Antiviral isoflavonoid sulfate and steroidal glycosides from the fruits of Solanum torvum

The C-4 sulfated isoflavonoid, torvanol A (1), and the steroidal glycoside, torvoside H (3), together with the known glycoside, torvoside A (2), were isolated from a MeOH extract of Solanum torvum fruits. Upon enzymatic hydrolysis with beta-glucosidase, torvoside A (2) and torvoside H (3) yielded the corresponding acetal derivatives 4 and 5, respectively. Torvanol A (1), torvoside H (3) and compound 5 exhibited antiviral activity (herpes simplex virus type 1) with IC(50) values of 9.6, 23.2 and 17.4 microg/ml, respectively. Compounds 1-5 showed no cytotoxicity (at 50 microg/ml) against BC, KB and Vero cell lines.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

Arabidopsis thaliana beta-Glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides

In higher plants, beta-glucosidases belonging to glycoside hydrolase (GH) Family 1 have been implicated in several fundamental processes including lignification. Phylogenetic analysis of Arabidopsis thaliana GH Family 1 has revealed that At1g61810 (BGLU45), At1g61820 (BGLU46), and At4g21760 (BGLU47) cluster with Pinus contorta coniferin beta-glucosidase, leading to the hypothesis that their respective gene products may be involved in lignification by hydrolysing monolignol glucosides. To test this hypothesis, we cloned cDNAs encoding BGLU45 and BGLU46 and expressed them in Pichia pastoris. The recombinant enzymes were purified to apparent homogeneity by ammonium sulfate fractionation and hydrophobic interaction chromatography. Among natural substrates tested, BGLU45 exhibited narrow specificity toward the monolignol glucosides syringin (K(m), 5.1mM), coniferin (K(m), 7mM), and p-coumaryl glucoside, with relative hydrolytic rates of 100%, 87%, and 7%, respectively. BGLU46 exhibited broader substrate specificity, cleaving salicin (100%), p-coumaryl glucoside (71%; K(m), 2.2mM), phenyl-beta-d-glucoside (62%), coniferin (8%), syringin (6%), and arbutin (6%). Both enzymes also hydrolysed p- and o-nitrophenyl-beta-d-glucosides. Using RT-PCR, we showed that BGLU45 and BGLU46 are expressed strongly in organs that are major sites of lignin deposition. In inflorescence stems, both genes display increasing levels of expression from apex to base, matching the known increase in lignification. BGLU45, but not BGLU46, is expressed in siliques, whereas only BGLU46 is expressed in roots. Taken together with recently described monolignol glucosyltransferases [Lim et al., J. Biol. Chem. (2001) 276, 4344-4349], our enzymological and molecular data support the possibility of a monolignol glucoside/beta-glucosidase system in Arabidopsis lignification.     

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.     

An enzyme-polymer film prepared with the use of poly(vinyl alcohol) bearing photosensitive aromatic azido groups

Photochemical reaction of poly(vinyl alcohol) bearing aromatic azido groups was applied for immobilization of beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21.) in poly(vinyl alcohol) film. Photo-crosslinking and immobilization reactions proceeded by light irradiation for 25 min in air. The immobilized enzyme showed approx. 40% of its native enzyme activity with an apparent Michaelis constant of 3.9 mM. The Michaelis constant of the native enzyme was 2.3 mM. Some properties of the immobilized and native enzyme are compared.     

A H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates

Background

The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).

Methods

Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.

Results

The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.

Conclusions and general significance

The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.     

Functional expression of human liver cytosolic beta-glucosidase in Pichia pastoris. Insights into its role in the metabolism of dietary glucosides.

Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic beta-glucosidase (CBG) that hydrolyses various beta-d-glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated a detailed assessment of the enzyme specificity towards dietary glycosides. A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of approximately 10 mg x L-1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-beta-D-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (beta-D-fucosides, alpha-L-arabinosides, beta-D-glucosides, beta-D-galactosides, beta-L-xylosides, beta-D-arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (K(m)) and specificities (k(cat)/K(m)) for dietary xenobiotics than for other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.     

The action on cellulose and its derivatives of a purified 1,4-beta-glucanase from Trichoderma koningii.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.     

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.     

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.     

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis palustris</Emphasis> grown on microcrystalline cellulose

An extracellular beta-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC-MS/MS suggested that the protein has high homology with fungal beta-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-beta-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified beta-glucosidase was observed at pH 4.5 and 70 degrees. The F. palustris protein exhibited half-lives of 97 h at 55 degrees and 15 h at 65 degrees, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-beta-lactoside, p-nitrophenyl-beta-xyloside, p-nitrophenyl-alpha-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the beta-glucosidase from F. palustris can be classified as an aryl-beta-glucosidase with cellobiase activity.     

Purification and Properties of beta-Glucosidase from Aspergillus terreus

A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification.     

Production, Purification, and Properties of a Thermostable beta-Glucosidase from a Color Variant Strain of Aureobasidium pullulans

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced beta-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular beta-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mumol . min . mg of protein under these conditions. The purified beta-glucosidase was active against p-nitrophenyl-beta-d-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with K(m) values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K(i) = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified beta-glucosidase (0.21 U/ml) from A. pullulans.     

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.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Purification of an isoflavonoid 7-O-beta-apiosyl-glucoside beta-glycosidase and its substrates from Dalbergia nigrescens Kurz

A beta-glycosidase was purified from the seeds of Dalbergia nigescens Kurz based on its ability to hydrolyse p-nitrophenyl beta-glucoside and beta-fucoside. This enzyme did not hydrolyze various glycosidic substrates efficiently, so it was used to identify its own natural substrates. Two substrates were identified, isolated and their structures determined as: compound 1, dalpatein 7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside and compound 2, 6,2',4',5'-tetramethoxy-7-hydroxy-7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside (dalnigrein7-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside). The beta-glycosidase removes the sugar from these glycosides as a disaccharide, despite its initial identification as a beta-glucosidase and beta-fucosidase.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).