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.     

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.     

Two β-glucosidase activities in Fibrobacter succinogenes S85

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides ) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated β -glucosidase active against cellobiose. The properties of the β -glucosidase activity have been investigated with the chromogenic substrate β -nitrophenyl β -D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the β -glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl β -D-glucoside, 5-bromo-4-chloro-3-indolyl β -D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated β -glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.     

Cell wall-associated isoflavonoids and β-glucosidase activity in Lupinus albus plants responding to environmental stimuli

Plants respond to various biotic or abiotic stimuli with different mechanisms and the mobilization of phenolic compounds is one of them. In white lupin (Lupinus albus L.) plants, isoflavones and their glucosides were localized in cell walls where the high constitutive activity of β‐glucosidase (EC 3·2·1·21) was also identified. The enzyme was partially purified from root cell walls. Its polymeric active form has 180 or 200 kDa as determined by non‐denaturing electrophoresis and gel filtration, respectively, and the isoelectric point is at pH 6·9. The enzyme is an exoglucosidase, preferentially hydrolysing conjugates of phenolic compounds with β anomers of glucose. It is not active against purified lupin cell walls. The specific β‐glucosidase activity varies in different tissues with the highest one in roots, and always higher in cell walls than in protoplast. The cell wall location of the enzyme was confirmed biochemically by its activity in intercellular washing fluids. Both aglycones and glycosides were also present in these fluids. The specific β‐glucosidase activity correlated well with the isoflavonoid aglycone/glycoside ratios in various tissues: the higher β‐glucosidase activity, the higher relative aglycone content. β‐glucosidase activity was found to be inducible under conditions of yeast elicitor treatment. Induction of the enzyme was accompanied by changes in the isoflavone secretion and accumulation, suggesting the regulatory role of β‐glucosidase activity in root exudation of isoflavonoids. No correlation however, was found between changes in β‐glucosidase activity and the presence of isoflavonoids in root exudates of plants grown under various nitrogen nutrition regimes.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

Abstract Using ferritin-conjugated antibodies as an electron microscopic marker, β-glucosidase was localized within the cell walls of the imperfect fungus Trichoderma reesei QM9414. With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chalcea , β-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane.     

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.     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

Tissue localization of β-glucosidase in rye, maize and wheat seedlings

The localization of β -glucosidase was determined at the tissue level in roots and shoots of rye, wheat and maize seedlings, using an immunohistochemical approach with antibodies directed against purified maize β -glucosidase as the primary antibody. In the roots, the β -glucosidase was found in the epidermis and the underlying cell layer. In the leaves, staining was seen in the epidermis (rye and wheat) and nearby vascular tissue (rye, wheat and maize). In all 3 species, β -glucosidase activity was highest in the coleoptile. Here the enzyme was restricted to the epidermis in wheat and to cells near the vascular tissue in maize, but was found in the whole tissue, except the vascular tissue, in rye. Maize, wheat and rye all contain hydroxamic acid glucosides and results are discussed in relation to a proposed role of β -glucosidase as part of a defense system releasing hydroxamic acid aglucone upon herbivore attack, pathogen penetration or aphid infestation.     

Characterization of pedicel β‐glucuronidase activity in developing maize (Zea mays) kernels

A conspicuous endogenous maize (Zea mays L.) β-glucuronidase (GUS) activity was observed in histochemical assays of non-transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue-specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p-nitrophenyl-β-D -glucuronide or 4-methylumbelliferyl-β-D -glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG-based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with K_m values for MUG of 2.2 and 2.7 µM for the early- and late-eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native M_r of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid-1,4-lactone. Only the early-eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5-bromo-4-chloro-3-indoyl-β-D -glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid-1,4-lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli-derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.     

Thiamine increases β-glucosidase production in the newly isolated strain of Fomitopsis pinicola

Aims:  To isolate a high β-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain.
Methods and Results:  A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l⁻¹) improved BGL production in F. pinicola cultures by 3·7-fold to give a specific activity of 114·4 μmol min⁻¹ mg⁻¹ protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported.
Conclusion:  Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812.
Significance and Impact of the Study:  Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.     

Hydrolysis of vicine and convicine from fababeans by microbial β-glucosidase enzymes

A.M. MCKAY. 1992. The toxic glycosides vicine and convicine which are present in fababeans have been implicated in favism, an anaemic disease of humans. Vicine and convicine concentrations are reduced by growth of Lactobacillus plantarum on fababean suspensions. The glycosides are eliminated from the fababean substrate by the growth of the filamentous fungus Fusarium graminearum. Incubation of fababean suspension with concentrated culture filtrate of Aspergillus oryzae , induced for extracellular β-glucosidase production, results in complete degradation of the glycosides. This study suggests a potential use of micro-organisms or microbial enzymes for detoxification of fababeans.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Influence of various bile salts on ββ-glucuronidase activity of intestinal bacteria

While the decrease of the β-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.     

Physical characterization of isozymes of endo-β-1,4-glucanase and β-1,4-glucosidase from Aspergillus species

Electrophoretic data revealed the presence of various isozymes of endoglucanase and beta-glucosidase, the number of which varied from one to three in various species of the genus Aspergillus. pH 5.0 was optimum for all the isozymes whereas metal ion treatment showed complete inhibition of almost all the isozymes by Hg2+ and partial inhibition by Ca2+ and Co2+ of isozymes of both the enzymes. An alteration in the electrophoretic mobility of isozymes of beta-glucosidase was also noticed in some species with Hg2+ treatment.     

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.     

Characterization of β-mannanase and β-mannosidase secreted from the yeast Trichosporon cutaneum JCM 2947

Y. ODA AND K. TONOMURA. 1996. β-Mannanase and β-mannosidase were purified from the culture fluid of the yeast Trichosporon cutaneum JCM 2947 (= T. beigelii CBS 5790). The molecular weights of the two enzymes were estimated to be 49 900 and 114000 by SDS-PAGE and 4500 and 193 000 by gel filtration, respectively. β-Mannanase contained 43% molecular weight as carbohydrate. The K _m and V _max values of β-mannanase for konjac glucomannan were 2.7 (mg ml^-1) and 10.6 (U mg protein^-1), and those of β-mannosidase for p -nitrophenyl β-D-mannopyranoside were 0.25 (mmol l^-1) and 91.7 (U mg protein^-1). Maximal activities were observed between pH 4.0 and 6.5 at 50°C for β-mannanase and around 6.5 at 40°C for β-mannosidase.