Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

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.     

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.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

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.     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.     

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.     

Stable β-glucosidase from Aureobasidium

A crude extract from Aureobasidium had β-glucosidase activity, hydrolysing cello-biose, methyl-β-D-glucoside, lactose, carboxymethylcellulose, avicel, o -nitrophenyl-β-D-glucoside and p -nitrophenyl-β-D-glucoside, and had favourable properties such as high pH and thermal stabilities. The optimum pH and temperature of the cello-biase activity were 4 and 80°C, respectively. The cellobiase activity was stable at pH 3–7 to 7.8 for at least 3 h, and retained 34 and 78% of its original activity at pH 1.5 and 9, respectively. Cellobiase activity was stable at 80°C for 15 min, and retained 81% of its original activity at 85°C.     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

β-Xylosidase and xylanase characterization and production by Streptomyces sp. CH-M-1035

Extracellular xylanase activity and cell-bound β-xylosidase production by a selected strain of Streptomyces sp. CH-M-1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell-bound β-xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β-xylosidase activity was obtained when Streptomyces sp. CH-M-1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH-M-1035 was grown on xylans, but not on agroindustrial residues.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

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.     

Proteolytic activity from a thermophilic Streptomyces megasporus strain SDP4

Multiple proteases secreted by a thermophilic actinomycete Streptomyces megasporus SDP4 after 18 h of growth at 55 °C are reported. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 6–12 and 25–85 °C, respectively. Optimum activity was observed at pH 8·0, pH 10·0 and 55 °C and was calcium independent. Thermostability was enhanced in the presence of 0·01 mol l⁻¹ calcium ions and half-life was 30 min at 85 °C. The enzyme was active in the presence of SDS. Both, EDTA and PMSF were partially inhibitory, indicating the presence of serine and metal requiring proteases. Three active zones in the range of 90–30 kDa were detected post-electrophoretically.     

Hyper production of β-glucosidase by an Aspergillus sp.

Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.