Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.     

Production and partial purification of invertase using <Emphasis Type="Italic">Cympopogan caecius</Emphasis> leaf powder as substrate

The present study investigates the efficiency of Aspergillus niger to produce invertase, an industrially important enzyme by using powdered stem of Cympopogan caecius (Lemon grass) as sole substrate and sole carbon source for the microorganism. The molecular weight of invertase was estimated to be 66–70 kDa by sodium do decyl sulphate poly acrylamide gel electrophoresis (SDS PAGE). The production of the enzyme was studied at different pH scales ranging from pH 4.0 to 7.0 at a constant temperature of 30°C and 2% substrate concentration. The maximum production of invertase (specific activity −0.0516 μk/mg protein) was obtained at pH 5.5 at 30°C temperature, and incubation for 48 h. The activity was found to be stable at pH 5.5 for 30 min. The enzyme was found to be stable in the temperature range of 20–55°C. The effect of divalent metal ions Cu²⁺, Fe²⁺, Co²⁺ on the activity of the enzyme invertase showed that these ions affected the activity by a certain factor. The study can be further industrially exploited in a country-like India where lemon grass is found in plenty and can be used as substrate for enzyme production. Moreover, the preparation of the substrate is also a simple process.     

Isoforms of trehalase and invertase of Fusarium oxysporum

Enzymatic assays and native PAGE were used to study trehalase and invertase activities, depending on culture age and different sugar conditions, in cell-free extracts, culture filtrates and ribosomal wash of Fusarium oxysporum. The activity of invertase preceded that of trehalase; in the exponential phase of growth, mainly invertase activity was produced, whereas trehalase activity was high in the stationary phase. In this last phase of growth, the activity of intracellular trehalase was repressed by monosaccharides, whereas disaccharides, especially lactose and starch, enhanced the activity of intracellular and extracellular trehalase. However, invertase activity was not repressed under these conditions and had the maximal activity in the presence of saccharose. Intracellular trehalase appeared in a single, high-molecular weight (120 kDa) form, whereas the extracellular enzyme appeared in a single, low-molecular weight (60 kDa) form. The activity pattern of invertase isoforms indicated the occurrence of three forms of intracellular enzyme with the main activity band at 120 kDa and two isoforms of extracellular enzyme. In the ribosomal wash, high-molecular weight isoforms of both trehalase and invertase were identified. A possible role of trehalase and invertase in carbohydrate metabolism of fungal pathogens is also discussed.     

Priming Aphytis: behavioral modification of host selection by exposure to a synthetic contact kairomone

The introduced parasitoid, Aphytis melinus DeBach (Hymenoptera: Aphelinidae), utilizes a kairomone, O-caffeoyltyrosine, to recognize California red scale, Aonidiella aurantii (Maskell) (Homoptera: Diaspididae). Wasps used in augmentative release programs for California red scale on California citrus are reared on oleander scale, Aspidiotus nerii Bouché (Homoptera: Diaspididae), themselves reared on squash. The goals of this study were: 1) to determine if long-term rearing on oleander scale had caused A. melinus to develop a preference for oleander scale, and 2) to determine if the preference of oleander-reared A. melinus for California red scale might be enhanced by exposing them to synthetic O-caffeoyltyrosine prior to release. Wasps that were removed from their hosts as pupae and allowed to emerge as adults isolated from their hosts retained a strong preference for California red scale regardless of rearing host. This preference was reduced if wasps were allowed to emerge from oleander scale, thus acquiring early adult experience with oleander scale. The preference for California red scale was restored, however, by exposing wasps reared on oleander scale to synthetic O-caffeoyltyrosine prior to bioassay. Exposure of A. melinus reared in commercial insectaries to O-caffeoyltyrosine prior to release may be a means to improve the effectiveness of such wasps in augmentative release programs to control California red scale.     

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.     

Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Désirée, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.     

Isolation, Mapping, and Characterization of Trehalaseless Mutants of Neurospora crassa

Mutant strains of Neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group I. Maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. Heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. In addition, strains with duplications heterozygous for trehalase produce less than 1% of the activity of the standard strain. An inhibitor of trehalase has been found in tre(-) strains; its sensitivity to heat and proteolysis, and its nondialyzability suggest that this substance is a protein. The mig gene, which determines the rate of migration of trehalase on acrylamide gels, has been shown to be less than 1 map unit away from the tre gene.     

Absorption of toxic beta-glucosides produced by plants and their effect on tissue trehalases from insects

Trehalases present in body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor (Coleoptera), Musca domestica (Diptera), Spodoptera frugiperda and Diatraea saccharalis (Lepidoptera) were assayed in the presence and absence of toxic beta-glucosides produced by plants or their aglycones. The glucosides used were phlorizin, amygdalin, prunasin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with and without esculin. More than 60% of total trehalase activity was found in the midgut of these insects. As a rule, trehalases present in each insect were inhibited by at least two of the glucosides. Prunasin was the best inhibitor in tissues with highest trehalase activity. S. frugiperda beta-glucosidases were not able to hydrolyze esculin. Nevertheless, their larval midguts absorb the intact glucoside that is recovered from the fat body, Malpighian tubules and mainly from haemolymph. Mature larvae fed on a diet containing 3 mM (0.1%) esculin have 0.2 mM esculin in their haemolymph, and weigh 60% of control larvae. In vitro, haemolymph trehalase activity is abolished by 0.5 mM esculin. This inhibition may play a role in the decrease of body weight and in animal survival. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activity in tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why the S. frugiperda development is not impaired by 1% dietary amygdalin, in contrast to what is observed when insects are reared in 0.1% esculin. The data suggest that many plant beta-glucosides are toxic because they inhibit trehalase, a key enzyme controlling glucose availability in insects.     

Acid-stable α-Amylase of Black Aspergilli

When black Aspergilli were cultivated in appropriate condition, culture filtrate showed dextrinizing activity even after acid treatment such as pH 2.5, at 37°C for 30 minutes. It suggested the existence of acid-stable dextrinizing amylase. To isolate this enzyme paper el-ectrophoretic procedure was carried out and the spot which showed acid-stable dextrinizing activity was obtained in addition to α-amylase and glucoamylase spots. This new amylase was purified by fractional precipitation with ammonium sulfate, rivanol and acetone, and was obtained in a crystalline form.     

Purification and characterization of polygalacturonase produced by thermophilic Thermoascus aurantiacus CBMAI-756 in submerged fermentation

An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C. The apparent K _m with citrus pectin was 1.46 mg/ml and the V _max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity at pH 9.0. Metal ions, such as Zn⁺², Mn⁺², and Hg⁺², inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing mono, di and tri-galacturonic acids within 10 min of hydrolysis.     

Origin of intestinal disaccharidases of Ascaris suum

Disaccharidases from the gut of Ascaris suum were investigated to determine whether they were synthesized by the worm or whether they were host enzymes adsorbed to the worms' intestinal cells. Alpha-d-glucoside glucohydrolase (maltase) (EC 3.2.1.20), Beta-d-fructofuranoside fructohydrolase (invertase) (EC 3.2.1.26) and 1-glucohydrolase (trehalase) (EC 3.2.1.28) from Ascaris were studied in both a membrane (brush border)-bound and solubilized form with regard to temperature stability and pH optima. Data collected were compared to similar data on hog intestinal enzymes. Worm maltase and trehalase were relatively heat labile, whereas the hog enzymes were more stable to heat inactivation. Worm invertase was heat stable in comparison to the hog enzyme. The pH optima for Ascaris maltase and invertase were different from those of hog disaccharidases, whereas the pH optimum for trehalase from both parasite and host were similar. Tissue homogenates of second-stage larvae contained measurable maltase, but not sucrase, or trehalase activity. Results suggested that Ascaris intestinal disaccharidases represent three distinct enzymes of parasite rather than host origin.     

Increased parasitization of California red scale in the field after exposing its parasitoid, Aphytis melinus, to a synthetic kairomone

The introduced parasitoid, Aphytis melinus DeBach (Hymenoptera: Aphelinidae), utilizes a kairomone, O-caffeoyltyrosine, to recognize California red scale, Aonidiella aurantii (Maskell) (Homoptera: Diaspididae). Wasps used in augmentative release programs for California red scale on California citrus are reared on oleander scale, Aspidiotus nerii Bouché (Homoptera: Diaspididae), themselves reared on squash. Previous laboratory studies demonstrated that wasps reared on oleander scale but exposed to O-caffeoyltyrosine before bioassay more readily accepted California red scale covers for probing than wasps reared similarly but not exposed to O-caffeoyltyrosine. The goal of this study was to determine if wasps reared on oleander scale but exposed to O-caffeoyltyrosine actually parasitized a greater number of field-reared California red scale than unconditioned wasps. In order to distinguish between progeny of conditioned and unconditioned wasps in the field, we utilized two A.melinus strains that carried different electrophoretically distinguishable alleles. In half of the releases, Strain A was conditioned to O-caffeoyltyrosine and Strain B served as the control, while this pattern of conditioning was reversed for the remaining releases. Although there were differences in the rates of population growth of the two strains, whichever strain was the conditioned strain left a greater than expected number of progeny compared to the control strain in all comparisons. Exposure of A. melinus reared in commercial insectaries to O-caffeoyltyrosine prior to release may be a means to improve the effectiveness of such wasps in augmentative release programs to control California red scale.     

Co-purification of glucanase with acid trehalase–invertase aggregate in Saccharomyces cerevisiae

An electrophoretically homogenous aggregate of acid trehalase, invertase and an unidentified 37–41 kDa protein was purified from Saccharomyces cerevisiae. N-terminal analysis of the protein revealed an amino acid sequence identical to that of Bgl2p (endo-β-l,3-glucanase) of S. cerevisiae. Acid trehalase activity with co-eluted glucanase activity was observed from late growth phase through early stationary phase. Pools with high percentage of Bgl2p corresponded with high acid trehalase activity. A BGL2 deletion strain had lower acid trehalase activity. The 37–41 kDa protein represents Bgl2p which, besides imparting glucanase activity, could also be acting as a regulator for the acid trehalase activity by association in the enzyme aggregate.     

Characteristics of trehalase inCandida tropicalis

Summary The trehalase content of different yeasts varies widely. A strain ofCandida tropicalis was found to be the best source of this enzyme among the yeasts tested. The trehalase activity in this yeast could be increased 8.5 times by growing it on trehalose rather than glucose. Thus trehalase is an adaptive enzyme inC. tropicalis. It was found that the amount of trehalase which could be solubilized increased with increasing pH during autolysis of the cells, none being released from the cell debris at pH 4.5 and most at pH 6.3. Some evidence was obtained to show that the solubilization was caused by an enzyme. The stability of trehalase under various conditions was studied. A partial purification was achieved by precipitation with 40% ethanol at a temperature of −18°C. The maximum temperature of the enzyme was 48°C., and the optimum pH ranged from 4.1 to 5.3     

小麦几种主要次生物质对麦长管蚜几种酶活力的影响

借助麦蚜人工饲料研究明确了小麦几种主要次生物质单宁酸、总酚（没食子酸）和香豆素对麦长管蚜Sitobion avenae的存活、生长和发育有明显的抑制作用,其抗蚜阈值浓度分别为0.06%、0.08%和0.065%。用昆虫酶系体外抑制法,研究上述三种次生物质的抗蚜阈值浓度对麦长管蚜的糖转化酶和解毒酶活力的影响,结果表明: 0.06%单宁酸强烈抑制麦长管蚜蔗糖酶、海藻糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力;0.08%没食子酸显著抑制蔗糖酶、羧酸酯酶和谷胱甘肽S-转移酶的活力;0.065%香豆素极显著地抑制谷胱甘肽S-转移酶活力。     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

Population dynamics ofHemisarcoptes coccophagus Meyer (Astigmata: Hemisarcoptidae) attacking three species of armored scale insects (Homoptera: Diaspididae)

The parasitic miteHemisarcoptes coccophagus Meyer (Astigmata: Hemisarcoptidae) feeds on armored scale insects (Homoptera: Diaspididae). Three host scales were studied: chaff scale (Parlatoria pergandii Comstock andP. cinerea Hadden) infesting citrus (grapefruit and orange) orchards, latania scale [Hemiberlesia lataniae (Signoret)] and olcander scale (Aspidiotus nerii Bouche) occurring on acacia (Acacia cyanophylla) branches and leaves. The overall dynamics of mite populations were similar on the different scale species, although the level of attack on each sampled diaspidid host was different. Rates of young mite stages were highly variable on all three hosts throughout the sampling period while adult rates were stable, constituting around 20% of all mites. No oviposition by mites occurred in mid-winter on any scale species, on any plants or parts, at all sampling sites. Of the three diaspidids, highest mite prevalence (parasitization rate) occurred on latania scale. Mite prevalence on oleander scale infesting mature leaves was higher than on young leaves.H. coccophagus apparently first attacks latania scale on branches, it then disperses to the same host inhabiting mature leaves, from there to co-occurring oleander scale and finally to oleander scale infesting young leaves. Mite prevalence on chaff scale was influenced by host plants and climatic regions: in the Negev it was higher than in the coastal plain, with scales on grapefruit being more heavily attacked at the former site but not on coastal plain. Our data suggest thatH. coccophagus prefers ovipositing scale females. The decreasing order of preference for other scale stages was: young females immature males, 2nd-instar nymphs females.