ß- Glucosidases from leaves and roots of the common beet,Beta vulgaris

?-Glucosidases detected in the leaves and roots of the common beet,Beta vulgaris, have been demonstrated to catalyze hydrolysis of native betacyanins. A method is described for the isolation and purification of ?glucosidase from the roots, which involves ammonium sulfate precipitation, DEAE-cellulose chromatography, and Sephadex gel filtration. Maximum activity of the enzyme is detected at 50°C and pH 8.0; it retains stability within the pH range from 5.1 to 9.2. In leaves, ?-glucosidase is associated with chloroplast membranes; solubilization of the membranes results in enzyme inactivation.     

Podophyllum peltatum possesses a beta-glucosidase with high substrate specificity for the aryltetralin lignan podophyllotoxin

A beta-glucosidase with high specificity for podophyllotoxin-4-O-beta-D-glucopyranoside was purified from the leaves of Podophyllum peltatum. The 65-kDa polypeptide had optimum activity at pH 5.0 and was essentially inactive at pH 6.5 or above. Maximum catalytic activity of this glucosidase was obtained at 45 degrees C, but the enzyme was not heat stable. This beta-glucosidase displayed higher substrate specificity for podophyllotoxin-4-O-beta-D-glucopyranoside than for the other lignans tested, and for the (1-->3) linkage of laminaribiose than for other glucosidic linkages.     

An isoflavone conjugate-hydrolyzing beta-glucosidase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and cellular localization

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 '-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.     

Study on the production of acetyl esterase and side-group cleaving glycosidases of ammonia fungi

Acetyl esterase was found to be widely distributed in ammonia fungi in a screen comprising 26 species (71 strains). No great differences appeared in enzyme production for acetyl esterase, beta-xylosidase, alpha-arabinosidase, or beta-glucosidase between different strains of the same species, but differences were detected between different genera. Acetyl esterase of Coprinus phlyctidosporus and Lyophyllum tylicolor may act cooperatively with beta-glucosidase. An increase in urea concentration significantly affected enzyme activity. It was supposed that urea used as 20 mg/g litter may solubilize leaf nutrients. At 20 mg urea added/g litter, a sizable increase in beta-glucosidase activity of C. phlyctidosporus and L. tylicolor was found, whereas a decrease in enzyme production of alpha-arabinosidase and beta-xylosidase was detected in some strains. Acetyl esterase and beta-glucosidase of C. phlyctidosporus, L. tylicolor, C. leucocephala 589, and C. rhombisperma 248 were most active in acidic conditions (pH 5.3-6.3), whereas acetyl esterase of L. nuda 561 and L. tarda 564 was most active in alkaline conditions (pH 8.3).     

Sugar transport: Occurrence of trehalase activity in sugar cane

Summary Trehalase activity was detected in extracts of roots, leaves, and stalk tissue from sugar cane. The enzyme was not bound to cell particulates, and had a pH optimum of 6.2 and a Michaelis constant for trehalose of 1×10^-4 M. The level of enzyme detected in mature stalk tissue was too low to account for glucose transport into tissue slices. The enzyme level was high in immature stalk tissue in which the vacuolar sugar pool turns over rapidly. Trehalose synthesis and breakdown may be part of a system for transport of hexose out from the vacuole.     

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.     

日本根霉IFO5318胞外β—葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe~(3 )、Hg~2 )等对酶活力有抑制作用。     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.     

Association of beta-glucosidase with intact cells of Thermoactinomyces.

The location of the B-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little beta-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude beta-glucosidase preparation were determined to be pH 6.5 and 50--55 degrees C. Enzyme activity studies indicate that the same enzyme(s) accounts for the beta-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.     

Purification and characterization of a beta-glucosidase from Trichoderma reesei

A beta-glucosidase has been purified from culture filtrates of the fungus Trichoderma reesei QM9414 grown on microcrystalline cellulose. The beta-glucosidase was purified using two successive DEAE-Sephadex anion-exchange chromatography steps, followed by SP-Sephadex cation-exchange chromatography and concanavalin-A--agarose chromatography. Evidence for homogeneity is provided by polyacrylamide disc gel electrophoretic patterns, which show a single protein band. Sedimentation equilibrium analysis yielded a molecular mass of 74.6 +/- 2.4 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis yielded a single protein band with a molecular mass of 81.6 kDa. Thus, the enzyme appears to be a single, monomeric polypeptide. The beta-glucosidase is isoelectric at pH 8.5. The enzyme is rich in basic amino acids and contains few half-cystine and methionine residues. The purified beta-glucosidase contains less than 1% by weight of neutral carbohydrate. The beta-glucosidase catalyzes the hydrolysis of cellobiose, p-nitrophenyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside; the values of V/Km for each substrate were determined to be 2.3 X 10(4), 6.9 X 10(5) and 2.9 X 10(6) M-1 S-1 respectively. The enzyme is optimally active from pH 4.5 to 5.0 and is labile at higher hydrogen ion concentrations. The beta-glucosidase has an unusually high affinity for D-glucose (Ki = 700 microM). Comparison of inhibition constants for cello-oligosaccharides suggests that the substrate-binding region of the beta-glucosidase comprises multiple subsites.     

One-step purification and characterization of an intracellular β-glucosidase from Metschnikowia pulcherrima

A collection of 60 non-Saccharomyces yeasts isolated from grape musts in Uruguayan vineyards was screened for beta-glucosidase activity and Metschnikowia pulcherrima was the best source of this enzyme activity. Its major beta-glucosidase was successfully purified to homogeneity by ion-exchange chromatography on amino-agarose gel. The enzyme exhibited an optimum catalytic activity at 50 degrees C and pH 4.5 and was active against (1 --> 4)-beta and (1 --> 2)-beta glycosidic linkages. In spite of preserving 100% of its activity and stability in the presence of 12% (v/v) ethanol and 5 g glucose/l, the enzyme was unstable below pH 4. We characterized the beta-glucosidase from M. pulcherrima with a view to its potential applications in wine-making.     

Characterization of beta-glucosidase from corn stover and its application in simultaneous saccharification and fermentation

Purification and characterization of beta-glucosidase from corn stover was performed and the enzyme was tried in SSF to evaluate the suitability of plant glycosyl hydrolases in lignocellulose conversion. A beta-glucosidase with M(w) of 62.4 kDa was purified to homogeneity from post-harvest corn stover. The following physicochemical and kinetic parameters of the beta-glucosidase were studied respectively: optimum temperature, thermal stability, optimum pH, pH stability, K(m), V(max), V(i), cellobiose inhibition, tryptic peptide mass spectrometry and effect of metal ions and other reagents on the activity. The beta-glucosidase activity on salicin was optimal at pH 4.8 and 37 degrees C. The unique property of optimum temperature makes the beta-glucosidase potentially useful in SSF. In SSF of steam explosion pretreated corn stover, the supplementation of the purified beta-glucosidase was more effective than Aspergillus niger beta-glucosidase.     

The beta-glucosidase in the gut contents of the snail Achatina achatina.

1. The enzyme beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from the gut contents of active Achatina achatina exists in two molecular forms, beta-glucosidase C (mol.wt. about 82000) and D (mol.wt. about 41000). 2. Only the lower-molecular-weight species was found in the gut contents of aestivating snails or in extracts from their digestive glands and washed gut walls. 3. On re-activation of some aestivating snails, betion of ATP and Mg2+ to the isolated gut contents or to extracts from washed gut walls led to the formation of higher-molecular-weight forms of the enzyme, beta-glucosidase A (mol.wt. about 329000) and beta-glucosidase B (mol.wt. about 165000). 5. All these forms of the enzyme have similar pH optimum (pH 5.0-5.6). 6. The Michaelis constants (Km) and heat stability of the enzyme increased with increasing molecular complexity.     

Purification and characterization of a ribonuclease from barley roots

A ribonuclease isolated from barley malt roots exhibited characteristics that conformed to those of RNase I (EC 3.1.27.1). It differed from RNase I from barley leaves and barley seeds in its action on polynucleotides and on 3′,5′-dinucleoside monophosphates, and from barley seed RNase I in its optimum pH. Gel electrophoresis indicated that the enzyme was present in the embryo, roots, shoot and endosperm of germinating barley. The enzyme showed pH optimum at 5.0, isoclectric pH at 4.5, a thermal optimum of 50°, and an apparent molocular weight of 19 000.     

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.     

Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification

Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.     

Influence of ammonium and nitrate nutrition on enzymatic activity in soybean and sunflower

Under conditions of controlled pH, nitrate and ammonium are equally effective in supporting the growth of young soybean (Glycine max var. Bansei) and sunflower (Helianthus annuus L. var., Mammoth Russian) plans. Soybean contains an active nitrate reductase in roots and leaves, but the low specific activity of this enzyme in sunflower leaves indicates a dependency upon the roots for nitrate reduction. Suppression of nitrate reductase activity in sunflower leaves may be due to high concentrations of ammonia received from the roots. Nitrate reductase activity in leaves of nitrate-supplied soybean and sunflower follows closely the distribution of nitrate reductase. For the roots of both species, glutamic acid dehydrogenase activity was greater with ammonium than with nitrate. The glutamic acid dehydrogenase of ammonium roots is wholly NADH-dependent, whereas that of nitrate roots is active with NADH and NADPH. In leaves, an NADPH-dependent glutamic acid dehydrogenase appears to be responsible for the assimilation of translocated ammonia and ammonia formed by nitrate reduction.     

Photomodulation of strigolactone biosynthesis and accumulation during sunflower seedling growth

Present investigations report the presence of strigolactones (SLs) and photomodulation of their biosynthesis in sunflower seedlings (roots, cotyledons and first pair of leaves) during early phase of seedling development. Qualitative analyses and characterization by HPLC, ESI-MS and FT-IR revealed the presence of more than one type of SLs. Orobanchyl acetate was detected both in roots and leaves. Five-deoxystrigol, sorgolactone and orobanchol were exclusively detected in seedling roots. Sorgomol was detectable only in leaves. HPLC eluted fraction from seedling roots and leaves co-chromatographing with GR24 (a synthetic SL) could also bring about germination in Orobanche cernua (a weed) seeds, which are established to exhibit SL – mediated germination, thereby indicating the SL identity of the eluates using this bioassay. SLs accumulation was always more in the roots of light-grown seedlings, it being maximum at 4 d stage. Although significant activity of carotenoid cleavage dioxygenase (CCD, the enzyme critical for SL biosynthesis) was detected in 2 d old seedling roots, SLs remained undetectable in cotyledons at all stages of development and also in the roots of 2 d old light and dark-grown seedlings. Roots of light-grown seedlings showed maximum CCD activity during early (2 d) stage of development, thereby confirming photomodulation of enzyme activity. These observations indicate the migration of a probable light-sensitized signaling molecule (yet to be identified) or a SL precursor from light exposed aerial parts to the seedling roots maintained in dark. Thus, a photomodulation and migration of SL precursor/s is evident from the present work.     

Production and Characterization of Cellulase and beta-Glucosidase from a Mutant of Alternaria alternata

A mutant of Alternaria alternata excreted enhanced levels of carboxymethylcellulase and particularly beta-glucosidase when grown in cellulose liquid media. Both enzymes were purified two- to four-fold by ammonium sulfate precipitation and gel filtration, and the kinetic data showed K(m) values of 16.64 mg/ml of culture fluid for carboxymethylcellulase and 0.14 mM p-nitrophenyl-beta-d-glucoside and 0.81 mM cellobiose for beta-glucosidase at pH 5. Carboxymethylcellulase and extracellular beta-glucosidase functioned optimally at pH 5 to 6 and 4.5 to 5 and at temperatures of 55 to 60 and 70 to 75 degrees C, respectively. Both temperature optima and thermostability of beta-glucosidase were among the highest ever reported for the same enzyme excreted from cellulase and beta-glucosidase hyperproducing microorganisms.     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.