A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Extracellular and mycelial amylases of the thermophilic fungus Malbranchea sulfurea

The thermophilic fungus Malbranchea sulfurea produces extracellular -amylase whereas -glucosidase is mainly cell bound. Extraction of the cell bound enzyme was maximum with one molar NaCl, followed by Triton ×100 and Urea-Na₂SO₃ extractants. Supplementation of Triton ×100 in growth medium significantly affected the presence of enzymes at various locations. A role for cell bound -amylase and -glucosidase has been suggested in rapid starch utilization by the fungus during early growth phase.     

The temperature influences the ratio of glucosidase and galactosidase activities of β-glycosidases

The selectivity for the glycon part of a donor substrate of -glycosidases from almond, a mesophilic (Kluyveromyces fragilis) and three highly thermophilic organisms (Caldocellum saccharolyticum, Sulfolobus solfataricus and Pyrococcus furiosus) was investigated at various temperatures (25–90 °C). On the basis of kinetic constants, the selectivity was calculated as the specificity constant (V _max /K _m ) ratio or V _max ratio of glucoside to galactoside donor. In the almond -glucosidase and the mesostable enzyme one enzyme activity dominated whereas the thermostable enzymes expressed both high -glucosidase and high -galactosidase activities. Surprisingly, for -glycosidases from almond, K. fragilis, and C. saccharolyticum the donor selectivity decreased as the temperature increased. In contrast, two of the highly thermostable enzymes (from S. solfataricus and P. furiosus) had constant donor selectivity as the temperature increased. The results thus showed -glycosidases of differing origins to differ markedly in their substrate specificity and in the extent to which their selectivity for the glycon part of the donor substrate is influenced by the temperature.     

Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1

Two intracellular -glucosidases (E.C. 3.2.1.21) were purified from the filamentous fungus Neurospora crassa, mutant cell-1 (FGSC no. 4335) and characterized. The extent of purification were 2.55- and 28.89-fold for -glucosidase A and -glucosidase B, respectively. -Glucosidase A was a dimeric protein, and B a monomeric protein, with molecular masses of 178 and 106 kDa, respectively. Both isoenzymes were glycoproteins with relatively high carbohydrate contents (-glucosidase A, 29.2%; -glucosidase B, 34.2%). The isoelectric points determined by IEF were 6.27 and 4.72, respectively. pH optima for activity were determined to be 5.0 and 5.5, and temperature optima to be 55 and 60 °C, for -glucosidases A and B, respectively. Both purified -glucosidases. especially -glucosidase B, showed relatively high stability against pH and temperature. Both enzymes were stable in the pH range of 5.0–9.0. The activities were completely retained up to 48 h at temperatures below 40 °C. At higher temperatures, enzymes were relatively unstable and lost their activities at 60 °C after 24 h. Both -glucosidases were highly activated by CuCl₂, and inhibited by SnCl₂ and KMnO₄. Hg²⁺ and Ag⁺ also inhibited severely -glucosidase B. The K _m and V _max values of the isoenzymes against cellobiose as substrate were 1.50 mM and 12.2mol min^–1 mg^–1 for -glucosidase A and 2.76 mM and 143.5 mol min^–1 mg^–1 for -glucosidase B.     

Degradation of bromoxynil by resting and immobilized cells of Agrobacterium radiobacter 8/4 strain

The Agrobacterium radiobacter 8/4 strain capable of degradation of bromoxynil, ioxynil and dichlobenil, arylnitrile herbicides was isolated from soil and cell entrapment was investigated. Three immobilization techniques was used: Caalginate, Ca-pectate and -carrageenan technique, and resting cells. The highest degradation rates were obtained with Ca-alginate and -carrageenan entrapped cells.     

Retention of lead within the digestive vacuole inTetrahymena

Summary The ciliateTetrahymena pyriformis was exposed to lead acetate. Cell proliferation in the presence of 0.1% lead salt (with or without EDTA) equaled, after a variable lag period, that of the control cells. The lead (550 ppm) forms a fluffy precipitate with the organic growth medium; this was in part prevented by addition of EDTA. The cells primarily ingested the fluffy precipitate whereby they became exposed to large amounts of lead. Within the digestive vacuole, the fluffy precipitate became converted into refractile structures (3 m in diameter) which were egested and accumulated at the bottom of the culture flask. The lead content of these defecation balls was higher than that of the fluffy precipitate. In addition to the lead-containing vacuoles, the cells contained small, refractile granules. The apparent, high tolerance ofTetrahymena towards lead is believed to be due in part to the low ionic concentration of lead under the present conditions and in part to a detoxication mechanism consisting of retention of lead within the digestive vacuoles and perhaps of accumulation of lead within the small, refractile granules.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

Summary Thermostable, extracellular -amylase and -glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an -amylase and an -glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The -amylase and -glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The -glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl--d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the -amylase and -glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.     

Purification and properties of an extracellular β-glucosidase from Lenzites trabea

Summary When culturing the cellulolytic-active Basidiomycete and brown-rot fungus Lenzites trabea A-419 in submerged culture with glucose and cellulose as a carbon source, the fungus only excreted -glucosidase (EC 3.2.1.21) and an endo-1,4--glucanase (EC 3.2.1.4).No evidence for C₁ activity (EC 3.2.1.91) was found in the culture filtrate or in the ultra concentrate. -Glucosidase could be separated from endoglucanase by chromatography on Sepharose 6-B. Further fractionation of the -glucosidase on DEAE-Sephadex A-25 resulted in a 525-fold purification. The molecular weight of the isolated -glucosidase was determined by co-chromatography on Sephadex G-200 to be 320,000 daltons. The enzyme developed maximum activities at pH 4.5 and 75°C. The enzyme does not act on crystalline cellulose or CMC, but it hydrolyzes cellotriose,-tetraose, and-pentaose to cellobiose and glucose. -glucosidase activity was strongly inhibited by the reaction product, glucose. A K_i value of 2.7×10^–3 (M) for noncompetitive inhibition was found.     

Bacillus stearothermophilus ATCC12016 α-glucosidase specific for α-1,4 bonds of maltosaccharides and α-glucans shows high amino acid sequence similarities to seven α-d-glucohydrolases with different substrate specificity

We have sequenced the gene encoding Bacillus stearothermophilus ATCC12016 -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) specific for non-reducing terminal -1,4 bonds of maltosaccharides and -glucans. The amino acid sequence of the enzyme deduced from the nucleotide sequence of the gene (1665 base pairs) consisted of 555 residues with a molecular mass of 65233. The enzyme showed 40%–57% sequence similarities to -d-glucohydrolases with very different substrate specificity, such as Bacillus cereus ATCC7064 oligo-1,6-glucosidase, Bacillus thermoglucosidasius KP1006 oligo-1,6-glucosidase, Saccharomyces carlsbergensis CB11 -glucosidase, Bacillus sp. F5 -glucosidase, Streptococcus mutans (Ingbritt strain) dextran glucosidase, Bacillus sp. SAM1606 -glucosidase and Escherichia coli ECL116 trehalose-6-phosphate hydrolase. All these enzymes had sequences equivalent to secondary elements revealed in B. cereus oligo-1,6-glucosidase by X-ray crystallography. We have suggested that the B.stearothermophilus enzyme adopts the same polypeptide folding, i.e. an (/)₈-barrel in the N-terminal active-site domain, as the B.cereus enzyme and other -glucohydrolases.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Production of cellulases by thermophilic fungi grown on Leptochloa fusca straw

Seven indigenous thermophilic fungi were screened for cellulase and xylanase production when grown on Leptochloa fusca (kallar grass) straw. Aspergillus fumigatus produced the highest activities of 0.4, 2.5, 3.5 and 0.14 U/ml of filter paper cellulase, CM-cellulase, xylanase and -xylosidase, respectively. Sporotrichum thermophile produced 0.47 -glucosidase/ml. Chaetomium thermophile, Humicola grisea and Torula thermophila had lower activities than the other thermophilic fungi.The authors are with the National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Cytochemischer Nachweis von Hydrolasen in Pilzzellen

Zusammenfassung Die Anwendbarkeit der Tetrazoliumsalz-Methode (Lojda, 1965) und der Brom-naphthyl-glykosid-Methode (Cohen u. Mitarb., 1952; Rutenburg u. Mitarb., 1958, 1960) zum cytochemischen Nachweis von -Galactosidase (E.C. 3.2.1.23), -Glucosidase (E.C. 3.2.1.20) und -Glucosidase (E.C. 3.2.1.21) in den Zellen der Pilze Neurospora crassa, Aspergillus oryzae und Saccharomyces cerevisiae (Vorkultur auf Nährböden mit Zusätzen von Enzyminduktoren) wurde untersucht. In allen Fällen waren die TS-Medien — was die Art der Enzymlokalisation, die Empfindlichkeit und Spezifität betrifft — dem Brom-naphthylglykosid-Verfahren überlegen. Bei N. crassa und A. oryzae ließen sich -Galactosidase, - und -Glucosidase, in den Zellen von S. cerevisiae jedoch nur die letzten beiden Enzyme nachweisen. Ergebnisse von Kontrollreaktionen untermauern die Spezifität der Nachweisreaktionen. In den Konidien von N. crassa und A. oryzae trat auch bei Kontrollen Pormazanbildung auf; die möglichen Ursachen werden diskutiert. Aus dem Reaktionsbild kann keine Aussage über die Art der intrazellulären Lokalisation der untersuchten Glycosidasen gemacht werden.

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Cytochemical detection of hydrolases in fungus cellsI. Glycosidases

Summary The usefullness of the tetrazolium salt-method (Lojda, 1965) and the bromo-naphthyl-glycoside-method (Cohen et al., 1952; Rutenburg et al., 1958, 1960) for the intracellular detection of -galactosidase (E.C. 3.2.1.23), -glucosidase (E.C. 3.2.1.20) and -glucosidase (E.C. 3.2.1.21) in the cells of the fungi Neurospora crassa, Aspergillus oryzae and Saccharomyces cerevisiae (cultivation on media containing enzyme inductors) was examined. In all cases the tetrazolium salt-media gave far better results concerning enzyme localization, sensitivity and specificity. In N. crassa and A. oryzae -galactosidase, - and -glucosidase could be detected, in the cells of S. cerevisiae only the two latter enzymes. The results of control reactions demonstrate the specificity of the cytochemical reactions. In the conidia of N. crassa and A. oryzae formazan deposition was observed even in control reactions; the possible reasons are discussed. The reaction pictures give no suggestions concerning the exact intracellular localization of the glycosidases tested.

     

Synthesis and activity of α-glucosidase produced byBifidobacterium pseudolongum

Bifidobacterium pseudolongum NCFB 2244 grew on starch as sole source of carbon and energy, but cell yields and specific growth rates were considerably lower than on glucose (=0.19±0.04 and 0.38±0.09 respectively). Amylase activity was not detected in cultures of this bacterium, but cell-associated -glucosidase was constitutively produced. Analysis of -glucosidase activity from cell extracts by preparative isoelectric focusing gave two peaks of activity with apparent isoelectric points of 3.9 (Enzyme I) and 4.2 (Enzyme II), corresponding to threefold and fourfold purification factors respectively. No -glucosidase activity was detected with Enzyme I after gel-filtration chromatography on Sephadex G150. However, activity was recovered in samples containing Enzyme II, indicating the protein had a molecular mass of approximately 126 kDa. This was subsequently confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results show that the restricted ability ofB. pseudolongum to utilize starch as a carbohydrate source is owing to synthesis of at least one, and possibly two, -glucosidase(s).     

On the mechanism of direct conversion of cellulose to ethanol by Fusarium oxysporum: effect of cellulase and β-glucosidase

Summary The effects of the three main enzymes involved in cellulose saccharification, namely cellobiohydrolase, carboxymethylcellulase and -glucosidase, on the direct conversion of cellulose to ethanol by Fusarium oxysporum F3 were investigated. Ethanol production was not affected when the activity of the former two enzymes was varied within a wide range. By contrast, -glucosidase markedly affected ethanol production showing an optimum level of 0.7–0.8 unit/ml growth medium. A significant decrease of cellulose bioconversion time to ethanol was obtained when -glucosidase activity was adjusted to this optimal level at the beginning of the fermentation process. Offprint requests to: B. J. Macris     

Production and characterization of β-glucosidases from different Aspergillus strains

-D-Glucosidase enzymes (-D-glucoside glucohydrolase, EC 3.2.1.21) from different Aspergillus strains (Aspergillus phoenicis, A. niger and A. carbonarius) were examined with respect to the enzyme production of the different strains using different carbon sources and to the effect of the pH and temperature on the enzyme activity and stability. An efficient and rapid purification procedure was used for purifying the enzymes. Kinetic experiments were carried out using p-nitrophenyl -D-glucopyranoside (pNPG) and cellobiose as substrates. Two different fermentation methods were employed in which the carbon source was glucose or wheat bran. Aspergillus carbonarius proved to be the less effective strain in -glucosidase production. Aspergillus phoenicis produced the highest amount of -glucosidase on glucose as carbon source however on wheat bran A. niger was the best enzyme producer. Each Aspergillus strain produced one single acidic -glucosidase with pI values in the range of pH 3.52–4.2. There was no significant difference considering the effect of the pH and temperature on the activity and stability among the enzymes from different origins. The enzymes examined have only -glucosidase activity. The kinetic parameters showed that all enzymes hydrolysed pNPG with higher efficiency than cellobiose. This shows that hydrophobic interaction plays an important role in substrate binding. The kinetic parameters demonstrated that there was no significant difference among the enzymes from different origins in hydrolysing pNPG and cellobiose as the substrates.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Production of cellulolytic enzymes from fungi and use in the saccharification of palm cake and palm fibre

Aspergillus niger ATCC 6275 possesses the highest carboxymethyl-cellulase, xylanase and -glucosidase activities under liquid and solid cultivations compared withMyceliophthora thermophila IFO 31843 and an isolate, F11. Palm cake proved to be a better substrate for enzyme production and saccharification than palm fibre. Saccharification of these two substrates, using crude enzyme solutions from three fungi and commercial enzymes, was investigated.     

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

Summary Thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released -glucosidase, -glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and -glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The -glucosidase, xylanase and cellulase from Thermotoga and two -glucosidases, a -glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.