Purified Enzyme Preparation Research Articles

Chitinase and β-1,3-glucanase in grapevine leaves: a possible defence against powdery mildew infection

Leaves were collected from 21 different grapevine (Vitis ssp.) genotypes with varying resistance to powdery mildew disease caused by Uncinula necator. For leaves collected from field-grown vines in spring there was a correlation between resistance rating and activity of chitinase and β-1,3-glucanase. The correlation was greater with the sum of the two enzyme activities. In contrast, no correlation was obtained for leaves collected during summer. With leaves from glasshouse grown vines, wounding or infection with powdery mildew increased both chitinase and β-1,3-glucanase activity. Light microscope examination of detached leaves inoculated with U. necator conidia showed that germination appeared to occur at the same rate on leaves of a susceptible (Sultana) and a resistant (Seyval) genotype. Subsequent development of mycelia was severely restricted on the resistant genotype but it was prolific on the sensitive genotype. A bioassay was developed based on germination and extension of the germ tube of U. necator conidia on agar plates. Agar preparations containing desalted crude extracts of grapevine leaves inhibited growth and caused the tips of the hyphae to rupture. The effect was not observed with boiled extracts and was greater with extracts from resistant genotypes. Chitinase and β-1,3-glucanase were purified 760-fold and 46-fold respectively from leaves of Seyval grapevines. The purified enzyme preparations inhibited germ tube growth, with the effect being more prominent in the presence of both enzymes. The results demonstrate that these two enzymes have antifungal activity against U. necator, and are consistent with these pathogenesis-related proteins having a role in defence of grapevines against powdery mildew.

Purification and some characteristics of phosphatase of a psychrophile.

The phosphatase of a psychrophile was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, butyl-Cellulofine, Sephacryl S-100, and Mono-Q columns. The purified enzyme preparation was found to be electrophoretically homogeneous on native- and SDS-PAGE, and its molecular mass was determined to be 38.4 kDa by MALDI-TOF mass spectrometry. Maximal activity was observed at 30 degrees C and pH 6.0. Furthermore, the activity of this enzyme at 0 and 5 degrees C was 27 and 28%, respectively, of that at 30 degrees C. The enzyme was stable in the pH range of 6.0 to 8.0 and up to 20 degrees C. The enzyme was affected by metal ions; the activity was enhanced by Mg2+ and Ca2+ ions, but depressed by Zn2+ ions. Analysis of the amino acid composition indicated that this phosphatase contains no S-S bond, and only a few prolyl residues necessary to retain the rigid structure of a protein molecule. The phosphatase shows typical features of a cold enzyme; high catalytic activity at low temperature and rapid inactivation at an intermediate temperature.

Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation.

A cDNA of pea (Pisum sativum L.) RbcS 3A, encoding a small subunit protein (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), has been expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter, and the transcript and mature S protein were detected. Specific antibodies revealed two protein spots for the four Arabidopsis S and one additional spot for pea S. Pea S in chimeric Rubisco amounted to 15 to 18% of all S, as judged by separation on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels from partially purified enzyme preparations and quantitation of silver-stained protein spots. The chimeric enzyme had 11 +/- 1% fewer carbamylated sites and a 11 +/- 1% lower carboxylase activity than wild-type Arabidopsis Rubisco. Whereas pea S expression, preprotein transport, and processing and assembly resulted in a stable holoenzyme, the chimeric enzyme was reproducibly catalytically less efficient. We suggest that the presence of, on average, one foreign S per holoenzyme is responsible for the altered activity. In addition, higher-plant Rubisco, unlike the cyanobacterial enzyme, seems to have evolved species-specific interactions between S and the large subunit protein that are involved in carbamylation of the active site.

A Newly Identified Methyl-branched Chain Fatty Acid Synthesizing Enzyme from Mycobacterium tuberculosis var. bovis BCG

Mycobacterium bovis BCG produces a variety of methyl-branched fatty acids. They include C28 to C32 mycocerosic acids esterified to phthiocerol and phenolphthiocerol and the shorter (C22 to C26) mycocerosic acids esterified to phthiocerol. A mycocerosic acid synthase gene-disrupted mutant was still able to produce the shorter mycocerosic acids. The enzyme short chain mycocerosic acid synthase (SMAS), that catalyzes the synthesis of such acids, was purified using anion exchange and red-agarose chromatography. Gel filtration showed the native enzyme to be a 537-kDa protein. Since SDS-polyacrylamide gel electrophoresis of the purified enzyme showed a 280-, 170-, and 100-kDa protein and they cross-reacted with antibodies prepared against the 280- or 100-kDa protein, this enzyme is composed of the three subunits or two 280-kDa monomers with an unusual susceptibility to a proteolytic nick. SMAS utilizes methylmalonyl-CoA with C12 to C20 acyl-CoA as primers and with either NADH or NADPH as the reductant to synthesize the short mycocerosic acids. The Km values for NADH and NADPH were 93 and 90 microM, respectively. Antibodies raised against either the 280- or 100-kDa protein inhibited the incorporation of methylmalonyl-CoA into fatty acids by SMAS. The enzyme is not immunologically closely related to mycocerosic acid synthase or fatty acid synthase.

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants.

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.

Tomoxiprole Selectively Inhibits Cyclooxygenase-2

Tomoxiprole is a nonsteroidal anti-inflammatory compound that was reported to have low ulcerogenic potential, a quality that would be expected of a cyclooxygenase-2-selective inhibitor, and, in fact, we find it is selective for this isozyme. In stably transfected COS cells, the compound inhibits recombinant human cyclooxygenase-2 (IC 50 = 7 nM) more potently than recombinant cyclooxygenase-1 (IC 50 = 240 nM), and similar results are obtained with partially pure ovine enzyme preparations. The compound is thus a very potent as well as selective inhibitor of cyclooxygenase-2. As is true of some other cyclooxygenase-2-selective inhibitors, tomoxiprole inhibition of cyclooxygenase-2 but not cyclooxygenase-1 is time-dependent.

Chemoenzymatic approaches to SCH 56592, a new azole antifungal

AbstractChemoenzymatic approaches to the synthesis of two key chiral‐precursors of a new azole antifungal agent, SCH 56592, are described. In particular, the enzymatic diastereoselective acylation of 2‐benzyloxy‐3‐pentanol (7) was developed to produce (2S;3R)‐7 in >97% diastereomeric excess (de) from otherwise unusable mixtures of (2S,3R)/(2S,3S)‐7 (40–80% de). The selectivity and reactivity of commercially available Candida rugosa and Mucor miehei lipases are compared for the acylation of 7 and the hydrolysis of the corresponding butyrate 16a. Of the 17 C. rugosa enzyme preparations that were examined for acylation of 7, two purified enzyme preparations showed no reactivity, five enzymes showed high diastereoselectivity with preference for the (2S,3R)‐isomer, and seven showed a slight preference for the (2S,3S)‐isomer.

Synthesis and biological evaluation of 6′,7′-dihydroxybergamottin (6,7-DHB), a naturally occurring inhibitor of cytochrome P450 3A4

Abstract The recently isolated inhibitor of cytochrome P450 CYP3A4, 6′,7′-dihydroxybergamottin (6,7-DHB), was synthesized by an efficient route that is readily adaptable to the production of analogues. The compound was evaluated as an inhibitor of 3A4 in a purified enzyme preparation, as well as against human liver microsomes and human 3A4 expressed in Escherichia coli membrane. In each case, 6,7-DHB proved to be a potent NADPH- and time dependent inactivator of 3A4.

Heterologous expression of carbonyl reductase: Demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese Superoxide dismutase.

Overproduction and purification of an agarase of bacterial origin

The agarase gene from Streptomyces coelicolor has been cloned in the non-producer bacterium Streptomyces lividans under the control of its own set of promoters and under the control of a heterologous promoter that is functional only during exponential growth. The best level of overproduction was obtained when the strain containing the natural gene was cultivated in fed batch with mannitol as carbon source. The protein, with a relative molecular mass of 32 kDa, has been purified following an affinity purification method. Contaminating activities seem to be absent from the purified enzyme preparation that can be used to purify DNA from agarose gels.

A Study on the Complexes between Human Erythrocyte Enzymes Participating in the Conversions of 1,3-Diphosphoglycerate

The ability ofd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH·2,3-DPGM and GAPGH·3-PGK complexes were detected. TheKdvalues of the complexes determined with purified enzyme preparations were in the range 2.40–2.48 μmfor both the GAPDH·2,3-DPGM and GAPGH·3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, theKdfor the GAPDH·2,3-DPGM complex was found to be 0.54 μm, and theKdfor the GAPDH·3-PGK complex was 0.49 μm. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein–protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

Acetyl Coenzyme A Acetyltransferase of Rhizobium sp. (Cicer) Strain CC 1192

To investigate why Rhizobium sp. (Cicer) strain CC 1192 cells accumulate poly-R-3-hydroxybutyrate in the free-living state but not as bacteroids in nodules on chickpea (Cicer arietinum L.) plants, we have examined the kinetic properties of acetyl coenzyme A (acetyl-CoA) acetyltransferase (also known as acetoacetyl-CoA thiolase and 3-ketothiolase [EC 2.3.1.9]) from both types of cells. The enzyme had a native molecular mass of 180 (plusmn) 4 kDa, and the subunit molecular mass was 44 (plusmn) 1 kDa. The seven amino acids from the N terminus were Lys-Ala-Ser-Ile-Val-Ile-Ala. Thiolysis and condensation activity of the enzyme from free-living CC 1192 cells were optimal at pHs 7.8 and 8.1, respectively. The relationship between substrate concentrations and initial velocity for the thiolysis reaction were hyperbolic and gave K(infm) values for acetoacetyl-CoA and CoA of 42 and 56 (mu)M, respectively. The maximum velocity in the condensation direction was approximately 10% of that of the thiolysis reaction. With highly purified preparations of the enzyme, a value of approximately 1 mM was determined for the apparent K(infm) for acetyl-CoA. However, with partially purified enzyme preparations or when N-ethylmaleimide was included in reaction mixtures the apparent K(infm) for acetyl-CoA was close to 0.3 mM. In the condensation direction, CoA was a potent linear competitive inhibitor with an inhibition constant of 11 (mu)M. The much higher affinity of the enzyme for the product CoA than the substrate acetyl-CoA could have significance in view of metabolic differences between bacteroid and free-living cells of CC 1192. We propose that in free-living CC 1192 cells, the acetyl-CoA/CoA ratio reaches a value that allows condensation activity of acetyl-CoA acetyltransferase, but that in CC 1192 bacteroids, the ratio is poised so that the formation of acetoacetyl-CoA is not favored.

Alterations in Superoxide Dismutase Isozymes by Methylmercury

A study of alterations in two mammalian superoxide dismutases (SODs), Cu,Zn-SOD and Mn-SOD, caused by methylmercury has been reviewed. Mechanisms for the isozyme-selective decrease in MnSOD activity by the organometallic compound observed in vivo have been extensively examined by experiments with purified enzyme preparations. © 1997 John Wiley & Sons, Ltd.

Alterations in Superoxide Dismutase Isozymes by Methylmercury

A study of alterations in two mammalian superoxide dismutases (SODs), Cu,Zn-SOD and Mn-SOD, caused by methylmercury has been reviewed. Mechanisms for the isozyme-selective decrease in MnSOD activity by the organometallic compound observed in vivo have been extensively examined by experiments with purified enzyme preparations. © 1997 John Wiley & Sons, Ltd.

Heme Biosynthesis by the Malarial Parasite: IMPORT OF δ-AMINOLEVULINATE DEHYDRASE FROM THE HOST RED CELL

The mouse and human malarial parasites, Plasmodium berghei and Plasmodium falciparum, respectively, synthesize heme de novo following the standard pathway observed in animals despite the availability of large amounts of heme, derived from red cell hemoglobin, which is stored as hemozoin pigment. The enzymes, delta-aminolevulinate dehydrase (ALAD), coproporphyrinogen oxidase, and ferrochelatase are present at strikingly high levels in the P. berghei infected mouse red cell in vivo. The isolated parasite has low levels of ALAD and the data clearly indicate it to be of red cell origin. The purified enzyme preparations from the uninfected red cell and the parasite are identical in kinetic properties, subunit molecular weight, cross-reaction with antibodies to the human enzyme, and N-terminal amino acid sequence. Immunogold electron microscopy of the infected culture indicates that the enzyme is present inside the parasite and, therefore, is not a contaminant. The parasite derives functional ALAD from the host and the enzyme binds specifically to isolated parasite membrane in vitro, suggestive of the involvement of a receptor in its translocation into the parasite. While, ALAD, coproporphyrinogen oxidase, and ferrochelatase from the parasite and the uninfected red cell supernatant have identical subunit molecular weights on SDS-polyacrylamide gel electrophoresis and show immunological cross-reaction with antibodies to the human enzymes, as revealed by Western analysis, the first enzyme of the pathway, namely, delta-aminolevulinate synthase (ALAS) in the parasite, unlike that of the red cell host, does not cross-react with antibodies to the human enzyme. However, ALAS enzyme activity in the parasite is higher than that of the infected red cell supernatant. We therefore conclude that the parasite, while making its own ALAS, imports ALAD and perhaps most of the other enzymes of the pathway from the host to synthesize heme de novo, and this would enable it to segregate this heme from the heme derived from red cell hemoglobin degradation. ALAS of the parasite and the receptor(s) involved in the translocation of the host enzymes into the parasite would be unique drug targets.

Isoforms of Linamarase in Cassava (Manihot esculenta)

Linamarase (EC. 3.2.1.21) was purified from different tissues of cassava (leaf, rind and tuber) to compare the kinetic properties and characteristics of the enzyme in these tissues. Purified enzyme preparation appeared as single band of average molecular size 70 kD in SDS-PAGE gels. The kinetic properties of linamarase with respect to pH and temperature indicated that tuber linamarase possessed a broader pH optimum and higher temperature stability as compared to leaf and rind enzymes. Differences in Km values for linamarin were observed with leaf linamarase having the highest Km value (500 μM) followed by rind (400 μM) and then tuber (250 μM) linamarases. Rind enzyme appeared to be less susceptible to urea denaturation than the leaf enzyme. Comparison of elution profiles from DEAE-Cellulose indicated that the relative amounts of the various ionic forms of the enzyme differed in the case of each tissue. Elution patterns of the enzyme from Con A-Sepharose also differed, suggesting difference in glycosylation among leaf, rind and tuber enzymes. This was confirmed by carbohydrate analysis which showed that the tuber linamarase contained significantly higher amount of protein bound carbohydrate. These results suggest the possible occurrence of different forms of linamarase in cassava.

Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Fructan biosynthesis in excised leaves of Lolium temulentum

SUMMARYA partially purified enzyme preparation from leaves of Lolium temulentum L. was previously shown to catalyse the net synthesis of oligofructans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magnitude and properties of these hydrolytic activities have been determined. The significance of these catabolic activities for studies of fructan polymerization both in vitro and in tissues in a physiologically anabolic state are discussed.The preparation hydrolysed 1‐kestose, 6‐kestose, neokestose, inulin oligosaccharides of low degree of polymerization (DP 4 and 5) and endogenous oligofructans from L. temulentum, with the concomitant release of monosaccharide. The preparation also released reducing sugar at low rates from high molecular weight inulin but had no detectable activity against bacterial levan. Simultaneous incubation of sucrose and Neosugar (a commercially available mixture of predominantly β‐2, 1 linked tri‐, tetra‐ and penta‐saccharides) showed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentrations > 30 mol m−3. The kinetic properties for hydrolysis of both sucrose and Neosugar were determined. For sucrose and Neosugar respectively, Michaelis constants at 30°C and pH 6.0 were 7.7±0.5 and 14.1± 1.1 mol m−3 (as terminal fructose) and maximum velocities were 6.5 ± 0.1 and 2.7 ± 0.1 mg g−1 fr. wt h−1 (equivalent to 10.0 and 4.2 nkat g−1 as reducing sugar release). Maximal temperatures for activity were 45 and 44°C, and Arrhenius activation energies were 39.9 and 46.9 kJ mol−1. Preincubations for 1 h at 49 and 48°C caused 50% loss of activity in subsequent assays at 30°C. The pHs for maximal activity for the two substrates were 5.2 ± 0.1 and 5.5±0.1.Using size exclusion chromatography (SEC), an activity catalysing the formation of fructan oligosaccharides and another catalysing sucrose hydrolysis, were not fully resolved, but exhibited distinct profiles of elution indicating Mr of 57 and 133 kD respectively. When assayed for the hydrolysis of Neosugar, the SEC eluate exhibited two peaks of activity indicative of Mr values of 57 and 133 kD.

Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan‐polymerizing enzyme preparation

A partially purified enzyme preparation from leaves of Lolium temulentum L. was previously shown to catalyse the net synthesis of oligofructans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magnitude and properties of these hydrolytic activities have been determined. The significance of these catabolic activities for studies of fructan polymerization both in vitro and in tissues in a physiologically anabolic state are discussed.The preparation hydrolysed 1‐kestose, 6‐kestose, neokestose, inulin oligosaccharides of low degree of polymerization (DP 4 and 5) and endogenous oligofructans from L. temulentum, with the concomitant release of monosaccharide. The preparation also released reducing sugar at low rates from high molecular weight inulin but had no detectable activity against bacterial levan. Simultaneous incubation of sucrose and Neosugar (a commercially available mixture of predominantly β‐2, 1 linked tri‐, tetra‐ and penta‐saccharides) showed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentrations >30 mol m−3. The kinetic properties for hydrolysis of both sucrose and Neosugar were determined. For sucrose and Neosugar respectively, Michaelis constants at 30°C and pH 6·0 were 7·7±0·5 and 14·1±1·1 mol m−3 (as terminal fructose) and maximum velocities were 6·5±0·1 and 2·7±0·1 mg g−1 fr. wt h−1 (equivalent to 10·0 and 4·2 nkat g−1 as reducing sugar release). Maximal temperatures for activity were 45 and 44°C, and Arrhenius activation energies were 39·9 and 46·9 kJ mol−1. Preincubations for 1 h at 49 and 48°C caused 50% loss of activity in subsequent assays at 30°C. The pHs for maximal activity for the two substrates were 5·2±0·1 and 5·5±0·1.Using size exclusion chromatography (SEC), an activity catalysing the formation of fructan oligosaccharides and another catalysing sucrose hydrolysis, were not fully resolved, but exhibited distinct profiles of elution indicating Mr of 57 and 133 kD respectively. When assayed for the hydrolysis of Neosugar, the SEC eluate exhibited two peaks of activity indicative of Mr values of 57 and 133 kD.

The 2,4-dichlorophenol hydroxylase of Alcaligenes eutrophus JMP134 is a homotetramer.

2,4-Dichlorophenol hydroxylase (DCP-hydroxylase) is a key enzyme in the pathway for degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) in many bacteria. In Alcaligenes eutrophus JMP134, DCP-hydroxylase was reported to consist of two dissimilar types of subunit of 66 and 45 kDa, a structure which is different from that in other bacteria. Using a different procedure involving affinity purification and ion-exchange chromatography, we have purified active enzyme from JMP134 and show that it has a native molecular mass of approximately 245 kDa and consists of a single type of subunit of 66 kDa, similar to all other flavoprotein monooxygenase enzymes. A 45-kDa polypeptide, found in partially purified enzyme preparations, was not required for enzyme activity but had some serologic and N-terminal amino acid sequence similarity to the 66-kDa enzyme subunit.