucrose influx into the treated leaves was similar to the controls, although sucrose levels increased from 2.1 to 5.3 micromoles per milligram chlorophyll. However, after a 34-hour treatment, sucrose levels increased from 2.1 to 11.5 micromoles per milligram chlorophyll. In this instance, sucrose influx decreased relative to the untreated controls. Decreasing sugar levels by DCMU treatment resulted in a small stimulation of sucrose influx. A similar DCMU treatment applied to leaves of Allium cepa also resulted in an increase in sucrose influx. However, in A. cepa we could not attribute this increase to a lowering of sugar levels, as the kinetic profiles obtained from control leaves did not vary from each other throughout the day, despite considerable changes in sugar levels. Additionally, it appeared that sucrose uptake in onion may be set at some point and remains invariant throughout the day. Similar studies were also conducted on discs cut from mature leaves of Spinacia oleracea var America. Between 1 and 8 hours after the onset of the photoperiod, the sucrose content of the spinach leaves increased from 2.6 to 9.3 micromoles per milligram chlorophyll. A comparison of the kinetic profiles obtained from leaf discs, taken at these times, indicated that sucrose uptake was not influenced by these changes in internal sugar levels. The relationship between the above findings and `trans' inhibition of exogenous sucrose uptake is discussed. Although intermediate changes in sugar levels in sugar beet leaves did not appear to affect sucrose influx, autoradiographic studies revealed that these changes dramatically affected the partitioning of exogenously supplied [¹⁴C]sucrose. Our results indicate that while intermediate changes in internal sugar levels have little effect on sucrose influx across the plasmalemma, they may dramatically affect partitioning between the phloem and the mesophyll vacuole.     

Cellulase genes of Cellulomonas uda CB4. I. Cloning and expression of β-glucosidase genes in Escherichia coli

Two β-glucosidase genes in Cellulomonas uda CB4 were cloned in Escherichia coli with pAT325 constructed from pAT153 and pBR325. Plasmids pCC1 and pCG1 were isolated from the transformants producing β-glucosidase, and the β-glucosidase genes cloned were in 6.1 and 8.1 kb BamHI fragments, respectively. The amount of β-glucosidase expressed in E. coli harboring pCCl and pCGI was 1.2 and 4.0 times that in the present strain. E. coli harboring pCCl grew efficiently on cellobiose.     

Purification and characterization of an isoflavones conjugate hydrolyzing β-glucosidase (ICHG) from Cyamopsis tetragonoloba (guar)

A β-glucosidase with high specific activity towards isoflavone glycosidic conjugates was purified from seeds of Guar (Cyamopsis tetragonoloba) by ammonium sulphate precipitation followed by size exclusion and ion exchange chromatography. The pH and temperature optima of the purified Isoflavones conjugate hydrolyzing β-glucosidase (ICHG) were found to be pH 4.5 and 37 °C, respectively. The enzyme was relatively stable at higher temperatures. Effect of different divalent metal ions was studied and it was found that Cobalt and Mercury ions completely inhibited the enzyme activity. K_m and V_max of the purified isoflavones conjugates hydrolyzing β-glucosidases (ICHG) was 0.86 mM and 6.6 IU/mg respectively. The enzyme was most likely a trimer (approximate Mr 150 kDa) with potential subunits of 50 kDa. The purified enzyme showed activity against isoflavone conjugate glycosides viz daidzin and genistin but was inactive towards other flavonoid conjugates. The product conversion was confirmed by HPTLC and HRMS analysis. The MALDI-TOF analysis of the ICHG showed a score greater than 78 with 20 matches in MASCOT software. The five resultant peptides obtained had highest similarity in sequence with β-glucosidase from Cicer arietinum. The β-glucosidase from the C. arietinum has also been reported to exhibit the isoflavone conjugate hydrolyzing properties thus confirming the nature of the enzyme purified from the Guar seeds.