Purification and properties of glutamate synthase fromStreptomyces lincolnensis

Glulamale synthase (EC 1.4.1. 14) was purified to homogeneity from 8 cell-free extract ofStreptomyces lincolnensis by precipitation with streptomycin sulfate and ammonium sulfate, and column chromatography on DEAE cellulose, Sepharose 6B, DEAE-sephadex A-50, hydruxyapatite and Sephadex G-150. The enzyme activity is stabilized by addition of α-ketoglutarate, PMSF, EDTA, β-mercaptoethanol and glycerol. The native enzyme has a molecular weight of 138 000 and is composed of two nonidentical subunits with molecular weights of 81 000 and 57 000. Spectroscopic exarnination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The enzyme shows optimum activity at pH 7.2 and 30°C. Km values for α-ketoglutarate, L-glutamine and NADH were 417, 435, and 52.1 μmol/L, respectively. When NADPH was substituted lor NADH as reductant, there was approximately 13% of the control activity. The activity of this glutamate synthase is inhibited by its products (i.e. glutamare and NAD), several metal ions, amino acids and tricarboxylic acid cycle intermediates.     

Production and properties of fibrinolytic enzyme fromStreptomyces sp. NRC 411

Of 16Streptomyces spp. investigated for the production of extracellular fibrinolytic enzyme, one species was chosen as the most promising producer. Using shaken cultures grown for 7 days, optimal conditions for enzyme production were pH 6.0, 5% (w/v) starch as carbon source, (NH₄)₂SO₄ and soybean flour as nitrogen sources and KH₂PO₄ at 1.2 g/l. Maximal activity of the crude enzyme was at pH 6.0 and 45°C. Holding the enzyme at 37°C for 2 h decreased the activity by only 10%.     

Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utilizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The Km value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35 degrees C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30 degrees C but was progressively less stable at 40 and 50 degrees C.     

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Purification and properties of 16 alpha-hydroxyprogesterone dehydroxylase from Eubacterium sp. strain 144

Eubacterium sp. strain 144 converts 16 alpha-hydroxyprogesterone to 17-isoprogesterone. The first step of this reaction is catalyzed by 16 alpha-hydroxyprogesterone dehydroxylase (16 alpha-dehydroxylase). This enzyme was purified 40-70-fold and characterized. 16 alpha-Dehydroxylase was found to be active in two molecular weight forms of Mr 181 000 and 326 000. A subunit relative molecular weight of 42 400 was determined by sodium dodecyl sulfate gel electrophoresis of the purified enzyme. Although active with both 16 alpha-hydroxyprogesterone and 16 alpha-hydroxypregnenolone, the affinity of 16 alpha-dehydroxylase for the latter steroid was twice that of the former based on the apparent Km values. Evidence of possible substrate inhibition at high concentrations was seen with 16 alpha-hydroxypregnenolone. 16-Ketoprogesterone was found to be a competitive inhibitor of 16 alpha-dehydroxylase with respect to both steroid substrates. Although generally unaffected by low concentrations of non-ionic detergents, 16 alpha-dehydroxylase activity was stimulated 3-7-fold by sodium dodecyl sulfate and inhibited strongly by cetyltrimethylammonium bromide.     

Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601.

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.     

Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23.

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase activity were 50 degrees C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from family F.     

Purification and properties of glutathione reductase from the cyanobacterium Anabaena sp. strain 7119.

An NADPH-glutathione reductase (EC 1.6.4.2) has been purified 6,000-fold to electrophoretic homogeneity from the filamentous cyanobacterium Anabaena sp. strain 7119. The purified enzyme exhibits a specific activity of 249 U/mg and is characterized by being a dimeric flavin adenine dinucleotide-containing protein with a ratio of absorbance at 280 nm to absorbance at 462 nm of 5.8, a native molecular weight of 104,000, a Stokes radius of 4.13 nm, and a pI of 4.02. The enzyme activity is inhibited by sulfhydryl reagents and heavy-metal ions, especially in the presence of NADPH, with oxidized glutathione behaving as a protective agent. As is the case with the same enzyme from other sources, the kinetic data are consistent with a branched mechanism. Nevertheless, the cyanobacterial enzyme presents three distinctive features with respect to that isolated from non-photosynthetic organisms: (i) absolute specificity for NADPH, (ii) an alkaline optimum pH value of ca. 9.0, and (iii) strong acidic character of the protein, as estimated by column chromatofocusing. The kinetic parameters are very similar to those found for the chloroplast enzyme, but the molecular weight is lower, being comparable to that of non-photosynthetic microorganisms. A protective function, analogous to that assigned to the chloroplast enzyme, is suggested.     

Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3

alpha-Pinene oxide is an intermediate in the degradation of alpha-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with alpha-pinene and constituted 6 to 7% of the soluble protein of cell extracts. The apparent molecular weight of the native enzyme was 50,000 by ultracentrifugal analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave two dissimilar subunits with apparent molecular weights of 17,000 and 22,000. The enzyme was devoid of prosthetic groups, had no cofactor requirement, and had a broad pH activity range, a Km for alpha-pinene oxide of 9 microM, and a turnover number of 15,000. Inhibitors included sulfhydryl reactive compounds, terpene epoxides, and pinane derivatives with substituent groups at carbon 3. A mechanism for the concerted reaction has been proposed in which decyclization is initiated by donation of a proton from the catalytic center to the oxygen of the epoxide with consequent destabilization. In vitro the enzyme was inactivated during catalysis, and a reactive cationic intermediate may be responsible for this phenomenon. The enzyme should be classified as a lyase EC 4.99.-.-.     

Biotransformation of 3-methylphthalate by Micrococcus sp. strain 12B

When Micrococcus strain 12B grown on o-phthalate was incubated with 3-methylphthalate, three compounds accumulated. These were shown to be 2-pyrone-3-methyl-4,6-dicarboxylic acid, 3,4-dihydroxy-6-methylphthalic acid, and 5-hydroxy-3-methyphthalic acid, all previously undescribed. A pathway for the formation of these compounds is proposed.     

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase fromSynechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacteriumSynechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6–8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min^–1 at 40° C. The activation energy was 12.4 Kcal for t>29° C and 20.6 Kcal for t<29° C. The specific absorption coefficient, A _{280 mm} ^{1% 1cm} of the pure enzyme in phosphate buffer at pH 6.8 was 15.2.By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer.When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.     

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.     

Purification of ACV synthetase fromStreptomyces clavuligerus

Summary By a combination of protamine sulfate treatment, ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography, an active -(L--aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from the prokaryoteStreptomyces clavuligerus was purified 135-fold to give a single major protein band on SDS-PAGE. Its size appears to be approximately 360 kDa which is very similar to that of the enzyme from the eukaryote,Cephalosporium acremonium.     

Purification and properties of mycodextranase from Streptomyces sp. J-13-3.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.     

Purification and properties of 5-keto-d-fructose reductase from a mutant strain derived from Corynebacterium sp.

5-Keto-d-fructose reductase was purified about 300-fold from a mutant strain derived from Corynebacterium sp. SHS 0007 (ATCC 31090). The enzyme appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The enzyme converted 5-keto-d-fructose to l-sorbose in the presence of NADPH. The reduction did not occur in the presence of NADH. The reverse reaction was not observed. The molecular weight of the enzyme was estimated to be about 33,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme appeared to be monomeric. The optimum pH was 6.0–7.0 for the reductase. The K_m value (pH 7.0, 30°C) of the enzyme for 5-keto-d-fructose was 5.9 mM. The enzyme was relatively inactive on 2, 5-diketo-d-gluconate in the presence of NADPH.     

Purification and some properties of carbon monoxide dehydrogenase from Acinetobacter sp. strain JC1 DSM 3803.

A brown carbon monoxide dehydrogenase from CO-autotrophically grown cells of Acinetobacter sp. strain JC1, which is unstable outside the cells, was purified 80-fold in seven steps to better than 95% homogeneity, with a yield of 44% in the presence of the stabilizing agents iodoacetamide (1 mM) and ammonium sulfate (100 mM). The final specific activity was 474 mumol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, NAD(P), flavin mononucleotide, flavin adenine dinucleotide, and ferricyanide were not reduced by the enzyme, but methylene blue, thionin, and dichlorophenolindophenol were reduced. The molecular weight of the native enzyme was determined to be 380,000. Sodium dodecyl sulfate-gel electrophoresis revealed at least three nonidentical subunits of molecular weights 16,000 (alpha), 34,000 (beta), and 85,000 (gamma). The purified enzyme contained particulate hydrogenase-like activity. Selenium did not stimulate carbon monoxide dehydrogenase activity. The isoelectic point of the native enzyme was found to be 5.8; the Km of CO was 150 microM. The enzyme was rapidly inactivated by methanol. One mole of native enzyme was found to contain 2 mol of each of flavin adenine dinucleotide and molybdenum and 8 mol each of nonheme iron and labile sulfide, which indicated that the enzyme was a molybdenum-containing iron-sulfur flavoprotein. The ratio of densities of each subunit after electrophoresis (alpha:beta:gamma = 1:2:6) and the number of each cofactor in the native enzyme suggest a alpha 2 beta 2 gamma 2 structure of the enzyme. The carbon monoxide dehydrogenase of Acinetobacter sp. strain JC1 was found to have no immunological relationship with enzymes of Pseudomonas carboxydohydrogena and Pseudomonas carboxydovorans.     

Heterotrophic sulfur reduction by Thermotoga sp. strain FjSS3.B1

Thermotoga sp. strain FjSS3.B1 was able to reduce sulfur to sulfide when grown on a mineral medium with glucose as the sole carbon and energy source. There was no increase in specific growth yield coupled to sulfur reduction, but the specific growth rate, final growth yield, and tolerance of H2 were all increased in the presence of sulfur. At dissolved H2 concentrations, of 550 to 600 mumol/l (at 77 degrees C) growth was not possible unless sulfur was added. Glucose was fermented via the Embden-Meyerhof-Parnas pathway to lactate, acetate, H2 and CO2 (and other unidentified minor products). The thermodynamic problems associated with the relatively high redox potential electrons from the 1,3-bisphosphoglycerate/glyceraldehyde 3-phosphate couple (E'0 = -350 mV) are overcome by reducing sulfur to sulfide (E'0 = -270 mV) rather than the energetically unfavourable production of H2 (E'0 = -414 mV). Under high hydrogen partial pressures there was increased production of lactate as an alternative electron sink. The results indicate that sulfur reduction operates primarily as an electron sink rather than as a detoxification reaction or energy-generating mechanism.     

Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1.

An alkaliphilic Bacillus sp. strain, 41M-1, isolated from soil produced multiple xylanases extracellularly. One of these xylanases was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The moleculr mass of this enzyme (xylanase J) was 36 kDa, and the isoelectric point was pH 5.3. Xylanase J was most active at pH 9.0. The optimum temperature for the activity at pH 9.0 was around 50 degrees C. The enzyme was stable up to 55 degrees C at pH 9.0 for 30 min. Xylanase J was completely inhibited by the Hg2+ion and N-bromosuccinimide. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. The apparent Km and Vmax values on xylan were 3.3 mg/ml and 1,100 micromol-1 mg-1, respectively. Xylanase J showed high sequence homology with the xylanases from Bacillus pumilus and Clostridium acetobutylicum in the N-terminal region. Xylanase J acted on neither crystalline cellulose nor carboxymethyl cellulose, indicating a possible application of the enzyme in biobleaching processes.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Purification of DNA-dependent RNA polymerase fromStreptomyces granaticolor

RNA nucleotidyltransferase (EC 2.7.7.6) of Streptomyces granaticolor was purified by precipitation with polymin P and ammonium sulphate, affinity chromatography on DNA- cellulose and gell filtration on Biogel A 1.5 m. SDS-polyacrylamide gel electrophoresis revealed 8 protein bands of molar mass ranging from 37 to 130 kg/mol. Proteins of molar mass of 130 and 120 kg/mol were identified to be beta and beta subunits, respectively. The role of other subunits of the enzyme is discussed.