A simple method for the purification of over-expressed extracellular sucrase of Zymomonas mobilis from a recombinant Escherichia coli

The over-expressed extracellular sucrase (SacC) of Zymomonas mobilisfrom a recombinant Escherichia coli (pZSP62) carrying the sacC gene was purified partially by repeated cycles of freezing and thawing. This method separated the highly expressed recombinant protein from the bulk of endogenous E. coli proteins. The enzyme was further purified 14 fold with a 55% yield from the cellular extract of E. coli by hydroxyapatite chromatography. The purified enzyme had a Mr of 46 kDa by SDS-PAGE. Its km value for sucrose was 86 mM and was optimal at pH 5.0 and at 36°C.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

Purification and properties of a thermostable pullulanase from Clostridium thermosulfurogenes EM1 which hydrolyses both α-1,6 and α-1,4-glycosidic linkages

Summary A novel thermostable pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes EM1, was purified and characterized. Applying anion exchange chromatography and gel filtration the enzyme was purified 47-fold and had a specific activity of 200 units/mg. The molecular mass of this thermostable enzyme was determined to be 102 000 daltons and consisted of a single subunit. The enzyme was able to attack specifically the -1,6-glycosidic linkages in pullulan and caused its complete hydrolysis to maltotriose. Surprisingly and unlike the enzyme from Klebsiella pneumoniae, the purified enzyme from this anaerobic thermophile exhibited, in addition to its debranching and pullulanase activity, an -1,4 hydrolysing activity as well. By the action of this single polypeptide chain various branched and linear polysaccharides were completely converted to two major products, namely maltose and maltotriose. The K _m values of this enzyme for pullulan and amylose were determined to be 1.33 mg/ml and 0.38 mg/ml, respectively. This debranching enzyme displays a temperature optimum at 60°–65° C and a pH optimum at 5.5–6.0. The application of this new class of pullulanase (pullulanase type II) in industry will significantly enhance the starch saccharification process. Offprint requests to: G. Antranikian     

Bovine testicular heparin sulfamidase

Heparin sulfamidase was purified 2200-fold in 7% yield from bovine testis by a procedure which consisted of homogenization of the tissue in the presence of Triton X-100, ammonium sulfate fractionation, chromatography on concanavalin A-Sepharose, chromatography on heparin-Sepharose, and chromatofocusing. As shown by SDS-PAGE, the most highly purified preparation contained one major component, which had a mobility corresponding to a molecular weight of 55 000. Chromatography of the active enzyme on Sephacryl S-300 indicated a molecular weight of 100 000, suggesting that the native enzyme consisted of two subunits. K_M for the purified enzyme was 1.6 M, assayed with [N-³⁵S]heparin as a substrate; V_max was 4.4 pmol of sulfate released per min per g of protein. A pH of 6.7 was estimated from the elution profile on chromatofocusing and was corroborated by cold focusing electrophoresis. The enzyme was optimally active between pH 4.8 and 5.1. Maximum activity was observed in the presence of 0.175 M NaCl, while lower activities were observed upon addition of other salts.     

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.     

Isolation and Characterization of Chitosanase from the Strain Bacillus sp. 739

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 was determined. Maximum enzyme activity was observed in a medium containing biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity by chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. The temperature and pH optima of the purified chitosanase were in the ranges 45–55°C and 6.0–6.5, respectively. Time to half-maximum inactivation of the enzyme at 50°C was equal to 1 h. With colloidal chitosan as the substrate, the value of K of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

Purification of α-amylase from Bacillus licheniformis by chromatofocusing and gel filtration chromatography

A combination of chromatofocusing and gel filtration chromatography resulted in a simple purification of -amylase from Bacillus licheniformis. The purification was approximately 77-fold. Identification of the purity was established by SDS–PAGE. Molecular weight and isoelectric point of the purified enzyme were 58 kDa and 7.18 respectively. Western blot analysis confirms the specificity of antibody raised against purified -amylase.     

An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans

Summary An acetyl esterase was purified from Trichoderma reesei by cation and anion exchange chromatography. The enzyme had a molecular weight of 45 000 as determined by SDS-electrophoresis, or 67 000 as determined by gel filtration. In chromatofocusing the enzyme was shown to consist of two isoenzymes with isoelectric points of 6.8 and 6.0. The enzyme showed activity towards naphthyl acetate, triacetin and glucose-and xylose acetates. However, it liberated acetic acid from acetylated xylo-oligomers only to a small extent. The liberation of acetic acid from the oligomeric substrate was enhanced by addition of endoxylanase and -xylosidase.     

Characterization of pullulan-hydrolysing enzyme from an alkalopsychrotrophic Micrococcus sp.

Summary A pullulan-hydrolysing enzyme of Micrococcus sp. 207 was purified to an electrophoretically homogenous state by chromatography on DEAE-Toyopearl, -cyclodextrin-Sepharose and Asahipak GS-520P. The purified enzyme was free of -amylase activity. The molecular weight of the enzyme as estimated by SDS-PAGE was 120,000 and the pI value as determined by isoelectric focusing was 4.9. The enzyme was most active at pH 8.0 and 50°C. The enzyme was activated by the addition of CaCl₂, but its thermoresistance increased after removing free Ca²⁺ ions. The enzyme could hydrolyse the -1,6-linkages of amylopectins, glycogens and pullulan and the K _m value for pullulan was about 0.018%. Pullulan at concentrations above 0.012% inhibited the enzyme activity and the activity was competitively inhibited by cyclodextrins. Offprint requests to: K. Horikoshi     

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.The authors contributed equally to this work.     

Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the strain

Summary A cholesterol-oxidase-producing microorganism, strain COX629, isolated from soil was identified as Pseudomonas sp. The cholesterol oxidase produced by Pseudomonas sp. strain COX629 was purified 2400-fold to homogeneity in an overall yield of 60% from culture broth. The enzyme was a monomer with a molecular weight of 56 000, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Sephadex G-150 gel column chromatography. The enzyme showed optimum activity at pH 7.0 and was stable over a rather wide pH range of 4.0 to 11.0. The enzyme showed a high substrate specificity for 3-hydroxysteroids and the K _m value for the oxidation of cholesterol by this enzyme was about 0.2 mM. A characteristic of the enzyme is marked stability at high temperature.     

Characterization of alginate lyase (ALYII) from Pseudomonas sp. OS-ALG-9 expressed in recombinant Escherichia coli

An alginate lyase named ALYII was purified to homogeneity from Escherichia coli JM109 carrying a recombinant plasmid, pJK26 harbouring the alyII gene from Pseudomonas sp. OS-ALG-9 by column chromatography with DEAE-cellulose, CM-Sephadex C-50, butyl-Toyopearl 650 M and isoelectric focusing. The molecular size of the purified ALYII was estimated to be 79 kDa by SDS-PAGE and its pI was 8.3. The enzyme was most active at pH 7.0 and 30 °C. Its activity was completely inhibited by Hg²⁺. The enzyme was poly -D-1, 4-mannuronate-specific rather than -D-1, 4-guluronate-specific and it showed a promotion effect in alginate degradation by combination with ALY, an another poly -D-1, 4-mannuronate-specific alginate lyase from the same strain.     

Purification and Characterization of Serine Hydroxymethyltransferase from Mitochondria of Euglena gracilis z

Mitochondrial serine hydroxymethyltransferase, l-serine: tetrahydrofolate 5,10-hydroxymethyl-transferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with l-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88,000 by gel filtration through Sephadex G-200, and 44,000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5′-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had l-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity.     

Partial purification of γ-glutamyltransferase from human brain microvessels

Two forms of gamma-glutamyltransferase from human brain cortex microvessels were partially purified by gel permeation and ion-exchange and group-affinity chromatography. The specific activity of the purified preparations was 320-fold (detergent form) and 830-fold (proteolytic form) higher than that of the enzyme in the brain cortex homogenate. The relative molecular mass of the proteolytic form of the enzyme was about 90,000 as determined by gel permeation chromatography. The major part of the enzyme (about 80%) was absorbed on Con A-Sepharose 4B. The pH optima for transfer reactions with -glutamyl-4-nitroanilide as donor and glycylglycine andl-cystine as acceptors were in the range of 8.2 to 9.0. The studied enzyme was inhibited by a mixture ofl-serine and borate and by bromcresol green.