α-Glucosidase Activity Located at the Cell Surface in Callus of Convolvulus arvensis

Cell wall preparations of Convolvulus callus were found to contain α-glucosidase activity, the bulk of which could be solubilized by solutions of high ionic strength. Callus tissue incubated in 0.5 M KC1 released α-glucosidase activity into the washing medium as distinct from tissue incubated in 1.0 M sorbitol. The wall-bound activity of KCl-treated tissue was found to be less than that of sorbitol-treated tissue, while the difference between both activities proved to be equal to the enzyme activity found in the washing medium of KCl-treated tissue. Since no trace of cell leakage was observed, it is concluded that α-glucosidase activity is located at the cell surface. The level of this surface-located enzyme was not affected by the presence of maltose in the nutrient medium.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

Hydrolytic Enzymes of Euglena gracilis: Characterization and Activity as a Function of Culture Age and Carbon Deprivation*†

SYNOPSIS. Optimal assay conditions are described for 8 hydrolases of Euglena gracilis var. bacillaris, SM-L1 (streptomycinbleached) strain, 7 of which have an acid pH-optimum. Acid phosphatase, β-galactosidase, β-glucosidase, β-fucosidase, cathepsin D, RNase, DNase, and an esterase are active in cell homogenates. Amylase has very low activity, and β-glucuronidase, arylsulfatase, β, N-acetyl-glucosaminidase, α-fucosidase, and α- and β-mannosidase are inactive. Hydrolase activity increases as a culture proceeds from the midexponential to the late stationary-phase of growth, being most pronounced in the case of β-glucosidase. In cultures deprived of a utilizable carbon source, the specific activities of the hydrolases (per mg total protein or dry weight) increase. When expressed on a per cell basis, however, the activities of DNase decrease while those of β-galactosidase, cathepsin D, and RNase increase. The hydrolases appear to be involved in the adaptation of Euglena to the metabolic demands imposed by different conditions of growth.     

Stability and expression of a cloned α-glucosidase gene inZymomonas mobilis grown in batch and continuous culture

Summary Strains ofZymomonas mobilis containing an -glucosidase gene cloned fromBacillus brevis strain 27-7 (NRRL B-4389) on the plasmid pNSW358 showed varying degrees of stability in batch culture under non-selective conditions. After 45 generations of growth in continuous culture, pNSW358 was stable inZ.mobilis strain ZM6100 and the specific activity of -glucosidase in these cells was 2.7 nmol/min/mg protein. Lysed cell extracts confirmed the activity of the -glucosidase enzyme in ZM6100(pNSW358) with 21 g/1 ethanol in 50 (82% theoretical conversion of maltose to ethanol). ZM6100(pNSW358) whole cells showed a very slow conversion rate on maltose as a sole carbon source with only 5.3 g/1 ethanol after 30 days on 100 g/l maltose medium.     

Cloning and functional expression of thermostable β-glucosidase gene from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis>

A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.     

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.     

Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non-photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton-symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH-dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha-phenylglucoside, carbonyl cyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane-spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Métabolisme de l’Acide 2-Aminoéthylphosphonique (Ciliatine) chez le Poulet

For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.     

New antihyperglycemic, α-glucosidase inhibitory,and cytotoxic derivatives of benzimidazoles

Glycosidases play an important role in a wide range of physiological and pathological conditions, and have become potential targets for the discovery and development of agents useful for the treatment of diseases such as diabetes, cancer, influenza, and even AIDS. In this study, several benzimidazole derivatives were prepared from o-phenylenediamine and aromatic and heteroaromatic carboxaldehydes in very good yields, using PdCl₂(CH₃CN)₂ as the most efficient catalyst. Synthesized compounds were assayed for their activity on yeast and rat intestinal α-glucosidase inhibition and cytotoxic activity against colon carcinoma cell line HT-29. Compound 3e exhibited 95.6% and 75.3% inhibition of yeast and rat intestinal α-glucosidase enzyme, while showing 74.8% cytotoxic activity against the HT-29 cell line at primary screening concentrations of 2.1?mM for yeast and rat intestinal α-glucosidase enzyme and 0.2?mM for cytotoxic activity against the HT-29 cell line, respectively. Compound 3c displayed 76% and 34.4% inhibition of yeast and rat intestinal α-glucosidase enzyme, and 80.4% cytotoxic activity against the HT-29 cell line at similar primary screening concentrations. The IC₅₀ value for the most potent intestinal α-glucosidase inhibitor compound 3e was found to be 99.4?μM. The IC₅₀ values for the most active cytotoxic compounds 3c and 3e were 82?μM and 98.8?μM, respectively. Both compounds displayed significant antihyperglycemic activity in starch-induced postprandial hyperglycemia in rats. This is the first report assigning yeast and rat intestinal α-glucosidase enzyme inhibition, cytotoxic activity against the HT-29 cell line, and antihyperglycemic activity to benzimidazole compounds 3c and 3e.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

Distribution of Yeast Cell Wall Lytic Activity among Microorganisms

An α-glucosidase has been isolated from the mycelia of Penicillium purpurogenum in electrophoretically homogeneous form, and its properties have been investigated. The enzyme had a molecular weight of 120,000 and an isoelectric point of pH 3.2. The enzyme had a pH optimum at 3.0 to 5.0 with maltose as substrate. The enzyme hydrolyzed not only maltose but also amylose, amylopectin, glycogen, and soluble starch, and glucose was the sole product from these substrates. The Km value for maltose was 6.94×10^?4 m. The enzyme hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. The enzyme had α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme also catalyzed the transfer of α-glucosyl residue from maltose to riboflavin.     

Consequences of disrupting the gene that encodes α-glucosidase II in the N-linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum

We have identified and disrupted the gene coding for α-glucosidase II in Dictyostelium discoideum. This enzyme is responsible for removing two α1,3-linked glucose residues from N-linked oligosaccharides on newly synthesized glycoproteins. Mutagenesis by restriction enzyme-mediated integration (REMI) generated a clone, DG1033, which grows well but forms abnormal fruiting bodies with short, thick stalks. The strain lacks α-glucosidase II activity and makes incompletely processed N-linked oligosaccharides that are abnormally large and have fewer sulfate and phosphate esters. The morphological, enzymatic, and oligosaccharide profile phenotypes of the disruption mutant are all recapitulated by a targeted disruption of the normal gene. Furthermore, all of these defects are corrected in cells transformed with a normal, full-length copy of the gene. The phenotypic characteristics of DG1033 as well as chromosomal mapping of the disrupted gene indicate that it is the site of the previously characterized modA mutation. The Dictyostelium gene is highly homologous to α-glucosidase II genes in the human and the pig, C. elegans, and yeast. Although various cell lines have been reported to be defective in α-glucosidase II activity, disruption of the Dictyostelium gene gives the first example of a clear developmental phenotype associated with loss of this enzyme. Dev. Genet. 21:177–186, 1997. © 1997 Wiley-Liss, Inc.     

Cloning of the α-Amylase cDNA of Aspergillus shirousamii and Its Expression in Saccharomyces cerevisiae

α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3? non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Purification,characterization and gene analysis of a new α-glucosidase from <Emphasis Type="Italic">shiraia</Emphasis> sp. SUPER-H168

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe²⁺, Cu²⁺, and Ag⁺ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K _m, V _max, and k _cat/K _m of the α-glucosidase were 0.52 mM, 3.76 U mg^?1, and 1.3?×?10⁴ L s^?1 mol^?1, respectively. K _m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.     

α-glucosidase in Mycoplasma mycoides subspecies capri

Abstract Mycoplasma mycoides subsp. capri utilisede maltose in medium lacking serum and hence serum saccharolytic enzymes. The presence of α-glucosidase activity was demonstrated by p-nitrophenyl-α- d -glucoside hydrolysis in toluene-treated cells. Specific activities were approx. 4-fold higher in cells grown in the presence of maltose than in cells grown with other sugars or with glucose plus maltose. Extracellular activity was < 2% of cellular activity in growing cultures. α-Glucosidase activity was also demonstrated in cells grown in medium containing serum. It is suggested that the presence of α-glucosidase might be of value in mycoplasma chatacterisation; in a previous study, α-glucosidase activity was not detected in Mycoplasma mycoides subsp. mycoides .     

Enhancement of maltose utilisation by Saccharomyces cerevisiae in medium containing fermentable hexoses

Saccharomyces cerevisiae are unable to maintain high rates of fermentation during transition from catabolism of hexoses to maltose. This phenomenon, termed ‘maltose lag’, presents problems for the baking, brewing and distilling industries, which rely on yeast catabolism of mixtures of hexoses and maltose. Maltose utilisation requires the presence of maltose permease and α-glucosidase (maltase), encoded by MAL genes. Synthesis of these is induced by maltose and repressed by glucose. One strain of baker’s yeast used in this work exhibited a marked maltose lag, whereas a second strain exhibited a shorter lag during conversion from hexose to maltose metabolism. The extent of the lag was linked to the levels of maltose permease and maltase in cells at the time of inoculation into mixed sugar medium. This view is supported by results showing that pulsing yeast with maltose to induce expression of MAL genes prior to inoculation into mixed sugar medium, enhanced sugar fermentation. Maltose pulsing of yeasts could therefore be useful for enhancing some fermentations relevant to baking and other yeast industries. Received 24 December 1988/ Accepted in revised form 18 March 1999     

Inducible α-glucosidase of Mucor rouxii: Effects of dimorphism on the development of the wall-bound activity

Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.