Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

Partial purification and biochemical properties of acid and alkaline phosphatases from Myxococcus coralloides D

F. GONZÁLEZ, M.E. FÁREZ-VIDAL, J.M. ARIAS AND E. MONTOYA. 1994. Acid phosphatase and alkaline phosphatase from vegetative cells of Myxococcus coralloides D were purified by two chromatographic steps. The molecular weights were estimated by gel filtration and SDS-PAGE. Optimum pH, stability, optimum temperature and thermal inactivation studies were made for both enzymes. EDTA and other chelating agents inhibited alkaline but not acid activity. Mg²⁺ activated the alkaline phosphatase, while the acid phosphatase was inhibited by fluoride. Both enzymes degraded a number of phosphomonoesters, but were unable to hydrolyse either polyphosphates or cAMP. The K _m values of the acid and alkaline phosphatases for p -nitrophenylphosphate were 5.0 times 10^-3 mol ***l^-1 and 1.5 times 10^-3 mol l^-1, respectively.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.     

Purification and properties of a chitinase from Penicillium oxalicum autolysates

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54 900 as estimated from SDS gel electrophoresis and 21 500 from gel filtration. The optimum pH and temperature were 5.0 and 35°C, respectively. The enzyme was stable at temperatures up to 45°C and in a pH range between 4.0 and 6.0. The K_m was 2.5 mg ml^-1 for colloidal chitin, Hg²⁺ and Ag⁺ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.     

Subject index volume 144

Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K _m and V _max values have been found to be 1.1 mM and 25.6 μmol min⁻¹(mg protein)⁻¹, respectively. Enzyme activity was inhibited by Hg²⁺, Ag²⁺, and Cu²⁺ at a concentration of 1 × 10⁻³ M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Characterization of L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.)

B.R. MOHAPATRA, R.K. SANI AND U.C. BANERJEE. 1995. The bacterial flora associated with an intertidal marine alga ( Sargassum sp.) were screened for the presence of extracellular L-asparaginase; one out of five Bacillus strains was found positive. The maximum L-asparaginase activity was found at 37°C and pH 8.0. The optimum NaCl concentration for enzyme activity was found to be 2% (w/v). The enzyme activity was not affected by the addition of different metal ions (Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺and Ni²⁺) at 10 mmol 1^-1, but was strongly inhibited by EDTA.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Changes in spectral properties of ageing and senescing maize and sunflower leaves

Activity and biochemical characteristic of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase from pear ( Pyrus communis cv. Blanquilla) was determined. The enzyme showed a low K_m (57.5 μM) for ACC and was dependent on O₂ (K_m 0.44% in atmosphere). It had an absolute requirement for Fe²⁺, ascorbate and CO₂ and was inhibited by α-aminoisobutyric acid (AIB: K₁ 4.2 m M ) and cobalt. ACC oxidase has an optimum pH of 6.7 and temperature maxima at 28 and 38°C and it is concluded that the activity of ACC oxidase from pear resembles authentic in vivo activity.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca²⁺ concentration required for PLI and PLII activity was determined to be 0·5 mmol l⁻¹. The Ca²⁺ requirement could not be replaced by other metal cations such as Mg²⁺, Cu²⁺, Zn²⁺, Fe³⁺ and Co²⁺. Optimal pH for activity was determined to be between 8·5 and 9·0. The K _m values for sodium polygalacturonate were 1·28 and 1·11 mg ml⁻¹ for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl₂ or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Purification and some properties of (1R,2S)-1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase from Comamonas testosteroni T-2

Abstract Inducible (1 R ,2 S )-1,2-dihydroxy-3,5-cyclohexadiene-l,4-dicarboxylate (diene-diol) dehydrogenase was found in extracts of Comamonas testosteroni T-2 grown in p -toluate-or terephthalate-salts medium and it was purified using anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is a homodimer with subunit M r 39000. It had a specific activity of 500 mkat/kg of protein and was activated by the addition of Fe²⁺. The dehydrogenase converted 1 mol diene-diol and 1 mol NAD⁺ to 1 mol protocatechuic acid, 1 mol NADH and 1 mol CO₂. Apparent K _m-values of 43 μM (NAD⁺) and about 90 μM (diene-diol) were determined. The hydride ion was transferred to the si face of NAD⁺.     

Partial purification and some biochemical properties of acid phosphatase in germinating chickpea (Cicer arietinum) seeds

An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K _m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg²⁺. Ca²⁺, Hg²⁺, Fe³⁺, arsenate, K⁺ and Zn²⁺ were inhibitory. Mn²⁺, dithiothreitol and EDTA had no effect, and polyamines were activators.     

Indole-3-acetaldehyde reductase in Phycomyces blakesleeanus. Characterization of the enzyme

lndole-3-acetaldehyde reductase (lAAld reductase EC 1.2.3.1) from Phycomyces blakesleeanus Bgff., a 38 kDa polypeptide as determined by gel filtration, is probably localized in the cytoplasm. The formation of indole-3-ethanol (lEt) is dependent on the presence of NAD(P)H. The enzymatic reduction of IAAId shows a pH optimum between 6 and 8 and a temperature optimum at 30°C. Enzyme activity follows Michaelis Menten kinetic (K_m= 200 μ M for IAAId; K_m= 24 μ M for NADPH). The isoelectric point of the IAAId reductase is at pH 5.4. Phenylacetaldehyde and benzaldehyde are competitive substrates. Hydroxymeihylindole promotes the reductive IEt formation, whereas NADP⁺ is a non-competitive inhibitor. Changes in lAAJd reductase activity correlate with certain developmental stages of the fungus.     

Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity

Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a p I of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co²⁺ and Zn²⁺, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. K _m values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.