Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Characterization of soluble rifamycin oxidase from Curvularia lunata var. aeria

Curvularia lunata var. aeria was grown on yeast extract, peptone and carboxymethylcellulose (YPC) medium for the production of extracellular rifamycin oxidase. The enzyme was partially purified through a Sephadex G-75 column. The half lives of rifamycin oxidase at 30° and 40°C were 9 d and 100 min, respectively. The activation and deactivation energies of the partially purified enzyme, calculated from Arrhenius plots, were 5.80 and 35.10 kcal mol^-1 respectively. The enzyme exhibited a K _m (rifamycin B) value of 0.67 mmol l^-1 and a V _max of 11 μmol h^-1 ml. Three metal ions, Fe²⁺, Ag⁺ and Hg²⁺, inhibited the enzyme in the 10–20 mmol l^-1 metal ion concentration range. Catalytic activity was not affected by the chelating agent, EDTA.     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.     

Isolation and characterization of cytoplasmic NADP+-malic enzyme from Lupinus luteus leaves

NADP⁺-dependent malic enzyme (L-malate : NADP⁺ oxidoreductase, decarboxylating, EC 1.1.1.40) was extracted from the leaves of yellow lupine. The purification procedure included fractionation with (NH₄)₂SO₄ and Sephadex G-25 chromatography, followed by purification on DEAE-cellulose and Sephadex G-200 columns. The enzyme was purified 122-fold. The enzyme affinity towards L-malate was found to be significantly higher with Mn²⁺ than with Mg²⁺. The Hill coefficient for Mg²⁺ depended on concentration and was 1.6 for the lower and 3.9 for the higher concentrations. The dependence of the enzyme activity on NADP⁺ followed a hyperbolic curve. K_m values and Hill coefficients for NADP⁺ were similar with both Mn²⁺ and Mg²⁺. The enzyme activity was strictly dependent on divalent cations and followed a sigmoidal curve at least for Mg²⁺. The enzyme had 4-fold higher affinity towards Mn²⁺ than towards Mg²⁺, the K_m values being 0.3 and 1.15 m M respectively. Of several tested organic acids, oxalate was the most effective inhibitor followed by oxaloacetate while succinate was the strongest activator.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Thermophilic glucoamylase from Talaromyces flavus

The heat-resistant mold, Talaromyces flavus , was found to produce a thermophilic glucoamylase that exhibited the highest activity at 50°C and in the pH range of 4.0–4.8. The K _m and V _max values of the crude enzyme for amylopectin were 0.21% and 16.7 mg glucose 1^-1, min^-1, respectively. The molecular weight of the enzyme as estimated by the gel filtration method was 42 kDa.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH₄)₂SO₄ precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)^-1 min^-1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn²⁺ or Mg²⁺). In the presence of MnCl₂₊ other divalent cations (Mg²⁺, Ca²⁺, Ba²⁺, Hg²⁺ and Cd²⁺) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The K_m for adenine was 4.5±1.5 μ M , the K_m for PRPP was 0.29±0.06 m M and the K_i for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The K_m for benzyladenine was approximately 0.73±0.06 m M     

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.     

Partial purification and some biochemical properties of nonspecific alkaline phosphatase in germinating chick-pea (Cicer arietinum) seeds

A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the K_m for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (K_m= 2.4 m M ), NADP⁺ (K_m= 4.0 m M ), 5'-AMP (K_m= 4.5 m M ), 5'-ADP (K_m= 6.1 m M ) and ribose-5P (K_m= 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn²⁺, K⁺, Hg²⁺ and Mo⁶⁺ were strong inhibitors, whereas F⁻ and Ca²⁺ inhibited weakly; Co²⁺ and Ni²⁺ were activators.     

Properties of glutamate dehydrogenase from developing maize endosperm

Glutamate dehydrogenase (EC 1.4.1.3) activity was assayed in homogenates of maize ( Zea mays L. inbred lines Oh43 and Oh43o₂) endosperm during development. During the period 20–35 days after pollination anabolic (aminative) activities were higher than catabolic (deaminating) ones. In order to study the regulation of GDH activity, glutamine or glutamate were injected into the ear peduncle before sample harvesting. The amination and deamination reactions showed similar behaviour with different nitrogen sources: glutamine increased, whereas glutamate decreased, both aminative and deaminative reactions. Partially purified enzyme was active with NADH and NADPH in a ratio 9:1. In Tris-HCl buffer a broad optimum at pH 7.6–8.9 and pH 6.8–8.9 was observed with NADH and NADPH, respectively, NADH activity was activated by Ca²⁺. Saturation curves for (NH₄)₂SO₄ and NADH showed normal Michaelis-Menten kinetics in the presence of 1 m M Ca²⁺, but substrate inhibition occurred without Ca²⁺. The enzyme was inactivated by EDTA. The effect of EDTA was reversed by Ca²⁺ and Mn²⁺, but not by Cu₂₊ and Mg²⁺.     

Purification and characterization of diacetyl reductase from Kluyveromyces marxianus

Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K _m and V _max values for diacetyl were 2.5 mmol 1^-1 and 0.026 mmol 1^-1 min^-1, respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa.