Extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6: preparation of it and its properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain, Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6-1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase from Streptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1-0.5% promoted the secretion of the extracellular glutamate oxidase.     

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep mutagenesis-selection procedure allowed a genetically stable strain,Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

beta-lactamase from Streptomyces cacaoi. Purification and properties

A beta-lactamase was purified to an apparently homogeneous state from Streptomyces cacaoi. The molecular weight calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000. pI was 4.7 and the optimal pH was 6.5. The optimum temperature was found to be between 40 degrees C and 45 degrees C, but the enzyme lost activity above 50 degrees C. N-Bromosuccinimide was the strongest inhibitor among the reagents tested, followed by iodine. p-Chloromercuribenzoate showed a weak inhibitory effect. Diisopropylfluorophosphate and sodium chloride did not show any inhibitory effect on the enzyme. The beta-lactamase catalyzed the hydrolysis of methicillin and cloxacillin at two-thirds to one-third the rate of benzylpenicillin. On the other hand, the enzyme hydrolyzed cephalosporins and 7-methoxycephalosporin only slowly. With benzylpenicillin as a substrate, the Km increased sharply with decreasing pH and the pK alpha estimated from the Km versus pH curve was 6.5 to 7.0. In contrast, with cloxacillin as a substrate, the Km showed a minimum at pH 7.5. The Vmax changed with pH in a bell-shaped curve in the case of benzylpenicillin, but the Vmax for cloxacillin changed only within a small range. In addition, the ratio of the hydrolysis rate of benzylpenicillin and cloxacillin at 30 degrees C and 20 degrees C (V30 degrees/V20 degrees) was found to be 1.23 and 1.55, respectively. These results indicate that the S. cacaoi beta-lactamase behaves differently toward benzylpenicillin and cloxacillin, although both are penicillins. S. cacaoi seems to release beta-lactamase into the culture medium soon after its biosynthesis without retaining it in the membrane and the soluble fraction. The possible relationships between beta-lactamases from Streptomyces and those from pathogenic bacteria are discussed.     

Arogenate dehydrogenase from Streptomyces phaeochromogenes. Purification and properties

Arogenate dehydrogenase, the terminal enzyme of tyrosine biosynthesis in Streptomyces phaeochromogenes, was purified to homogeneity by a five-step procedure. The enzyme is a dimer of Mr 57 600 as determined by dodecyl sulfate polyacrylamide gel electrophoresis after cross-linking of the monomers, or of 66 300 as found by gel permeation chromatography, and consists of two identical subunits of Mr 28 100. The pI of the enzyme is 4.45, and the Km values are 0.105mM for arogenate and 0.01 mM for NAD.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.     

Purification and properties of acetyl-CoA:L-glutamate N-acetyltransferase from human liver.

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km,acetyl-CoA 4.4 mM, Ki,acetyl-CoA 4.7 mM, Km,glutamate 8.1 mM, Ki,glutamate 8.8 mM; for the products, Ki,acetylglutamate 0.28 mM, Ki,CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Purification and properties of xanthine dehydrogenase from Streptomyces cyanogenus.

Xanthine dehydrogenase has been purified to a homogeneous state from cell-free extracts of a strain of Streptomyces. The enzyme has a molecular weight of 125,000 and consists of two subunits with a molecular weight of 67,000. The isoelectric point is at pH 4.4. The enzyme exhibits absorption maxima at 273, 355, and 457 nm and contains FAD, iron, and labile sulfide in a molar ratio of 1 : 7 : 1 per subunit. Little molybdenum could be detected. The enzyme is most active at pH 8.7 and at 40 degrees C, and is stable between pH 7 and 12 (at 4 degrees C for 24 h) and below 55 degrees C (at pH 9 for 10 min). The activity is stimulated by K+ at a concentration of 50 mM or more and also by keeping the enzyme at pH 9 to 11. The activity is inhibited by cyanide, Tiron, and p-chloromercuribenzoate and by adenine and urate. Among the compounds tested, hypoxanthine, guanine, xanthine 2-hydroxypurine, and 6,8-dihydroxypurine are oxidized at considerable rates; hypoxanthine is the best substrate. NAD+ is the preferred electron acceptor. Km values of the enzyme for hypoxanthine, guanine, xanthine, and NAD+ are 0.055, 0.015, 0.15, and 0.11 mM, respectively. Marked differences in the properties of this enzyme compared to others are the activity towards guanine, which has a higher affinity for the enzyme than hypoxanthine and xanthine, and a higher reactivity with hypoxanthine than xanthine. The organism has been identified as Streptomyces cyanogenus.     

Purification and properties of a xylanase from Streptomyces lividans.

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).     

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

Properties and prospects of practical use of extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6

The properties of extracellular L-glutamate oxidase, isolated and purified from Streptomyces sp. Z-11-6 (specific activity, 50.8 U/mg protein; yield, 40%), were studied. A photometrical method of determination of activities of alanine- and aspartate aminotransferases, based on the use of the L-glutamate oxidase and peroxidase, has been developed. This method is sufficiently sensitive to be used for the determination of aminotransferase activities in biological fluids. The presence of other amino acids did not interfere with the analysis and had no effect on the results of determination.     

Purification and properties of nitroalkane oxidase from Fusarium oxysporum.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(NO2)CH2CH3 + O2 + H2O leads to OHCCH2CH3 + HNO2 + H2O2. In addition to 1-nitropropane, 3-nitro-2-pentanol, 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavin adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavin 5'-phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and N-ethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavin adenine dinucleotide, 1.33 micrometer.     

Purification and properties of chitinase from Streptomyces cinereoruber

A chitinase (EC 3.2.1.14) was purified from the culture filtrate of Streptomyces cinereoruber, selected as a microorganism which produces enzymes lysing Aspergillus niger cell wall, by fractional precipitation with ammonium sulfate and column chromatographies on DEAE-cellulose, Sephadex G-100 and CM-Sephadex C-50. The final preparation was homogenous in polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was about 19,000 daltons and its isoelectric point was pH 8.6. The optimum pH and temperature for chitinase activity were 4.5 and at 50°C, respectively. The enzyme was stable in the pH range from 4.0 to 10.0. The activity was inhibited by Ag⁺, Hg⁺, Hg²⁺ and p-chloromercuribenzoate. Paper chromatographic analysis demonstrated that the hydrolytic products of colloidal chitin and chitotriose with the enzyme were N-acetylglucosamine and chitobiose. The lysis of A. niger cell wall with the enzyme is discussed.     

Purification and properties of alcohol oxidase from Poria contigua.

1. Alcohol oxidase (alcohol:oxygen oxidoreductase) was purified 22-fold from the brown rot fungus Poria contigua. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis, and by sedimentation in an ultracentrifuge. The molecular weight was calculated to be 610000 +/- 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels and electron microscopic analysis indicate that the enzyme is an octamer composed of eight probably identical subunits, each having a molecular weight of 79 000. The enzyme contains eight mol FAD/mol as the prosthetic group. 2. This alcohol oxidase oxidizes not only methanol but also lower primary alcohols (C2-C4), 2-propin-1-ol and formaldehyde. The apparent Km value for methanol is 0.2 mM, and that for formaldehyde 6.1 mM. Sodium azide was found to be a competitive inhibitor with respect to methanol. 3. The enzyme from the fungus Poria contigua is immunologically different from the alcohol oxidase isolated from the methanol-utilizing yeast Candida boidinii. Furthermore antiserum raised against this enzyme did not cross-react with the alcohol oxidase from the white rot fungus Polyporus obtusus.     

Purification and partial characterization of a cholesterol oxidase from Streptomyces fradiae

An extracellular cholesterol oxidase from Streptomyces fradiae (PTCC 1121) was purified in one step using DEAE-Sepharose. The purified enzyme had a molecular weight of 60 KDa. The optimum pH and temperature for activity was found to be 7 and 70 degrees C, respectively. This cholesterol oxidase was stable in pHs between 4-10 at 4 degrees C until 4 h. Thermal stability experiments showed that it has high stability and retains its full activity at 50 degrees C for 90 min. K(m) value for cholesterol oxidase was obtained to be about 7.06 x 10(-)(5) Mol.