Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties

Summary Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria.Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg²⁺ instability at 37 °C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides.In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S_0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 °C (P_0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S_0.5 and P_0.5 values were.In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S_0.5 as well as P_0.5 values do not vary significantly from one tissue to another.     

Deoxyguanosine kinase from human placenta

Deoxyguanosine kinase (ATP:deoxyguanosine 5'-phosphotransferase) has been purified up to a specific activity of 10.3 nmol/min per mg protein from human placenta. The enzyme appears to have a molecular weight of 58 000 from the results of Sephadex G-75 gel filtration. The enzyme catalyzed phosphorylation of deoxyguanosine and deoxyadenosine, but deoxycytidine was not phosphorylated. An apparent Km value for deoxyguanosine was 2.5 micro M. When ATP was used as a phosphate donor, the pH optimum was at pH 6.0, but the optimum was shifted to pH 6.8 by the addition of dTTP. At physiological pH, the activity was stimulated 3-4-fold by dTTP. dTTP was also an effective phosphate donor, but using dTTP as a phosphate donor, a broad pH optimum of 7.0 was observed. Two Km values of 0.13 and 2.2 mM were obtained for both MgATP2- and MgdTTP2-. The activity was strongly inhibited by dGTP and dGDP; 50% inhibition by 1.0 micro M dGTP and 2.1 micro M dGDP, respectively. The enzyme required the presence o Mg2+ or Mn2+.     

Production,purification and partial characterization of an endopolygalacturonase fromCryptococcus albidus var.albidus

Cryptococcus albidus var.albidus produced an extracellular endo-polygalacturonase (poly (1,4--d-galacturonide) glycanohydrolase EC 3.2.1.15) when grown in a synthetic medium containing one of a variety of pectic substances or galacturonic acid. The highest level of enzyme activity (15.5 VU-ml^–1) was obtained after 72 h of growth on 1.0% low-methoxyl pectin. The enzyme, purified by gel filtration (Sephadex G-100) after repeated ammonium sulphate precipitation and dialysis, showed only one band by polyacrylamide gel electrophoresis and had the following properties: mol wt (MW_r) 41000 dal; isoelectric point (pl) = 8.10 ± 0.10; optimum temperature and pH for activity around 37°C and pH 3.75, respectively; pH stability in the pH range 4.0 to 8.0; complete heat inactivation after 10 min at 55°C; Km and Vmax values 5.7· 10^–1 mg·ml^–1 and 5.1 · 10^–1 mmoles·min^–1, respectively.     

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.     

Linamarase from Fusarium equiseti

Summary Linamarase from Fusarium equiseti was purified 56-fold by ammonium sulphate fractionation, gel filtration on sephadex G-150 and ion-exchange chromatography on DEAE-sephadex A-50-120. The enzyme has Kms of 0.36 mM and 0.51 mM with respect to the artifical substrate, p-nitrophenyl--d-glucoside and its natural substrate, linamarin, respectively. -Gluconolactone is a strong competitive inhibitor of the enzyme with Ki of 0.2 mM when p-nitrophenyl--d-glucoside is the substrate. The pH optimum of the enzyme is 6.0 and further analysis indicates that the carboxyl of aspartate or glutamate, imidazole of histidine and the sulphhydryl group of cysteine may be involved in enzyme catalysis. The F. equiseti linamarase is significantly inhibited by such thiol-group reagents as iodoacetate, p-hydroxymercuribenzoate and iodosobenzoate indicating that sulphhydryl group may be present at the active site of the enzyme. The enzyme is stable up to 45°C which corresponds approximately with the optimum temperature of the linamarase-catalysed hydrolysis whose Ea was found to be 8.21 kcal/mole.     

Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe.

A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.     

Purification and some properties of two extracellular proteolytic enzymes produced byVibrio SA1

The purification and characterisation of an extracellular endo and aminopeptidase of the marineVibrio SA1 is described. The endopeptidase was purified by ammonium sulphate precipitation, gel filtration and affinity chromatography. It had a molecular weight of approximately 31 000, a pH optimum at 7.8 and a temperature optimum at 50 C. The enzyme was rapidly inactivated at 65 C.The aminopeptidase was purified by ammonium sulphate precipitation, gel filtration and preparative polyacrylamide gel electrophoresis. This enzyme had a molecular weight of approximately 21 000, a pH optimum at 8.6 and a temperature optimum at 60 C. Both proteases were inactivated by EDTA while reactivation occurred by Ca²⁺, Zn²⁺ and Mg²⁺ ions.The endopeptidase hydrolysed several peptide bonds in the oxidized B-chain of insulin, particularly those involving amino groups of hydrophobic amino acid residues with bulky side chains. It was unable to hydrolyse synthetic dipeptides, but a number of tripeptides were hydrolysed at a low rate. The aminopeptidase hydrolysed leucinamide and di- and tripeptides containing hydrophobic bulky amino acids as the N-terminal residue. It was concluded that the endopeptidase and the aminopeptidase ofVibrio SA1 possess complementary specificities.Abbreviations T= Triethylenetetramine - S= Succinic acid - PIPES= Piperazine-N,Nbis(2-ethane sulfonic acid) - HEPES= N-2-hydroxy-ethylpiperazine-N-2-ethane sulfonic acid Part of this study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

东亚钳蝎毒透明质酸酶的纯化和部分性质的研究

用CM-SephadexC50,CM-SephadexC25和SephadexG-75凝胶过滤,从东亚钳蝎毒中提纯蝎毒透明质酸酶,应用低pH系统不连续聚丙烯酰胺凝胶圆盘电泳,SDS-不连续聚丙烯酰胺凝胶垂直板电泳鉴定均为单一条带,活力提高34倍,产率为12％,纯品无出血活性,无神经毒性。用凝胶过滤法和SDS电泳法测得分子量为54000,PAS染色证实为糖蛋白。 纯化的透明质酸酶的最适pH为4.5～6.5,最适温度为37℃,该酶对热的稳定性比蛇毒透明质酸酶高一些,但在碱性环境中也易失活。0.15MNaCl对酶活性有明显稳定作用,Fe~(2+)、Fe~(3+)及肝素对酶活性有明显的抑制作用,Cu~(2+)对酶活力也有一定影响。     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.     

Purification and characterization of an extracellular chitobiase fromTrichoderma harzianum

Chitobiase (EC 3.2.1.29), from the culture filtrate ofTrichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose andp-nitrophenyl--N-acetyl glucosamine with apparent Km of 575 µM and 235 µM, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50°C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.     

Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis

Summary An extracellular -glucanase from Bacillus licheniformis has been isolated and characterized. Isolation has been performed by salting out and gel filtration chromatography, yielding a homogeneous active component with a molecular mass of 27 000–28 000 daltons and an isoelectric point of 4.7. In addition to being quite a thermostable protein (optimal temperature 55°C) the enzyme is active under a wide range of conditions including pH (4.0–10.5), and in the presence of a large number of metal ions, sodium dodecylsulphate and ethylenediaminetetraacetate. The simple purification procedure and useful properties make this enzyme suitable for brewing processes.     

Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L

Partial purification (approximately 270-fold) of sucrose synthase (EC 2.4.1.13) from developing cotyledons of Vicia faba L. cv Maris Bead was achieved by ammonium sulfate fractionation and hydrophobic, affinity, anion-exchange, and gel filtration chromatography. Further purification to homogeneity resulted from gel elution of single bands from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was identified as a homotetramer with a total molecular mass of 360 kD and subunits of 92 to 93 kD. Antibodies were raised to both native and denatured protein. The identity of the polypeptide was confirmed in western blots using antibodies raised against soybean nodule sucrose synthase. The enzyme has a pH optimum of 6.4 (cleavage direction) and an isoelectric point of 5.5. The affinity of the enzyme for sucrose (K_m) was estimated at 169 mm, and for UDP at 0.2 mm. With uridine diphosphate as the nucleoside diphosphate, the V_max is 4-fold higher than with adenosine diphosphate. Fructose acts as a competitive inhibitor with an inhibitor constant (K_i) of 2.48 mm.     

Purification and characterization of a β-cyclodextrin glucosyltransferase from an alkalophilic Bacillus sp.

Summary A -cyclodextrin glucosyltransferase was purified from alkalophilic Bacillus sp. No. 562 over 64-fold with a yield of 32%. Its molecular size was estimated to be 170 kDa by gel filtration and 82 kDa by SDS-PAGE, with a pI of 7.2. The enzyme showed optimum activity at 65 °C and pH 7.0. It was stable from 0 to70 °C and from pH 7.0 to 11.0. The enzyme was specifically inhibited by Fe2+ and Fe3+.     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Production and characteristics of an intracellularα-glucosidase from a color variant strain ofAureobasidium pullulans

Aureobasidium pullulans produced an intracellular-glucosidase. The enzyme was purified 124-fold by solubilization with Triton X-100, Q-Sepharose treatment, hydroxylapatite, octyl-Sepharose column chromatography, and gel filtration on Sephacryl S-200, and had a specific activity of 316.82 U/mg protein. The enzyme displayed an optimum pH for its action at 4.0 and was fully stable at pH 3.0–6.0 at 50°C. The-glucosidase was completely stable up to 60°C and had an optimum activity at 60°C. The partially purified enzyme preparation hydrolyzed maltose, isomaltose, sucrose, and trehalose at relative rates of 100, 60, 47, and 50, respectively, and had little or no activity on polysaccharides. TheK _m value for maltose hydrolysis at pH 4.0 and 50°C was 1.85mm. The enzyme was not adsorbed onto raw corn starch and showed little raw starch degradation. The-glucosidase did not require any metal ion for activity. This represents the first characterization of intracellular-glucosidase fromA. pullulans.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

5'-Nucleotidase from rat heart

5'-Nucleotidase has been extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent molecular weight of the subunit is 74 000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5'-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS, AMP, isoAMP, GMP, and IMP. It shows no activity with nucleoside 2'- and 3'-monophosphates, sugar phosphates, and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

Purification and properties of a heat-stable inulin fructotransferase from Arthrobacter ureafaciens

An inulin fructotransferase producing difructose dianhydride I (EC 2.4.1.200) was purified from Arthrobacter ureafaciens A51-1. It had maximum activity at pH 5.5 and 45 °C, and was stable up to 80 °C. This is the highest thermal stability for this enzyme reported to date. The molecular mass was estimated to be 38000 by SDS-PAGE, and 61000 by gel filtration. It was therefore estimated to be a dimer.     

Purification and Characterization of Lipase from Wheat (Triticum aestivum L.) Germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivumL.) is described. An electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 × 10^–3 mol/min per mg protein) was obtained after 61-fold purification. The molecular weight of the enzyme, determined by gel chromatography, was 143 ± 2 kDa. The optimal conditions for the enzyme were 37°C and pH 8.0. The homogeneous preparation of the lipase exhibited high thermal stability: over 20% of the original activity was retained after incubation of the preparation at high temperatures (60–90°C) for 1 h at pH 8.0.