A galactomannan-hydrolysing α-galactosidase from jack fruit (Artocarpus integrifolia) seed: Affinity chromatographic purification and properties

An acid α-galactosidase from the seeds of the jack fruit seed (Artocarpus integrifolia) has been purified to homogeneity by affinity chromatography on a matrix formed by cross-linking the soluble α-galactose-bearing guar seed galactomannan. The 35kDa enzyme was a homotetramer of 9.5kDa subunits. Its carbohydrate part (5.5%) was composed of galactose and arabinose. TheK _m withp-nitrophenyl α-D-galactoside as substrate was 0.35 mM. TheK _i values indicated inhibition by galactose, 1-O-methyl α-galactose and melibiose in the decreasing order. Among α-galactosides, the enzyme liberated galactose from melibiose, but not from raffinose or stachyose at its pH optimum (5.2). The guar seed galactomannan was however efficiently degalactosidated; limited enzyme treatment abolished the precipitability of the polysaccharide by the α-galactose-specific jack fruit seed lectin, and complete hydrolysis yielded insoluble polysaccharide. Though similar in sugar specificity and subunit assembly, α-galactosidase and the lectin coexisting in the jack fruit seed gave no indication of immunological identity.     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

α-Galactosidase of Bifidobacterium adolescentis DSM 20083

Bifidobacterium adolescentis was grown anaerobically in medium enriched with α-D-galactosides. α-Galactosidase (EC 3.2.1.22) was released from the cells by ultrasonic treatment and purified 36-fold by ultrafiltration, ammonium-sulphate precipitation, anion-exchange chromatography, and size-exclusion chromatography. Two protein bands were consistantly observed after sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretically homogeneous α-galactosidase was only obtained by electroelution. The enzyme had an apparent molecular mass of 344 kDa and 79 kDa as judged by size-exclusion chromatography and SDS-PAGE, respectively. Activity-staining after nondenaturing SDS-PAGE indicated an apparent molecular mass of 145 kDa. Thus, a tetrameric structure of the protein is suggested. The α-galactosidase showed optimal activity at pH 5.5 and 55°C. Lower pH values and higher temperatures rapidly inactivated α-galactosidase. The enzyme hydrolyzed specifically α-galactosidic linkages, and α-(1-3)-linkages were hydrolyzed at a higher rate compared to α-(1-6)-linkages. Hydrolysis of galactosides followed normal saturation kinetics; K_M-values for p-nitrophenyl-α-galactopyranoside (p-NPG) and raffinose were calculated with 0.957 mM and 4.12 mM, respectively. Received: 7 August 1998 / Accepted: 9 September 1998     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

α-L-rhamnosidase from Aspergillus clavato-nanicus MTCC-9611 active at alkaline pH

An a-L-rhamnosidase secreting fungal strain has been isolated and identified as Aspergillus clavato-nanicus MTCC-9611. The enzyme was purified to homogeneity from the culture filtrate of the fungus using concentration by ultrafiltration membrane and ion-exchange chromatography on CM-cellulose. The native PAGE analysis confirmed the homogeneity of the purified enzyme. The SDS-PAGE analysis of the purified enzyme revealed a single protein band corresponding to the molecular weight 82 kDa. The α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 had optimum at pH 10.0 and 50°C. The K _m values of the enzyme were 0.65 mM and 0.95 mM using p-nitrophenyl α-L-rhamnopyranoside and naringin as a substrates respectively. The enzyme transforms naringin to prunin at pH 10.0 and further hydrolysis of prunin to naringenin does not occur under these reaction conditions that makes α-L-rhamnosidase activity of Aspergillus clavato-nanicus MTCC-9611 promising enzyme to get prunin for pharmaceutical purposes.     

Purification and properties of α-glucose 1-phosphate-forming trehalose phosphorylase from a basidiomycete,Pleurotus ostreatus

Pleurotus ostreatus produced a high activity of α-glucose 1-phosphate (α-Glc 1-P) forming trehalose phosphorylase in vegetative mycelia and fruit-bodies. The enzyme was purified to homogeneity from the fruit-bodies by a procedure involving ammonium sulfate fractionation, DEAE-cellulose column chromatographies and cellulose phosphate column chromatographies. The enzyme catalyzes both the phosphorolysis of trehalose to produce α-Glc 1-P and glucose, and the synthesis of trehalose. It was not active toward other α- or β-glucosyl disaccharides and polysaccharides. The optimum pH was 7.0 for phosphorolysis and 6.4 for synthesis of trehalose. The Km values for trehalose and Pi in phospholytic reaction were 75 mM and 4.2 mM, respectively. Those for glucose and α-Glc 1-P in synthetic reaction were 505 mM and 38 mM, respectively. The estimated molecular mass by the sedimentation equilibrium method using an ultracentrifuge was 120 kDa. The molecular mass of the subunit (61 kDa) by SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a dimer of two identical subunits. The addition of glycerol higher than 25% into the enzyme solution stabilized its activity. The removal of phosphorus ions from the enzyme solution, by means of dialysis or electrophoresis, caused inactivation of the enzyme, probably by dissociation of the holoenzyme into the subunit proteins.     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999     

Expression and characterization of an <Emphasis Type="Italic">α</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 with potential for industrial application

In this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K _m and V _max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).     

Purification and characterization of cathepsin B from goat brain

Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withK _i value of 12.5 × l0⁻⁹M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.     

Purification and biochemical characterization of a soluble α-glucosidase from the parasite Entamoeba histolytica

A soluble α-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using a number of substrates indicated a preferential hydrolysis of isomaltose (α1,6) with much less activity on nigerose (α1,3) and maltose (α1,4). Trehalose (α1,1), kojibiose (α1,2) and cellobiose (β1,4) were not cleaved at all. As expected, isomaltose competed away hydrolysis of 4-methylumbelliferyl-α-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable K_i values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the α-glucosidase inhibitors 1-deoxynojirimycin, castanospermine and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited isomaltose hydrolysis in a competitive manner with K_i values of 1.2 and 1.5 μM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably involved in glycogen metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adverse effects of free fatty acid associated with increased oxidative stress in postischemic isolated rat hearts

The potential role of butyrate to modulate cellular metabolism through integrin receptor led to evaluation of its effect on collagen biosynthesis in cultured fibroblasts. Confluent human dermal fibroblasts were treated with 2 mM and 4 mM of sodium butyrate (NaB) for 48 h. It was found that butyrate induced collagen biosynthesis and prolidase activity independently of α₂β₁ integrin signaling. The expressions of both α₂ and β₁integrin subunits as well as integrin-induced activation of focal adhesion kinase (FAK) were not affected in the cells treated with NaB. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of Sos protein and MAP-kinases (ERK₁, ERK₂). The data suggests that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.     

<Emphasis Type="Italic">Dioscorea opposita</Emphasis> Thunb. α-mannosidase belongs to the glycosyl hydrolase family 38

α-Mannosidase (EC 3.2.1.24) was purified from ‘Iseimo’, a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting α-mannosidase activity. We purified α-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65°C. The K _m value for p-nitrophenyl-α-d-mannopyranoside was 0.35 ± 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved α-1,2 linkages preferentially to α-1,6 and α-1,3 linkages. The M _r of purified α-mannosidase was estimated to be 250–260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme’s properties indicated that Iseimo α-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

α-Chymotrypsin immobilized on ferromagnetic particles coated with titanium oxide: Production and catalytic characterization

Immobilization of α-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction. X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification. The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed by immobilized α-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The catalytic constant (25 s⁻¹) andK _M (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding characteristics for the free enzyme.     

Effect of ilvBN-encoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis

 Conversion of pyruvate to α-acetolactate, which is broken down to diacetyl and acetoin, can be catalysed by two α-acetolactate synthases in Lactococcus lactis. The enzyme encoded by the als gene (Als) has previously been shown to have a low affinity for pyruvate, which limits the formation of diacetyl. In this study we have expressed from a plasmid the ilvBN genes, which encode the other α-acetolactate synthase (IlvBN). This plasmid-directed enzyme expression provided up to 3.6-fold increased product formation in the L. lactis MG1363 and IL1403 backgrounds. Plasmid-based expression of the ilvBN genes, in an IL1403 derivative from which the leu.ilv.ald and flanking genes had been deleted, yielded up to 0.1 mM diacetyl whereas the host strain produced none. In addition, IlvBN, with a K _m value of 8.3 mM, was shown to have a greater affinity for pyruvate than does Als. Received: 28 June 1995 / Received revision: 26 September 1995 / Accepted: 29 September 1995     

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k ₁ but a larger k ₂. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.     

Effect of Methylglyoxal Modification of Human α-Crystallin on the Structure,Stability and Chaperone Function

α-Crystallin functions as a molecular chaperone and maintains transparency of eye lens by protecting other lens-proteins. Non-enzymatic glycation of α-crystallin by methylglyoxal, plays a crucial role on its chaperone function and structural stability. Our studies showed that methylglyoxal modification even in lower concentration caused significant decrease in chaperone function of α-crystallin as reflected both in thermal aggregation assay and enzyme refolding assay. Thermal denaturation studies showed drastic reduction of denaturation temperature with increase in the degree of modification. Thermodynamic stability studies by urea denaturation assay reflected a decrease of transition midpoint. Quantitatively we found that ΔG° of native α-crystallin decreased from 21.6 kJ/mol to 10.4 kJ/mol due to 72 h modification by 10 mM methylglyoxal. The surface hydrophobicity of α-crystallin after MG modification, was found to be decreased. Circular dichroism spectroscopy revealed conversion of β-sheet structure to random coil structure. Significant cross-linking was also observed due to methylglyoxal modification of human α-crystallin.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.