Immunogold labeling of CM-cellulase in Sinorhizobium fredii CCRC15769 by electron microscopy

The symbiotic roles of rhizobia with legumes have been extensively made use of in the agricultural field. An unresolved question about the infection process is the nature of the Rhizobium enzymes that degrade the plant cell wall. For the bacteria in the plant root-hair wall, carboxymethylcellulase (EC 3.2.1.4) could be one of the key enzymes in the early symbiotic process. Electron microscopic immunogold labeling experiments were performed with ultrathin sections of Sinorhizobium fredii CCRC15769. The results preliminarily indicated that antigenic determinants of membrane-bound carboxymethylcellulase are exposed, at the outside of the cytoplasmic membrane, to the periplasmic space; immuno-gold particles with 10nm diameter were hardly ever observed in the cytoplasm and other spaces.     

Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.     

Purification and characterization of extracellular invertase from the hypocotyls of mung bean (Phaseolus radiatus L.)

The extracellular invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by ammonium sulfate fractionation and sequential chromatography over diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A-Sepharose 4B affinity and Sephadex G-200. The overall purification was about 77-fold with a recovery of about 11%. The finally purified enzyme exhibited a specific activity of about 113 μmol of glucose produced mg^-1 protein min^-1 at pH 5.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The enzyme had the native molecular mass of 134 kD and subunit molecular weight of 67 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of homodimeric proteins. On the other hand, the enzyme appeared to be a glycoprotein containing mannosyl residues on the basis of its ability to interact specifically with the immobilized Con A and the separability of invertase-Con A complex by methyl-α-D-mannopyranoside. The enzyme had a K_m for sucrose of 3.4 mM and its pH optimum of 4.0. The enzyme showed highest enzyme activity with sucrose as substrate. Raffinose and cellobiose were hydrolyzed at a low rate, maltose and lactose were not cleaved by the enzyme. These results indicate the extracellular invertase is a β-fructofuranosidase.     

Purification and partial characterization of glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions has been purified to homogeneity. The purification procedure involves affinity chromatography on ADP-agarose type 2 as the major purification step. The recovery in the purification is 70%. The specific activity of the purified enzyme is about 10-times higher in the gamma-glutamyl transferase assay than in the coupled biosynthetic assay. The molecular weight was determined to 530,000 by native gradient polyacrylamide gel electrophoresis and to 500,000 by gel filtration. The subunits have an apparent molecular weight of 52,000. Glutamine synthetase isolated from Rsp. rubrum which had been exposed to ammonium ions ('switch-off') before harvest had about 20% of the transferase activity compared with the enzyme purified from nitrogen-starved cells. The low-activity form showed two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification and properties of urease fromSporobolomyces roseus

Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme fromSporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (± 4) kDa. The M _r of the native enzyme was estimated by size exclusion chromatography to be 340 (± 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 °C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen.     

Purification and properties of endo-1,3-α-D-glucanase from Pseudomonas

An endo-1, 3-α-D-glucanase (EC 3.2.1.59) was purified from cell-free culture supernatants of Pseudomonas NRRL-B-12324. The enzyme was purified 8.7-fold to a specific activity of 78.1 U/mg of protein. The enzyme was inducible and had an isolectric point of 4.6 and a K_m of 80.0 mM in terms of anhydroglucose units. Two distinct peaks of activity were resolved by gel filtration with two different supporting media, whereas only one peak of activity was resolved by isoelectricfocusing. The two peaks were assigned molecular weight values of 67 400 and 279 000. The pH optimum was near 5.0 and the temperature optimum was near 56°C. Additional gel filtration data indicated that the enzyme functions as an endohydrolase.     

N-terminal amino acid sequence of persimmon fruit beta-galactosidase.

beta-Galactosidase (EC 3.2.1.23) from persimmon fruit was purified 114-fold with a 15% yield using Sephadex G-100 gel filtration, CM-Sephadex ion exchange, and Sephacryl S-200 gel filtration chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The estimated molecular mass of the native beta-galactosidase by Sephacryl S-200 was 118 kD. After sodium dodecyl sulfate-PAGE of the enzyme electroeluted from native gels, two subunits with estimated molecular masses of 34 and 44 kD were observed, suggesting that the native enzyme was an aggregate of several subunits. Amino acid composition and N-terminal amino acid sequences of the two major subunits were different.     

Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14

Using five different steps, beta-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeneity with approximately 90-fold purification with a specific activity of 281 units mg-1 protein. A single band was observed in native PAGE. Activity staining of the native gel with 5-bromo 4-chloro 3-indoxyl beta-D-galactopyranoside (X-Gal) at pH 4.0 also produced a single band. Analytical gel filtration in Superdex G-75 revealed the molecular mass of the native protein to be approximately 75 kD. 10% SDS-PAGE under reducing conditions showed two subunits of molecular masses, 45 and 30 kD, respectively. Hence, beta-galactosidase from kidney beans is a heterodimer. A typical protein profile with lambda max at 280 nm was observed and A280/A260 ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86% sequence homology with an Arabidopsis thaliana and 85% with Lycopersicon esculentum putative beta-galactosidase sequences. The Electrospray Mass Spectrometric analysis of this band also revealed a peptide fragment that had 90% sequence homology with an Arabidopsis thaliana putative beta-galactosidase sequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometric analysis both by MALDI-TOF and ES MS revealed certain sequences that matched with phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0 and it hydrolysed o- and p-nitrophenyl beta-D galactopyranoside with a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively. The energy of activation calculated from the Arrhenius equation was 14.8 kcal/mol enzyme site. The enzyme was found to be comparatively thermostable showing maximum activity at 67 degrees C. Thermal denaturation of the enzyme at 65 degrees C obeys single exponential decay with first order-rate constant 0.105 min-1. Galactose, a hydrolytic product of this enzyme was a competitive inhibitor with a Ki of 2.7 mmol/L.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Properties of a partially purified acid phosphatase from pathogenic Nocardia brasiliensis

Like many other bacteria, Nocardia sp. possess acid phosphatase activity. In N. brasiliensis, a human and animal pathogen, this activity was resolved into two enzyme forms by native gel electrophoresis. One (isozyme I) was partially purified and characterized. It exhibited an estimated molecular weight on SDS-PAGE of 50 kDa, a pH optimum of 5.2, and a Km value of 1.25 mM for p-nitrophenylphosphate. The N. brasiliensis enzyme was stable at 4 °C for at least 24 h, but readily inactivated at 60 °C. Ammonium molybdate, sodium fluoride and L-(+)-tartrate were found to be potent inhibitors of the enzyme. Although its function is presently unknown, by analogy to other bacterial systems it could be envisioned to play an important role in the physiology and pathogenicity of the microorganism.     

Purification of iron superoxide dismutase from the cyanobacterium Anabaena cylindrica Lemm. and localization of the enzyme in heterocysts by immunogold labeling

Iron superoxide dismutase (Fe-SOD; EC 1.15.1.1) was isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica Lemm. Polyacrylamide gel electrophoresis separated the purified protein into three closely running, enzymatically active bands. The molecular weight of the enzyme was estimated by gel filtration to be about 40 kDa. Polyclonal antibodies were produced by immunization of rabbits with the isolated enzyme, and were purified on a column of protein A-Sepharose. The Fe-SOD antibody reacted with the purified Fe-SOD and also specifically recognized the protein in extracts of A. cylindrica. In the extracts, anti-Fe-SOD did not cross-react with Mn-SOD, an enzyme which belongs to an SOD class displaying high homology of primary and three-dimensional structure with respect to Fe-SOD. Iron superoxide dismutase was localized in heterocysts by immunogold labeling and transmission electron microscopy. These results are the first in-situ evidence for the presence of SOD in the cells specialized for nitrogenase activity.Abbreviations ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - SOD superoxide dismutase - PAGE polyacrylamide gel electrophoresis - pI isoelectric point This work was supported by a C.N.R. grant. We are grateful to Dr. A. De Martino for technical assistance.     

Isolation,Purification, and Properties of Cysteine Proteinase from <Emphasis Type="Italic">Bombyx mori</Emphasis> L. Eggs

Silkworm moth (bombyx) egg cysteine proteinase with maximal activity at pH 3.0 was purified by chromatography and isoelectrofocusing. On SDS-electrophoresis in polyacrylamide gel the purified enzyme showed a single band of molecular mass 50 kD. Isoelectrofocusing revealed that the bombyx egg cysteine proteinase exists in two forms with pI values of 5.95 and 6.43, respectively. The enzyme activity was sensitive to inhibition by iodoacetamide and p-chloromercuribenzoate but resistant to EDTA, pepstatin, and phenylmethylsulfonyl fluoride. The cysteine proteinase hydrolyzes storage proteins of bombyx eggs but it was inactive with respect to N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA). It is a cathepsin L-like enzyme.     

Cellulase-free xylanase production from an alkalophilicBacillus species

An alkalophilicBacillus (NCL-87-6-10, NCIM 2128), with a high productivity for extracellular xylanase (EC 3.2.1.8) and free of cellulase, was isolated from soil containing coconut fibre detritus. When grown on a wheat bran/yeast extract medium in submerged culture for 48 h, it produced 100 to 120 IU of enzyme activity per ml. The crude enzyme consists of two fractions of apparent mol sizes of approx 10.4 and 29 kDa in the proportion of 90:10, as determined by native gel exclusion chromatography. Optimum activity of the xylanase was at 60°C and pH 8.0. A two-fold increase in enzyme activity was obtained when reducing agents, thioethanol and dithiothreitol, were included in the assay.NCL Communication No. 5381.     

Purification and characterization of homodimeric methylmalonyl-CoA mutase from<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

High activity (>60 munit/mg protein) of 5-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000±5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0±2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH₂-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.Abbreviations AdoCbl 5-Deoxyadenosylcobalamin - KPB Potassium phosphate buffer - MCM Methylmalonyl-CoA mutase - PVDF Polyvinylidene difluoride     

Isolation of Hydroxyanthrones from the Roots of Rumex acetosa Linn

Microorganisms capable of producing high amounts of α-acetolactate decarboxylase (ALDC; EC 4.1.1.5) were screened for with stock type cultures. Brevibacterium acetylicum had the most potent enzyme activity among the strains tested. The productivity of ALDC by B. acetylicum was elevated by adding Zn²⁺ to the medium. ALDC was purified from the cell-free extract of B. acetylicum by a procedure involving ammonium sulfate fractionation, Sephadex G-100 gel filtration, and DEAE-cellulose and FPLC-MonoQ column chromatographies. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 62,000 by TSK-gel filtration and the subunit molecular weight was 31,000 by SDS polyacrylamide gel electrophoresis. The enzyme activity was inhibited by metal chelators such as diethyldithiocarbamate, 8-oxyquinoline, and o-phenanthroline. Analysis by atomic absorption spectrophotometry showed that zinc atoms were involved in the purified enzyme preparation.     

Studies on glutamine synthetase. Purification of the enzyme from mung bean (Phaseolus aureus) seedlings and modulation of the enzyme-antibody reaction by the substrates

Glutamine synthetase (L-glutamate : ammonia ligase, EC 6.3.1.2) fromPhaseolus aureus (mung bean) seedlings was purified to homogeneity by ammonium sulphate fractionation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and affinity chromatography on histidine-Sepharose. The enzyme had a molecular weight of 775,000 ± 25,000. The enzyme consisted of identical subunits with an approximate subunit molecular weight of 50,000. Hyperbolic saturation curves were obtained with the substrates, glutamate, ATP and hydroxylamine. Antibody, raised in the rabbit, against mung bean glutamine synthetase, completely inhibited the activity of the enzyme. Preincubation of the enzyme with glutamate and ATP, prior to the addition of the antibody, partially protected the enzyme against inhibition. TheK _m values of this enzyme-antibody complex and the native enzyme were identical (glutamate, 2.5mM; ATP, 1 mM; hydroxylamine, 0.5 mM). The Km values of the partially inhibited enzyme (the enzyme pretreated with antibody prior to the addition of substrates) were 2-fold higher than those of the native enzyme. These results suggested that the substrate-induced conformational changes in the enzyme were responsible for the protection against inhibition of the enzyme activity by the antibody.     

Immobilization and properties of β-D-galactosidase fromLactobacillus bulgaricus

β-D-galactosidase (EC 3.2.1.23) fromLactobacillus bulgaricus (1373) was immobilized by entrapment in a Polyacrylamide gel lattice. The enzymatic properties of the immobilized β-galactosidase were compared with those of the native enzyme. The temperature and pH optima were not affected by the immobilization. After entrapment of the enzyme no significant change was observed in its thermostability. The pH stability of the immobilized enzyme was higher than that of the native enzyme on the acidic side. TheK _m values for the immobilized and native β-galactosidase with both lactose ando-nitrophenyl-β-D-galactoside as substrates were comparable. The immobilized enzyme could be repeatedly used 12 times without any loss of activity. No loss in the activity of the immobilized β-galactosidase was found after its storage for 30 days at 4°C and for 20 days at 25°C.     

Rhizobial Surface Biopolymers and their Interaction with Lectin Measured by Atomic Force Microscopy

Summary Atomic force microscopy (AFM) was used to measure the morphology and physicochemical properties of rhizobia and to probe cell-surface polymers with tips modified with soybean agglutinin (SBA). AFM measurements of the length, width, and height of Sinorhizobium fredii CCRC15769 were 1580±450, 870±70, and 270±50 nm, respectively (means±SD). Different AFM image modes revealed the morphology, adhesion, viscoelasticity, and surface roughness of rhizobia in air using the tapping operation. Force–distance relationships between SBA-terminated AFM probes and Bradyrhizobium japonicum USDA110 were recorded and the retraction curves showed an unbinding force of 106±48 pN with a loading rate of 1 nN/s in PBS containing 0.1 mM Mn²⁺ and 0.1 mM Ca²⁺ (pH 7.2). The technique of AFM provides information about the morphology and molecular interaction forces of rhizobia under physiological conditions.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

L-asparaginase of Thermus thermophilus: Purification,properties and identificaation of essential amino acids for its catalytic activity

L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-asparaginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around 200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.