Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease.

Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease.     

β-Glucosidase Catalyzing Specific Hydrolysis of an Iridoid β-Glucoside from Plumeria obtusa

An iridoid β-glucoside, namely plumieride coumarate glucoside, was isolated from the Plumeria obtusa （white frangipani） flower. A β-glucosidase, purified to homogeneity from P. obtusa, could hydrolyze plumieride coumarate glucoside to its corresponding β-O-coumarylplumieride. Plumeria β-glucosidase is a monomeric glycoprotein with a molecular weight of 60.6 kDa and an isoelectric point of 4.90. The purified β-glucosidase had an optimum pH of 5.5 for p-nitrophenol （pNP）-β-D-glucoside and for its natural substrate. The Km values for pNP-β-D-glucoside and Plumeria β-glucoside were 5.04±0.36 mM and 1.02±0.06 mM, respectively. The enzyme had higher hydrolytic activity towards pNP-β-D-fucoside than pNP-β-D-glucoside. No activity was found for other pNP-glycosides. Interestingly, the enzyme showed a high specificity for the glucosyl group attached to the C-7＂ position of the coumaryl moiety of plumieride coumarate glucoside. The enzyme showed poor hydrolysis of 4-methylumbelliferyl-β-glucoside and esculin, and did not hydrolyze alkyl-β-glucosides, glucobioses, cyanogenic-β-glucosides, steroid β-glucosides, nor other iridoid β-glucosides. In conclusion, the Plumeria β-glucosidase shows high specificity for its natural substrate, plumieride coumarate glucoside.     

Galactosylsphingosine inhibition of the broad-specificity cytosolic beta-glucosidase of human liver

Glucosylsphingosine is a potent inhibitor of lysosomal glucocerebrosidase and the broad-specificity, cytosolic beta-glucosidase of human liver. In the present study, it was demonstrated that the broad-specificity beta-glucosidase was also inhibited by galactosylsphingosine. The inhibition was observed when the enzyme was assayed for beta-glucosidase, beta-galactosidase, beta-xylosidase, and alpha-arabinosidase activities. Inhibition was of the mixed-type and the degree of inhibition depended on the substrate. For example, galactosylsphingosine was a more potent inhibitor of beta-glucosidase activity (I0.5 = 0.3 mM) than beta-xylosidase activity (I0.5 = 1.2 mM). In addition, the observation that the broad-specificity, cytosolic beta-glucosidase was inhibited by hydrophobic glycosphingolipids prompted the definition of a revised purification procedure which took advantage of hydrophobic affinity chromatography. This revised purification scheme employed Octyl-Sepharose and yielded the largest (68,000 Da) and most active (470,000 nmol h-1 mg protein-1) beta-glucosidase preparation yet described. The beta-glucosidase preparation contained 19% serine and 17% glycine, while 24% of the total amino acids were hydrophobic. The results of this study document the presence of a sphingolipid binding site on the broad-specificity beta-glucosidase. The implications of galactosylsphingosine inhibition of cytosolic beta-glucosidase and the possible role of the enzyme in glycosphingolipid metabolism are discussed.     

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.     

Novel type of enzyme multimerization enhances substrate affinity of oat beta-glucosidase

Oat beta-glucosidase in plastid hydrolyzes avenacosides to C26-desgluco-avenacosides to combat against fungal infections. The enzyme has a unique quaternary protein structure of a three-dimensionally radiated assembly of long fibrillae. We elucidated the fibrillar assembly of oat type 1 beta-glucosidase by means of cryo-electron microscopy, enzyme kinetics and chemical modification. It was assembled by linear stacking of hollow trimeric units and the resulting fibril had a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. The enzyme active sites were located within the central tunnel. This unique multimer assembly increased enzyme affinity to avenacosides, in vivo substrates, and may function to discriminate avenacosides from many other kinds of beta-glucoside in oat. The fibrillar multimer of oat beta-glucosidase is a novel quaternary protein structure for enzyme supramolecular assembly that may have a functional role in the regulation of enzyme affinity.     

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.     

Sucrose gradient analysis of phospholipid-activated beta-glucosidase in type 1 and type 2 Gaucher's disease

Using sucrose density gradients, differences in delipidated lysosomal beta-glucosidase isolated from control spleen and spleen from patients with nonneurologic (type 1) and neurologic (type 2) Gaucher's disease have been examined. The three enzymes differ in sedimentation properties as well as in their responsiveness to activation by phosphatidylserine and heat-stable factor. The control beta-glucosidase sedimented as an apparent 45,000-Da species whose activity was dependent upon the inclusion of exogenous sodium taurodeoxycholate in the assay medium. Preincubation with a mixture of phosphatidylserine and heat-stable factor converted the control enzyme to a faster-sedimenting form which exhibited considerable activity in the absence of exogenous bile salt. Spleen beta-glucosidase from a patient with type 1 Gaucher's disease exhibited an apparent molecular weight of 154,000 on sucrose gradients. Like the control enzyme, the activity of this form was bile salt dependent. Upon preincubation with phosphatidylserine and heat-stable factor, beta-glucosidase from the type 1 case was also converted to a faster-sedimenting form which was more active in the absence of sodium taurodeoxycholate than in the presence of the bile salt. Spleen beta-glucosidase from the patient with type 2 Gaucher's disease sedimented as a broad peak of activity in the most dense regions of the sucrose gradients, appearing to be much larger than the beta-glucosidase from either the control or the type 1 Gaucher's disease patient. The activity of this large species was strongly dependent upon bile salt, and was not affected by preincubation of the enzyme with phosphatidylserine and heat-stable factor. Using the chaotropic salt, sodium thiocyanate (0.15 M), the spleen beta-glucosidase isolated from the type 1 Gaucher's disease case was converted to a slower-sedimenting species. The control enzyme sedimented slightly farther into the sucrose gradients upon treatment with the NaSCN. Thiocyanate treatment had no effect on the spleen beta-glucosidase isolated from the case of type 2 Gaucher's disease.     

A thermotolerant beta-glucosidase isolated from an endophytic fungi, Periconia sp., with a possible use for biomass conversion to sugars

A fungal strain, BCC2871 (Periconia sp.), was found to produce a thermotolerant beta-glucosidase, BGL I, with high potential for application in biomass conversion. The full-length gene encoding the target enzyme was identified and cloned into Pichia pastoris KM71. Similar to the native enzyme produced by BCC2871, the recombinant beta-glucosidase showed optimal temperature at 70 degrees C and optimal pH of 5 and 6. The enzyme continued to exhibit high activity even after long incubation at high temperature, retaining almost 60% of maximal activity after 1.5h at 70 degrees C. It was also stable under basic conditions, retaining almost 100% of maximal activity after incubation for 2h at pH8. The enzyme has high activity towards cellobiose and other synthetic substrates containing glycosyl groups as well as cellulosic activity toward carboxymethylcellulose. Thermostability of the enzyme was improved remarkably in the presence of cellobiose, glucose, or sucrose. This beta-glucosidase was able to hydrolyze rice straw into simple sugars. The addition of this beta-glucosidase to the rice straw hydrolysis reaction containing a commercial cellulase, Celluclast 1.5L (Novozyme, Denmark) resulted in increase of reducing sugars being released compared to the hydrolysis without the beta-glucosidase. This enzyme is a candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Purification, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp. alkalophilus.

An intracellular beta-glucosidase was purified from cell extracts of Bacillus circulans subsp. alkalophilus by NAD affinity and high-performance anion-exchange chromatographies. The enzyme was active against a wide range of aryl-beta-glucosides and beta-linked disaccharides. The structural gene for beta-glucosidase was cloned in Escherichia coli. The beta-glucosidase gene consisted of an open reading frame of 1,350 bp encoding a protein of 450 amino acids with a calculated M(r) of 51,303. The enzyme exhibited from 45 to 66% identity with five bacterial beta-glucosidases.     

Active site directed inhibition of a cytosolic beta-glucosidase from calf liver by bromoconduritol B epoxide and bromoconduritol F

Hydrolysis of p-nitrophenyl-beta-D-glucoside by cytosolic beta-glucosidase proceeds with retention of the anomeric configuration. Whereas inactivation of the enzyme by the glucosidase inhibitor conduritol B epoxide (CBE) was extremely slow (ki(max)/Ki 0.57 M-1 min-1) it reacted 130 times more rapidly with 6-bromo-6-deoxy-CBE (Br-CBE). The beta-glucosidase could be labeled with [3H]Br-CBE; incorporation of 1 mol inhibitor/mol enzyme resulted in complete loss of activity. Most of the bound inhibitor was released after denaturation and treatment with ammonia as (1,3,4/2,5,6)-6-bromocyclohexanepentol, thus demonstrating the formation of an ester bond with an active site carboxylate by trans-diaxial opening of the epoxide ring. It was concluded from the Ki values for the epoxide inhibitors and for coduritol B with the cytosolic enzyme and corresponding data for the lysosomal beta-glucosidase that the unusually low reactivity with CBE and Br-CBE is probably due to the inability of the cytosolic enzyme to effectively donate a proton to the epoxide oxygen. An extremely rapid inactivation of the cytosolic beta-glucosidase was caused by bromoconduritol F ((1,2,4/3)-1-bromo-2,3,4-trihydroxycyclohex-5-ene) with ki(max)/Ki 10(5) M-1 min-1. In contrast with the Br-CBE-inhibited enzyme the beta-glucosidase inhibited by bromoconduritol F was subject to spontaneous reactivation with t1/2 approximately 20 min.     

Specfic irreversible inhibition of sweet-almond beta-glucosidase by some beta-glycopyranosylepoxyalkanes and beta-d-glucopyranosyl isothiocyanate.

beta-D-Glucopyranosyl-(1S and 1R)-epoxyethanes (I and II), 1-(beta-D-glucopyranosyl)-(2R and 2S)-2,3-epoxypropanes (III and IV), beta-D-glucopyranosyl isothiocyanate (V) and beta-D-galactopyranosylepoxyethane (VI) are active-site-directed irreversible inhibitors of sweet-almond beta-glucosidase B (beta-D-Glucoside glucohydrolase, EC 3.2.1.21). Formation of the covalent bond is preceded by the binding of these inhibitors in the active site of the enzyme. This is testitified by the competitive character of inhibition of beta-glucosidase component B by compounds I-VI at the early period and by the protection of the enzyme from inactivation by its competitive inhibitors D-glucose and 1,5-D-gluconolactone. Epoxides I-IV are bound covalently with componet B at a molar ratio 1 : 1 as shown with the aid of 14C-labelled inhibitors. The release of the label from modified enzyme (E-I covalent) by treatment with hydroxylamine suggests the formation of an ester bond between inhibitors I-IV and the carboxyl group of the enzyme active site. The pH dependence curve of the inactivation rate of beta-glucosidase B is of a bell-shaped form for V and of a sigmoid character for I-IV and points to the involvement of the active site groups with pKa 5.6-5.9 and 4.2-4.4.     

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.     

Isozymes of β-Glucosidase in Dictyostelium discoideum

The activity of beta-glucosidase (EC 3.2.1.21) in extracts of Dictyostelium discoideum was investigated. The specific activity increased early in development, declined during pseudoplasmodium formation, and increased again during sorocarp formation. The beta-glucosidase which was present in growing amoebae and during the first stages of multicellular development was electrophoretically distinct from the enzyme which accumulated during the final stages of morphogenesis. Ribonucleic acid synthesis and protein synthesis during development were required for the accumulation of the later isozyme. Analysis of beta-glucosidase activity in a number of morphological mutants suggests that the enzyme which accumulates late in morphogenesis is developmentally controlled.     

Hydrolysis of a naturally occurring beta-glucoside by a broad-specificity beta-glucosidase from liver.

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.     

Human beta-glucosidase: inhibition by sulphates and purification by affinity chromatography on Dextran-sulphate-Sepharose

The acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) from human placenta is inhibited by sulphated macromolecules such as Dextran sulphate or chondroitin sulphate. This inhibition is alleviated by compounds such as crude taurocholate or phospholipids, which are known activators of acid beta-glucosidase. Partially-purified human beta-glucosidase will bind to Dextran sulphate linked to Sepharose 4B and can be eluted with low concentrations of crude sodium taurocholate. This procedure gives a 10-15 fold purification with good yield and has been included in a scheme giving an approx. 4000-fold purification of placental beta-glucosidase. Evidence is presented which suggests that phospholipids bind to beta-glucosidase by both ionic and hydrophobic interactions. The inhibition of enzyme activity caused by sulphated compounds and non-ionic detergents may be attributed to interference with, respectively, the ionic and hydrophobic binding of phospholipid to the enzyme.     

Heterogeneity in human acid beta-glucosidase revealed by cellulose-acetate electrophoresis

Cellulose-acetate gel electrophoresis, a technique commonly used for the separation of human acid hydrolases, was applied to study heterogeneity in acid beta-glucosidase (EC 3.2.1.45). With this technique, three forms of beta-glucosidase were distinguishable in extracts of several tissues. The most anodic beta-glucosidase activity (band 3) represents the broad-specificity beta-glucosidase that is not deficient in Gaucher disease and is not inhibited by conduritol B-epoxide (CBE). The beta-glucosidase activity was deficient in Gaucher disease. A third beta-glucosidase activity with an intermediate mobility (band 2) was also inhibited by CBE and deficient in Gaucher disease. Band 1 and band 2 beta-glucosidase thus represent different forms of glucocerebrosidase. By adding phosphatidylserine and sphingolipid activator protein (SAP-2), monomeric glucocerebrosidase could be completely converted into a form that comigrated with band 2 beta-glucosidase of tissue extracts. The addition of phosphatidylserine only also resulted in a changed mobility of the monomeric enzyme, but the migration in this case differed from that of band 2 beta-glucosidase of tissue extracts. The electrophoretic profile of beta-glucosidase activity of tissue extracts changed upon ethanol/chloroform extraction: the two glucocerebrosidase forms were converted into a band with a mobility identical to that of band 1 beta-glucosidase. Our findings indicate that the interaction of glucocerebrosidase with phospholipid and SAP-2 has major effects on the mobility of the enzyme in the cellulose-acetate gel electrophoresis system. The findings with the cellulose-acetate gel electrophoretic system are discussed in relation to the heterogeneity in glucocerebrosidase observed with sucrose density gradient analysis, immunochemical methods and isoelectric focussing studies.     

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.     

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.     

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).