Assay for the enzymatic conversion of indoleacetic acid to 3-methylindole in a ruminal Lactobacillus species

An assay to measure the rate of enzymatic formation of 3-methylindole (3MI) from indoleacetic acid (IAA) in Lactobacillus sp. strain 11201 was developed. The reaction mixture contained 50 micrograms of microbial protein per ml (range, 25 to 100 mg/ml), essential low-molecular-weight reaction ingredients, and radiolabeled IAA as substrate (range, 0 to 2 mM IAA). The reaction was anaerobic for 25 min at 39 degrees C. The apparent Michaelis-Menten constants were: Km, 0.14 mM IAA; and Vmax, 64 nmol 3MI.mg-1.min-1. The inhibitors avidin, aminopterin, and EDTA had no effect on the 3MI-forming enzyme. Dithionite stimulated the 3MI-forming enzyme. The product of the reaction, 3MI, acted as a noncompetitive inhibitor of the enzyme. Enzyme activity was associated with the cell wall fraction after sonication; treatment with the French press; or treatment with detergents, proteolytic enzymes, and EDTA.     

Effect of Indoleacetic Acid and Related Indoles on Lactobacillus sp. Strain 11201 Growth, Indoleacetic Acid Catabolism, and 3-Methylindole Formation

A study was conducted to determine the activity of the 3-methylindole (3MI)-forming enzyme in Lactobacillus sp. strain 11201. Cells were incubated anaerobically with 17 different indolic and aromatic compounds. Indoleacetic acid (IAA), 5-hydroxyindoleacetic acid, 5-methoxy-3-indoleacetic acid, indole-3-pyruvate, or indole-3-propionic acid induced 3MI-forming activity. The highest total enzyme activity induced by IAA was observed in cells incubated with an initial concentration of 1.14 mM IAA. Peak activity of the 3MI-forming enzyme occurred 4 h after bacteria were incubated with either 0.114 or 1.14 mM IAA. Enzyme activity peaked earlier (2 h) and disappeared more rapidly at 5.7 mM IAA than at other concentrations of IAA. The effects of IAA and 3MI on the growth of Lactobacillus sp. strain 11201 and formation of 3MI from IAA also were determined. Bacterial growth and 3MI formation from IAA were reduced in medium containing exogenous 3MI. The growth depression observed in medium containing 5.7 mM IAA appears to be due to the toxicity of 3MI rather than IAA. The formation of 3MI in this ruminal Lactobacillus sp. is mediated by an inducible enzyme, and as 3MI accumulates, bacterial growth and rates of 3MI formation from IAA are reduced.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Cyclic nucleotide phosphodiesterase from wheat sprouts. Purification, properties, effect of systemic fungicides

Cyclic nucleotide phosphodiesterase from wheat sprouts was isolated and partially purified. The molecular weight of the enzyme is about 83 000. The enzyme activity sharply rises as the inhibiting factors present in the homogenate are separated. The pH optimum of the enzymatic reaction is 4,8. Divalent cations (Mg2+, Mn2+, Cu2+) within the concentration range of 1--5 mM and complexons (EDTA, EGTA) at the concentration of 1 mM do not affect the PDE activity. The temperature optimum for the reaction is 60 degrees. The enzyme hydrolyzes 3' : 5'-AMP, 3' : 5'-GMP and 2':3'-AMP. The Km value for cAMP is 4 . 10(-3) M. The enzyme activity is inhibited by chemical agents possessing the fungicide activity, the strongest effect being exerted by anylate.     

Characterization of D-aminoacylase from Alcaligenes denitrificans DA181.

The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58,000 and 4.4, respectively. The apparent Km and kcat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 mM and 6.24 x 10(4) min-1, respectively. The optimum temperature was 45 degrees C. The enzyme was stable up to 55 degrees C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co2+ and partially by Zn2+.     

Partial Purification and Characterization of an Inducible Indole-3-Acetyl-L-Aspartic Acid Hydrolase from Enterobacter agglomerans

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH4)2SO4, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN₃, 5 mM -mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 g). It required 0.8 mM Ca²⁺ for half-maximal activation and 5 mM Ca²⁺ for optimal activation. Mg²⁺ (5 mM) was ineffective while Zn²⁺ and Hg²⁺ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.     

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

Proposed Enzymes of Auxin Biosynthesis and Their Regulation III. Some Properties of Pea Indolylacetaldehyde Oxidase

Indole-3-acetaldehyde oxidase (IAAld-oxidase) occurs in pea in two forms, of which the first, more active enzyme, has its pH optimum at 4.5, while the second, barely half as active, has a pH optimum at 7.0. Only the pH 4.5 oxidase can be resolved from the acetone powder. Besides IAA1d the more stable IA1d was used as substrate in testing the enzymatic activity. The pea enzyme seems not to be a dismutase since indolylmethanol or indolylethanol were not formed as products. Pyridine nucleotide coenzymes did not activate the partially purified enzyme. The pH 4.5 oxidase was inhibited by more than 50 % by IAA > L-asp > tryptophol > indoleacetylaspartic acid > 2,4-D (at 1 mM concentration). The pH 7.0 oxidase was inhibited relatively more weakly, a stronger than 50 % inhibition was caused only by NAA > L-asp. The oxidases were clearly distinguished by the response to L-asparagine (1 mM): the activity of the pH 4.5 oxidase was increased (+ 12 %), while the activity of the pH 7.0 oxidase was decreased (-71 %). In preliminaryin vitro experiments the phytohormones (1 mM) kinetin and GA₃ increased the conversion of IAAld to IAA, while ABA decreased it.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Initial characterisation of 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

Extracts of Pseudomonas sp. CBS3 converted 4-chlorobenzoate into 4-hydroxybenzoate. The enzyme responsible for this conversion was enriched by ammonium sulphate fractionation (30–60% saturation, 1.3-fold). The optimum conditions for the reaction were 30–35°C and pH 7–7.5. The enzyme was activated by Mn²⁺ (1 mM final concentration) up to 120-fold, and by Co²⁺ (1 mM final concentration) up to 60-fold. Other divalent ions had no effect. EDTA inhibited the enzyme. 4-Bromobenzoate and 4-iodobenzoate were substrates for the enzyme, but 4-fluorobenzoate was not converted.     

Changes in mitotic indices in roots of Vicia faba

Roots of Vicia faba were treated with solutions of colchicine or IAA or both. Mitotic indices and the frequencies of the different stages of mitosis were determined immediately after a three hour treatment or following a 24 hour period of recovery. Roots scored after treatment with colchicine for three hours showed several effects, none of which were reversed by simultaneous treatment with IAA. Treatment with IAA for three hours had little detectable effect on mitotic index (MI) on the frequencies of the various stages of mitosis. After a recovery period, following a three hour treatment, of 24 hours, colchicine treated roots showed a significant increase in their MI; this was due largely to an increase in the number of metaphases but it was also due in part to the presence of tetraploid cells in division. IAA treated roots revealed an inhibition of mitotic activity, which was most marked at 3.13–6.26×10^–4 M IAA. The results from roots treated with mixtures of colchicine and IAA for three hours and fixed 24 hours later showed: 1) the increase in MI induced by colchicine is reversed by IAA, the intensity of the reversal increasing with increasing concentrations of IAA; 2) reductions in the total numbers of cells in prophase or in metaphase occur after treatment with different concentrations of IAA; 3) IAA leads to a reduction in the number of tetraploid cells seen in division.It appears that colchicine induces a change in the pattern of mitotic activity 24 hours after the end of treatment and its effects are reversed by IAA. At 4.2×10^–4 M IAA a balance occurs between the opposing effects of colchicine and IAA and the MI is not significantly different from that of the controls. It is suggested that one result of a treatment with colchicine is a change in the level of growth factors in root meristems. This change, which appears to result in a temporary increase in MI is reversed by the addition of IAA. Thus one of the growth factors, the level of which has been affected, is replaceable by exogenous IAA.     

A soluble protein factor from chinese cabbage converts indole-3-acetaldoxime to iaa

A soluble enzyme preparation from Chinese cabbage seedlings (Brassica campestris ssp. pekinensis) which catalyses the conversion of indole-3-acetaldoxime (IAOX) to IAA was partially purified by ion exchange chromatography. After purification enzyme activity was stable for more than 6 hr. Substrate kinetics showed a K_m value of 50 μM; the pH optimum was 7. The conversion of IAOX to IAA was increased by NAD, NADP or FAD, but none of them seemed to be a preferential co-substrate. Besides IAA some labelled indole-3-acetaldehyde (IAALD) could be extracted from the reaction mixture. Addition of unlabelled IAALD at 100 nmol/ml led to a significant inhibition of IAA formation while some label accumulated in the aldehyde, Indole-3-acetonitrile was never detected as a reaction product. The results are compared with those from earlier in vivo experiments and are discussed in view of their significance for IAA biosynthesis in the Brassicaceae.     

Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621

Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K_m 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K_m 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu²⁺ and Pb²⁺ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Phytic acid-enhanced metal ion exchange reactions: the effect on carboxypeptidase A

On the basis of the known interaction of phytic acid to form soluble or insoluble complexes with cations, the effect of this naturally occurring polydentate ligand on carboxypeptidase A, a zinc-containing metalloenzyme, and its Co(II)-substituted derivative, has been studied. Under conditions of rigorous exclusion of adventitious metal ions, phytate showed no inhibitory effect. However, the addition of Cu(II) ions to form soluble phytate-Cu(II) complexes at pH 7.2 and 25 degrees C caused more than a 95% decrease in activity. The Cd(II) ion was nearly as effective but other ions showed only a small or no effect. In the absence of phytate, incubation of the enzyme with Cu(II) or Cd(II) at the same concentration produced only about a 25% reduction in activity. The decrease in activity followed first-order kinetics, and the rate constant was the same (1.2 x 10(-4) sec-1) as seen upon incubation with EDTA. However, in contrast to that observed upon incubation of the enzyme with phytate and Cu(II), exposure to EDTA produced a complete loss in activity which could be regained by addition of Zn(II) to the assay solution. In the former case, not only was there residual activity left after incubation at pH 7.2 for 24 hrs at 25 degrees C, but the initial activity could not be regained under similar assay treatment. An increase in either the Cu(II) or phytate concentration while the other was kept constant, yielded saturation curves with maximal effect at 3 x 10(-5) M for Cu(II) and at 5 x 10(-5) M for phytate (enzyme at ca. 10(-6) M). At these ratios, all of the cupric ions are completely bound to phytate as determined by ion-selective potentiometry. A preparative scale reaction of phytate and Cu(II) with carboxypeptidase A (kcat 8460 min-1; K'M 0.23 mM with CBZ-glycyl-glycyl-L-phenylalanine as substrate at pH 7.5, 25 degrees C) gave a product isolated in 95% yield but with lower activity (kcat 198 min-1; K'M 0.25 mM). A Cu(II)-carboxypeptidase preparation had similar kinetic parameters (kcat 207 min-1; K'M 0.34 mM). This near identity of constants suggested that a metal exchange reaction had occurred, i.e., incubation of Zn(II)-carboxypeptidase with a phytate-Cu(II) complex resulted in not only the removal of the zinc ion from the active site but also the sequential and rapid incorporation of a cupric ion into the apoenzyme so formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree,Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L‐tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L‐tryptophan. The preferred carbon and nitrogen sources were glucose and KNO₃ and the optimum concentrations 1% and 0.02%, respectively. FeSO₄ × 7 H₂O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO₄ × 7 H₂O (10 μg/ml), KNO₃ (0.02%) as well as EDTA (5 μg/ml) and L‐tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree‐Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.     

A biocatalyst for pyruvate preparation from -lactate: lactate oxidase in a Pseudomonas sp.

For the purpose of producing pyruvate from -lactate by enzymatic methods, four microorganism strains that produce lactate oxidase (LOD) were screened and isolated from many soil samples. Among them, strain SM-6, which showed high potential for pyruvate production, was chosen for further research. Physiological studies and 16S rDNA relationship reveal that SM-6 belongs to Pseudomonas putida. The optimized pH and temperature of the enzyme-catalyzed reaction were pH 7.2, and 39 °C, respectively. Low-concentration EDTA (1 mM) could improve the stability of pyruvate and conversion ratio of lactate oxidase. V_max and K_m value for -lactate were 2.46 μmol/(min mg) protein and 9.53 mM, respectively. On preparation scale, cell-free extract from SM-6, containing 300 mg/l of crude enzyme (4037 U/ml lactate oxidase), could convert 66% of 116 mM of -lactate into 76.6 mM pyruvate in 18 h, and 82% of substrate was transformed after 48 h, giving 95.0 mM (10.5 mg/ml) of pyruvate. The ratio of product to biocatalyst was 34.8:1 (g/g).