α-D-galactose-specific lectin from jack fruit (A rtocarpus integra) seed

An α-D-galactose-specific lectin from the seeds of jack fruit (Artocarpus integra) has been isolated in pure form by affinity chromatography on immobilised guar gum (a galactomannan). The lectin is shown to be a glycoprotein containing 3% carbohydrate and having a molecular weight of 39,500 as determined by gel filtration. Sodium dodecyl sulphate gel electrophoresis revealed a single polypeptide of 10,500 dalton, indicating that the native lectin is a tetrarner of identical subunits. The hemagglutinating activity of the lectin towards erythrocytes of all blood groups is found to be the same.     

A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties

An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60–80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-d-galactopyranoside, α-d-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-d-galactoside and 12 mM versus α-d-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg²⁺ and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of –SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.     

Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger

The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl α-galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k_cat/K_m values for hydrolysis of the branched α-galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched α-galactosides, but not linear raffinose family oligosaccharides.     

The purification and regulatory properties of α-oxoglutarate dehydrogenase from Acinetobacter lwoffi

The alpha-oxoglutarate dehydrogenase multienzyme complex was purified from Acinetobacter lwoffi to a high degree of homogeneity as shown by gel electrophoresis and analytical ultracentrifugation. Sedimentation-velocity analyses gave s(20,w) values which increased with increasing protein concentration, suggesting dissociation of the complex in dilute solution. The maximum s(20,w) value thereby obtained and the value determined by active enzyme centrifugation were both in the range 28-29S. Electron micrographs of the complex indicated a molecular diameter of 20-22nm (200-220A). The overall activity of the complex was inhibited by NADH, and kinetic studies indicated sites of action on the first and third enzyme components. AMP and ADP relieved this inhibition and also stimulated enzyme activity. Assays specific for the first enzyme component showed this to be the site of action of the adenylates. The activity of the complex varied with energy charge in a manner consistent with its role in energy metabolism.     

Production,purification, and properties of an α-l-arabinofuranosidase from Cihomitus squalens

A white-rot fungus Dichomitus squalens, when grown on 1% wheat-straw glucuronoarabinoxylan under aerated submerged conditions, secreted an α-l-arabinofuranosidase (4.3 nkat/mL). The enzyme was purified 70-fold by ammonium sulfate precipitation, chromatofocusing on PBE 94, gel filtration on Ultrogel AcA 54, rechromatofocusing on PBE 94, and lectin affinity chromatography on Concanavalin A-Ultrogel. The enzyme is a glycoprotein having a molecular weight of 60,000 and a pI of 5.1. The enzyme exhibited maximal activity at pH 3.5 and at 60°, and was fully inactivated within 30 min at 70°. The K_m value for p-nitrophenyl α-l-arabinofuranoside was 1.64mm. The α-l-arabinofuranosidase liberated arabinose from sugar-beet arabinan, wheat-straw and oat-spelt arabinoxylans, and wheat-bran heteroxylan, and was inactive towards gum arabic.     

Extracellular α-amylase from Thermus filiformis Ork A2: purification and biochemical characterization

An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH₂-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a K _m of 5.0 mg·ml⁻¹ and k _cat/K _m of 5.2 × 10⁵ ml·mg⁻¹ s⁻¹. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an α-amylase from a strain of the genus Thermus. Received: June 2, 1997 / Accepted: September 16, 1997     

Substrate-dependent production and some properties of a thermostable, α-galactosidase from Rhodothermus marinus

-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 U g^–1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85°C, with a half-life of 2 h at 75°C, and had a broad pH range (4–8).     

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     

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts – partial purification and biochemical properties

 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn²⁺ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K _m values for Mn²⁺, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000     

A novel metagenome-derived β-galactosidase: gene cloning, overexpression, purification and characterization

A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na⁺, K⁺, or Ca²⁺ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study.     

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the -galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH₂ terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH₂ terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/ factor and killer factor. A comparison of the derived amino acid sequence of this -galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the -galactosidase from Escherichia coli.     

Semisynthetic β-lactam antibiotics synthesizing enzyme from Acetobacter turbidans: purification and properties

Semisynthetic cephalosporin synthesizing enzyme has been purified from cell-free extract of Acetobacter turbidans ATCC 9325 by ion-exchange, hydrophobic chromatography and gel filtration. The purified enzyme migrated as two bands on SDS-gel electrophoresis and as six bands on native gel electrophoresis. This enzyme has an isoelectric point at 5.8 and contains most of the essential amino acids. The molecular weight was estimated to be 280 000 to 290 000 by gel filtration. Two different subunits of this enzyme having molecular weights of 70 000 and 72 000 have been identified in the presence of sodium dodecyl sulphate. The purified enzyme favours the synthetic reaction over the hydrolytic reaction by a factor of 2.6 times, as determined by the ratio of relative activities.     

A novel cold-adapted β-galactosidase isolated from Halomonas sp. S62: gene cloning, purification and enzymatic characterization

A novel 1,170 bp β-galactosidase gene sequence from Halomonas sp. S62 (BGalH) was identified through whole genome sequencing and was submitted to GenBank (Accession No. JQ337961). The BGalH gene was heterologously expressed in Escherichia coli BL21(DE3) cells, and the enzymatic properties of recombinant BGalH were studied. According to the polyacrylamide gel electrophoresis results and the sequence alignment analysis, BGalH is a dimeric protein and cannot be classified into one of the known β-galactosidase families (GH1, GH2, GH35, GH42). The optimal pH and temperature were determined to be 7.0 and 45 °C, respectively; the K _m and K _cat were 2.9 mM and 390.3 s^?1, respectively, for the reaction with the substrate ortho-nitrophenyl-β-d-galactopyranoside. At 0–20 °C, BGalH exhibited 50–70 % activity relative to its activity under the optimal conditions. BGalH was stable over a wide range of pHs (6.0–8.5) after a 1 h incubation (>93 % relative activity) and was thermostable at 50 °C and below (>60 % relative activity). The enzyme hydrolyzes lactose completely in milk over 24 h at 7 °C. The characteristics of this novel β-galactosidase suggest that BGalH may be a good candidate for medical researches and food industry applications.     

Xyloglucan mobilisation and purification of a (XLLG/XLXG) specific β-galactosidase from cotyledons of Copaifera langsdorffii

The storage xyloglucan of germinating seeds of Copaifera langsdorffii is degraded by the action of β-galactosidase, endo-β-glucanase, α-xylosidase and β-glucosidase, producing free galactose, glucose and xylose. One of the β-galactosidases from cotyledons of germinating seeds of C. langsdorffii was purified by ion exchange and gel chromatography (Biogel P-60), leading to a single polypeptide (molecular mass 40 kDa). The enzyme has optimum activity at pH 3.2 (stable from pH 2.3 to 6.0) and is active on p-NP-β-gal (K_m 3.5 mM) and lactose but not on o-NP-β-gal or p-NP-β-gal. Small amounts of galactose were released from xyloglucan of seeds of C. langsdorffii, Tamarindus indica and less from Hymenaea courbaril. No galactose was released after incubation with β-1,4-linked galactan from Lupinus angustifolius cotyledons. Much higher activity was observed on oligosaccharides obtained by hydrolysis of C. langsdorffii xyloglucan with Trichoderma viride cellulase. The purified β-galactosidase attacked XLLG and XLXG specifically, producing a mixture of XXXG and XXLG (unsubstituted glucose is assigned G; glucose branched with xylose is assigned X and if galactose is branching xylose, the trisaccharide is assigned L). Considering the recent discovery by Crombie and co-workers that (L) at the non-reducing end of the oligosaccharides prevents β-glucosidase from acting on GLXG or GLLG but not on GXLG or GXXG, the β-galactosidase isolated in this work seems to perform a key role in xyloglucan degradation since it is responsible for the retrieval of a major sterical hindrance (L) for further hydrolysis of the oligosaccharides and therefore essential for completion of xyloglucan mobilisation.     

Adenosine 5′-triphosphate–arginine phosphotransferase from lobster muscle: purification and properties

1. A simple chromatographic method is described for the purification of arginine kinase from lobster (Homarus vulgaris) muscle. 2. Some physical properties and the effects on enzyme activity of ionic strength, pH, buffer salts, metal ions and substrates are reported. 3. The kinetic parameters, evaluated by variation of the concentration of one of the substrates, are dependent on the concentration of the other substrate. 4. The properties of the enzyme are discussed in relation to previous findings about phosphagen phosphotransferases.     

Arabinoxylan-degrading enzyme system of the fungusAspergillus awamori: purification and properties of an α-l-arabinofuranosidase

An -l-arabinofuranosidase produced by the fungusAspergillus awamori had molecular mass of approximately 64 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) and was optimally active at pH 4.6 and 50°C. The enzyme, which chromatographed as a single component on SDS-PAGE, appeared to consist of two iso-enzymes of pI 3.6 and 3.2. Acting in isolation, the -l-arabinofuranosidase had only a very limited capacity to releasel-arabinose (less than 11%) directly from arabinoxylans that had been extracted from a number of plant cell wall preparations using 18% alkali, but a much higher proportion of thel-arabinose (46%) was released from a wheat straw arabinoxylan that had been isolated by steam treatment. There was a marked synergistic effect between the -l-arabinofuranosidase and an endo-(1 4)--d-xylanase produced byA. awamori in both the rate and extent of the release ofl-arabinose from both oat straw and wheat straw arabinoxylans, suggesting thatl-arabinose-substituted oligosaccharides generated by the endoxylanase action were better substrates for enzyme action. A novel property of the -l-arabinofurasidase was its capacity to release a substantial proportion (42%) of feruloyll-arabinose from intact wheat straw arabinoxylan. The concerted action of the -l-arabinofuranosidase and endoxylanase released 71% of the feruloyll-arabinose and 69% of thep-coumaroyll-arabinose substituents from the wheat straw arabinoxylan.     

Purification, cloning, and properties of α-galactosidase from Saccharopolyspora erythraea and its use as a reporter system

An -galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K_m for p-nitrophenyl--d-glucopyranoside (pNPG), K_m for melibiose and the V_max are similar to those of other studied -galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related -galactosidase enzymes. This fragment was used as a probe to clone the -galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique -galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The -galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl--d-galactosidase (X--gal) on agar media and pNPG in liquid media.     

Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase,and properties of the products

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.     

Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization,purification, and properties

Adenosine 5-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native M_r measured by gel-filtration was 350 000. The subunit M_r was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0–9.8 with a peak at pH 9.5. The apparentK _m value for adenosine 5-phosphosulfate (APS) was 2.1 M, when dithiothreitol was acceptor substrate. 3-Phosphoadenosine 5-phosphosulfate and inosine 5-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparentK _m = 1.5 mM) and dithioerythritol (apparentK _m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentK _m = 0.6 mM; the initial velocity was maximal at 3.0–4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25°C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na₂SO₄ or (NH₄)₂SO₄, and in the presence of APS or its analogs (AMP and -methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na₂SO₄, and by the analogs of APS.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - -m-APS -methylene-adenosine 5-phosphosulfate - DTT dithiothreitol - IPS inosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate We wish to thank Mr. I. Kashiwase, Mr. Y. Endo and Mr. Y. Mimura, School of Fisheries Sciences, Kitasato University, for their technical assistance in this study. The research described in this paper was partly supported by the Kitasato Research Grant (H5-9 and H6-13 to N.K.).