Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant ��-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10 min at 90 °C and pH 8. The enzyme was stable over a broad range of pH (pH 6-10) and showed good thermal stability when incubated at 70 °C. Chemicals like EDTA, Hg²⁺, Cu²⁺ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55 kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The K_M and V_max values of the enzyme were 4.4 mg/ml and 287 U/mg respectively. At high xylan concentrations (>70 mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.     

Comparative aspects of root and root nodule secondary metabolism in alfalfa

l-phenylalanine ammonia lyase (PAL), indoleacetic acid oxidase (IAA oxidase), O-methyltransferase, peroxidase, total phenolics and total indoles were compared in roots and root nodules of alfalfa. PAL, O-methyl-transferase, total phenolics and total indoles were higher in nodules than in roots. Isolated bacteroids were assayed for O-methyltransferase, PAL, peroxidase and total phenolics, but their levels were either low or not detectable. Nodule leghemoglobin was separated by disc gel electrophoresis and found to have IAA oxidase activity. Phenolics, IAA oxidase and leghemoglobin appear to be interrelated in regulating indole levels in the nodule.     

Purification and properties of the polyamine oxidase of barley plants

The polyamine oxidase of barley shoots is associated with a particle which sediments in low centrifugal fields. The enzyme was removed from these particles by washing in 0·5 M NaCl and then purified about 24-fold. The purified enzyme oxidized spermine stoicheiometrically to 1,3-diaminopropane and 1-(3-aminopropyl)pyrroline (pH optimum 4·0). Spermidine was oxidized to 1,3-diaminopropane and 1-pyrroline (pH optimum 6·6). At their respective pH optima, spermine is oxidized about 30 times faster than spermidine. Hydrogen peroxide was formed in the course of the polyamine oxidation. The enzyme was not sensitive to several copper chelating reagents but 2-hydroxyethylhydrazine caused 50% inhibition at 5 × 10⁻⁴ M. The enzyme was also present in particles in the roots of barley seedlings and in extracts of the leaves of oats, maize, rye and wheat.     

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.     

Yeast alpha glucosidase inhibitory and antioxidant activities of six medicinal plants collected in Phalaborwa,South Africa

Recent decades have experienced a sharp increase in the incidence and prevalence of diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as α-amylase and α-glucosidase and α-amylase. The aim of the current study was to screen six medicinal plant species, with alleged antidiabetic properties for α-glucosidase inhibitory activities. Powdered plant materials were extracted with acetone, and tested for ability to inhibit baker's yeast α-glucosidase and α-amylase activities. The largest mass (440 mg from 10 g) of the extract was obtained from Cassia abbreviata, while both Senna italica and Mormordica balsamina yielded the lowest mass of the extracts. Extracts of stem bark of C. abbreviata inhibited baker's yeast α-glucosidase activity with an IC₅₀ of 0.6 mg/ml. This plant species had activity at low concentrations, with 1.0 mg/ml and above resulting in inhibition of over 70%. The other five plant extracts investigated had IC₅₀ values of between 1.8 and 3.0 mg/ml. Senna italica only managed to inhibit the activity of enzyme-glucosidase at high concentrations with an IC₅₀ value of 1.8 mg/ml, while Tinospora fragosa extracts resulted in about 55% inhibition of the activity of the enzyme at a concentration of 3.5 mg/ml, with an estimated IC₅₀ value of 2.8 mg/ml. The bark extract of C. abbreviata was the most active inhibitor of the enzyme, based on the IC₅₀ values (0.6 mg/ml). The bark extract of C. abbreviata contains non-competitive inhibitor(s) of α-glucosidase, reducing V_max value of this enzyme from 5 mM·s^–1 to 1.67 mM·s^–1, while K_m remained unchanged at 1.43 mM for para-nitrophenyl glucopyranoside. Antioxidant activity of the extracts was also investigated. The C. abbreviata extract was more active as an antioxidant than the positive control, trolox. The extracts did not inhibit alphaamylase activity more than about 20% at the highest concentration tested.     

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Isolation,purification and characterization of phytase from germinating mung beans

Phytase isolated from mung bean cotyledons was purified about 80-fold with a recovery of 28%. The enzyme is stable at 0°, has a pH optimum at 7·5 and optimal temperature of 57°. The energy of activation is approximately 8500 cal/mole between 37° and 57°. Inhibition by P_i has been found to be competitive, the K_i value being 0·40–0·43 × 10⁻³ M; the K_m value with phytate is 0·65 × 10⁻³ M. Divalent cations are not required for activity. Lower members of inositol phosphates are better substrates, as shown by their V_max and K_m values. When subjected to polyacrylamide gel electrophoresis two bands have been resolved; one (major) corresponds to phytase and the other (minor) to phosphatase and pyrophosphatase activity. Filtration through Biogel P-200 partially resolves phytase from phosphatase and pyrophosphatase. The molecular weight of phytase is approximately 160,000.     

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.     

Kinetics of an extracellular exo-inulinase production from a 5-flourocytosine resistant mutant of Geotrichum candidum using two-factorial design

In the present study, eight different strains of Geotrichum candidum were isolated and screened for an extracellular exo-inulinase production using chemically enriched sucrose–mineral media. The isolate (Zool-3i) with a better enzyme activity (1.38 IU/ml) was subjected to induced mutagenesis using methyl methane sulphonate (MMS) and a mutant with an enzyme activity of 32.06 IU/ml was obtained. Further exposure to ethyl methane sulphonate (EMS) and ultraviolet (UV) radiations yielded a mutant exhibiting an improved activity of 39.34 IU/ml. The potential mutant was cultured overnight and plated on 5fc–YPR agar medium and thus made resistant against 5-flourocytocine. Over 50-fold enhancement in enzyme production (71.85 IU/ml) was achieved when the process parameters including incubation period (48 h), sucrose concentration (5.0 g/L), pH (6.0), inoculum size (2.0%, 16 h old) and urea (0.2%) were identified using Plackett–Burman design. On the basis of kinetic variables, notably Q_p (0.723 U/g/h), Y_p/s (2.036 U/g) and q_p (0.091 U/g cells/h), the mutant MEU-5fc-6 was found to be a hyper producer of exo-inulinase (HS, LSD 0.045, p ? 0.05).     

Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff: Characterization of the Enzyme

Indole-3-ethanol oxidase (IEt oxidase) from Phycomyces blakesleeanus Bgff.(P.b.) is a 56 kD polypeptide as determined by gel filtration. The reaction products are indole-3-acetaldehyde (IAAld) and, possibly, H₂O₂. Enzyme activity (33-45% ammonium sulfate fraction) shows a broad pH optimum and simple Michaelis-Menten kinetics (K_m 7 micromolar, Hill coefficient 0.95). Flavin adenine dinucleotide increases enzyme activity particularly under anaerobic conditions. Iodoacetate and HgCl₂ drastically inhibit the enzyme. With IAAld, product inhibition is observed at micromolar concentrations. IAA and some other acidic substituted indoles reduce enzyme activity but only at higher concentrations.     

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0–8.0) and temperatures below 50 °C. Metal ions including Na⁺, Mg²⁺, Mn²⁺, and Ca²⁺ notably increased the activity of the enzyme. With sodium alginate as the substrate, the K_m and V_max were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside

A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK_a of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters K_m of 200 μM and V_max of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment.     

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn²⁺ and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H₂O₂, were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mμ was reduced, while major absorption peaks appeared at 254 and 247 mμ. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mμ. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H₂O₂ (3 μmoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H₂O₂ inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H₂O₂ (0.03 μmole/ml). A comparison of K_m values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47 kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme.     

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.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

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.     

细菌黄嘌呤氧化酶的稳定性

研究pH、温度、金属离子和一些添加剂对黄嘌呤氧化酶稳定性的影响。结果表明：黄嘌呤氧化酶在pH4．5～7．5的范围内较稳定;反应最适温度为37℃。在常温25～35℃该酶比较稳定,经45℃处理2h．可保持50％左右,不同种类、不同浓度的金属离子对黄嘌呤氧化酶活性表现出程度不同的激活或抑制作用;添加谷氨酸和天门冬氨酸,能有效提高黄嘌呤氧化酶的存放稳定性.