Characterization of an endo-1,3(4)-β-d-glucanase gene from Cellvibrio mixtus

Abstract An endo-1,3(4)-β- d -glucanase gene ( cwd2 ) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb Pst I fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli , degraded both β-1,3 glucans and β-1,3–1,4 mixed-linkage glucans, was entohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40°C respectively. The M _r of specifically labelled Cwd2 was approximately 34 000. This gene was quite distinct from two other C. mixtus β-1,3 glucanases previously described.     

Isolation and analysis of a gene encoding α-glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan

This is the first report describing the analysis of a gene encoding an α-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for α-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The α-glucuronidase gene ( aguA ) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli , and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78 593 Da. The α-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS–PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (<630kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2- O -(4- O -methyl-α- d -glucopyranosyluronic acid)- d -xylobiose (MeGlcAX₂) to xylobiose and 4- O -methylglucuronic acid. The K _m for MeGlcAX₂ was 0.95mM. The pH optimum was 6.3. Maximum activity was measured at 85°C, about 25°C or more above the values reported for all other α-glucuronidases known to date. When incubated at 55–75°C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.     

Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity

Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a p I of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co²⁺ and Zn²⁺, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. K _m values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.     

Production of β-glucosidase (cellobiase) by Curvularia sp.

A Curvularia sp. isolated from soil was found to produce extracellular β-glucosidase activity when grown in yeast extract, peptone, carboxymethylcellulose (YPC) medium. An initial medium pH of 6·5 and cultivation temperature of 30°C were found to be most suitable for high enzyme productivity. The pH and temperature optima for the enzyme were 4·0 and 70°C, respectively. Under these conditions, the enzyme exhibited a K_m (0-nitrophenyl-β- d -glucoside) value of 0.20 mmol/l. Several divalent metal ions inhibited enzyme activity at high concentration. EDTA. also inhibited β-glucosidase activity.     

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice ( Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10–50°C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30–40°C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40°C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30–40°C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40°C were substantially higher than those grown at 10, 20 and 50°C. Furthermore, the activities of (1→3),(1→4)- β -glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1→3),(1→4)- β -glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30–40°C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of β -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of β -glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.     

Adenine Deaminase Activity of the yicP Gene Product of Escherichia coli

During previous work on deriving inosine-producing mutants of Escherichia coli, we observed that an excess of adenine added to the culture medium was quickly converted to hypoxanthine. This phenomenon was still apparent after disruption of the known adenosine deaminase gene (add) on the E. coli chromosome, suggesting that, like Bacillus subtilis, E. coli has an adenine deaminase. As the yicP gene of E. coli shares about 35% identity with the B. subtilis adenine deaminase gene (ade), we cloned yicP from the E. coli genome and developed a strain that overexpressed its product. The enzyme was purified from a cell extract of E. coli harboring a plasmid containing the cloned yicP gene, and had significant adenine deaminase [EC 3.5.4.2] activity. It was deduced to be a homodimer, each subunit having a molecular mass of 60 kDa. The enzyme required manganese ions as a cofactor, and adenine was its only substrate. Its optimum pH was 6.5-7.0 and its optimum temperature was 60°C. The apparent K_m for adenine was 0.8 mM.     

Evaluation of C-EC-agar, a modified mFC-agar for the simultaneous enumeration of faecal coliforms and Escherichia coli in water samples

A new medium, C-EC-agar (Biolife, Milan, Italy), was evaluated for the simultaneous enumeration by membrane filtration of faecal coliforms and Escherichia coli in water. The medium is a modification of m-faecal coliform agar, from which the aniline blue and lactose have been omitted and 4-methylumbelliferyl-β-D-glucuronide, 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside and isopropyl-β- d -thiogalactoside added. At 44°C E. coli gives blue-green colonies that fluoresce under u.v.-light (366 nm) and give a reddish-violet colour when Kovac's reagent is placed on the membrane. Under similar conditions, faecal coliform colonies do not fluoresce. To increase recovery on the medium, repair of sub-lethally injured cells by a 4-h incubation at 37°C on tryptic soy agar is recommended.     

Purification and properties of a BT-glucosidase of Hanseniaspora vineae Van der Walt and Tscheuschner with the view to its utilization in fruit aroma liberation

The β-glucosidase of Hanseniaspora vineae was purified by ion-exchange chromatography and gel filtration. Its molecular weight was 295000 PT 15000, its optimum pH was between 6 and 6–5, and its optimum temperature was 55°C. The enzyme was active against different soluble glucosides with β(1–2), β(1–3), β(1–4), β(1–6) and even aP(1–4) configurations. A glucosyltransferase activity appeared in the presence of ethanol. The enzyme was constitutive but its synthesis was repressed by glucose.     

β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.     

Purification and characterization of a β-glucosidase from the phytopathogenic fungus Fusarium oxysporum f. sp. melonis

T.M. ALCONADA AND M.J. MARTÍNEZ. 1996. Fusarium oxysporum f. sp. melonis produces cellulase and β-glucosidase activities in a medium with glucose and avicel as carbon source. A β-glucosidase from this crude material was purified by gel filtration and ion exchange chromatography successively. This enzyme is a unique band of protein in SDS-PAGE and isoelectric focussing. It had a molecular weight of 66000 and a pI of 5. Using p -nitrophenyl-β-D-glucopyranoside as substrate β-glucosidase shows a K _m of 210 μmol 1^-1, an optimum pH of 5.5 and an optimum reaction temperature of 60°C, being stable in a pH range of 5–7 for 48 h at room temperature.     

Expression and purification of a recombinant enantioselective amidase

Microbacterium sp. AJ115 metabolises a wide range of nitriles using the two-step nitrile hydratase/amidase pathway. In this study, the amidase gene of Microbacterium sp. AJ115 has been inserted into the pCal-n-EK expression vector and expressed in Escherichia coli BL21(DE3)pLysS. The expressed protein is active in E. coli and expression of the amidase gene allows E. coli to grow on acetamide as sole carbon and/or nitrogen source. Expression of active amidase in E. coli was temperature dependent with high activity found when cultures were grown between 20 and 30 degrees C but no activity at 37 degrees C. On induction, the amidase represents 28% of the total soluble protein in E. coli. The expressed amidase has been purified in a single step from the crude lysate using the calmodulin-binding peptide (CBP) affinity tag. The V(max) and K(m) of the purified enzyme with acetamide (50 mM) were 4.4 micromol/min/mg protein and 4.5mM, respectively. The temperature optimum was found to be 50 degrees C. Purified enzyme demonstrated enantioselectivity with the ability to preferentially act on the S enantiomer of racemic (R,S)-2-phenylpropionamide. S-2-phenylpropionic acid is produced with an enantiomeric excess of >82% at 50% conversion of the parent amide.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Characterization of a cell wall β-galactosidase of Cicer arietinum epicotyls involved in cell wall autolysis

β-Galactosidase (EC 3.2.1.23) has been established as the main enzyme involved in the autolytic process. The enzyme extracted from cell walls of epicotyls of Cicer arietinum L. cv. Castellana with 3 M LiCl is a 45 kDa protein composed of a single subunit, having an optimum pH of 4; an optimum temperature of 45°C and K_m and V_max of 1.72 m M and 18.5 nkat (mg protein)^–1 respectively, as evaluated against p -nitrophenyl-β- d -galactopyranoside. The enzyme is inhibited by Hg²⁺, d -galactono-1,4-lactone and galactose, substances that also inhibit the autolytic process. Ca²⁺ and EDTA, which do not affect the activity of the β-gaiactosidase, do however modify the hydrolysis of the cell wall mediated by the enzyme, and they also inhibit the autolytic process. Ca²⁺ decreased both processes, whereas EDTA increased them; and when both substances were added together, their individual effects were neutralized. The effects of both agents is probably due to modifications in the cell wall that prevent access of the enzyme to its substrate.     

Isolation and characterization of the enzymes involved in disintegration of the cell wall of Chlorella fusca

Using different chromatographical methods we have isolated an enzyme from the sporangia of Chlorella fusca Shihira et Krauss var. vacuolata , strain SAG 211–8b, which is responsible for the partial disintegration of the sporangium wall. We refer to it as carbohydrate-releasing activity (CRA). It is an endoenzyme and splits oligosaccharides from the inner layer of the cell wall. In appropriate tests it shows β- d -fucosidase activity (EC 3.2.1.,38): The protein has a molecular weight around 45 kDa and an isoelectric point of pH 4.3; maximum activity is found at pH 5.4 and 60°C, although this temperature inactivates the enzyme quickly. β- d -Mannosidase (EC 3.2.1.25) and β- d -glucosidase (EC 3.2.1.21) were also found in the presence of CRA. These glycosidases were identified as exoenzymes. They are involved in the further degradation of the liberated obligosaccharides.     

Detection of induced β-galactosidase activity in individual non-culturable cells of pathogenic bacteria by quantitative cytological assay

One Escherichia coli and two F lac ⁺ Salmonella strains were carbon and nitrogen stressed at 37°C over 35 days in the presence or absence of chloramphenicol; the number, activity and culturability of cells in the resultant populations were studied. Active cells were enumerated by fluorescence microscopy after treatment with the lac inducer IPTG and cytological assay for β-galactosidase. In all experiments, active and total cell counts remained within a three-fold range of each other and their initial values, while culturability fell by >10⁸-fold and 10³-fold in chloramphenicol-treated and untreated preparations, respectively. Quantitative image analysis revealed different distributions of cell-specific fluorescence and indicated a progressive decline in the levels of induced enzyme activity in both E. coli and Salmonella enteritidis . It was concluded that the non-culturable cells studied retained inducible enzyme activity and that this activity did not result from a starvation-induced programme of gene expression. Whether or not such active but non-culturable cells are viable, they are clearly responsive and have the potential to influence their environment. The assay described can be applied to heterogeneous populations and environments and shows considerable potential for the study of gene expression at the single cell level.     

Native β-glucuronidase activity in sugarbeet (Beta vulgaris)

β-Glucuronidase (EC 3.2.1.31) activity, initially thought absent from plants, has been found in a number of plant families. During an analysis of Agrobacterium -mediated transformation of sugarbeet ( Beta vulgaris L.), significant glucuronidase activity was observed in control (non-transformed) tissues when the fluorogenic substrates 4-methylumbelliferyl-β- d -glucuronic acid, resorufin glucuronic acid and 3-carboxyum-belliferyl-β- d -glucuronic acid were used to quantify β-glucuronidase activity under standard protocol conditions. Similarly, the colorigenic substrate p -nitrophenyl-β- d -glucuronide was hydrolyzed by this sugarbeet-derived glucuronidase. Biochemical and immunological data are presented to indicate significant differences between sugarbeet-derived glucuronidase and that from Escherichia coli (EC 3.2.1.31) encoded by gusA . These differences provide means of distinguishing between the two activities in extracts that contain a mixture of both. Use of X-glue, the substrate utilized in histochemical localizations of glucuronidase activity, gave no reaction product (i.e., indigo precipitate) at pH 7.0. However, at pH 3.0, 4.0 and 5.0 formation of the indigo precipitate was evident within 1 h at 37°C in sugarbeet callus and by 4 h in leaves and petioles. The specific activity of sugarbeet glucuronidase was observed to be strongly pH dependent, with an optimum near pH 4.0. The use of various β-glucuronidase assay techniques as applied to transformation of sugarbeet is discussed.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca

An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds, and was most active against cellobiose (V_max= 29, K _m = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose. Received: 21 August 2000 / Accepted: 4 October 2000