Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Purification of tobacco O-methyltransferases by affinity chromatography and estimation of the rate of synthesis of the enzymes during hypersensitive reaction to virus infection

The three tobacco (Nicotiana tabacum L.) S-adenosyl-L-methionine: o-diphenol-O-methyltransferases (OMTs; EC 2.1.1.6) were purified to homogeneity by affinity chromatography on adenosine-agarose. Amounts and catalytic actities of the enzymes were measured in tobacco leaves during the hypersensitive reaction to tobacco mosaic virus. The drastic increase in activity of each enzyme upon infection was shown to arise from the accumulation of enzymatic protein with constant specific enzymatic activity. Rates of OMT synthesis were determined from pulse-labeling experiments with L-[¹⁴C]leucine injected into the leaves. The specific radioactivities of the homogenous enzymes were compared in healthy and tobacco mosaic virus-infected tobacco. The results demonstrated that increase in OMT amounts is a consequence of de novo synthesis of the enzymes.Abbreviations DEAE diethylaminoethyl - OMT O-methyltransferase - SAM S-adenosyl-L-methionine - TMV tobacco mosaic virus     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q₁₀ (45–55° C) of 1.7 and an activation energy of 45 kJ · mol^–1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK _m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 M for phenylalanine and 176 M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.     

Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG polyethylene glycol - PMT putrescine-N-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from immunity to physiological susceptibility depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.Abbreviations BMT S-adenosyl-L-methionine:bergaptol O-methyltransferase - PAL phenylalanine ammonia-lyase - PBS phosphate-buffered saline     

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of =1.14 g·cm^–3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-l-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-l-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-l-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.Abbreviations BBE berberine bridge enzyme - STOX (S)-tetrahydroprotoberberine oxidase Dedicated to Professor Karl Decker, Freiburg, on the occasion of his 60^th birthday     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Purification and characterization of mouse hepatic enzyme that converts selenomethionine to methylselenol by its α,γ-elimination

The objective of this study was to purify and characterize a mouse hepatic enzyme that directly generates CH₃SeH from seleno-l-methionine (l-SeMet) by the α,γ-elimination reaction. The l-SeMet α,γ-elimination enzyme was ubiquitous in tissues from ICR mice and the activity was relatively high in the large intestine, brain, and muscle, as well as the liver. Aging and sex of the mice did not have any significant influence on the activity in the liver. The enzyme was purified from the mouse liver by ammonium sulfate precipitation and four kinds of column chromatography. These procedures yielded a homogeneous enzyme, which was purified approx 1000-fold relative to mouse liver extract. Overall recovery was approx 8%. The purified enzyme had a molecular mass of approx 160 kDa with four identical subunits. The K _m value of the enzyme for the catalysis of l-SeMet was 15.5 m M, and the V _max was 0.29 units/mg protein. Pyridoxal 5′-phosphate (pyridoxal-P) was required as a cofactor because the holoenzyme could be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of pyridoxal-P. The enzyme showed the optimum activity at around pH 8.0 and the highest activity at 50°C; it catalyzed the α,γ-elimination reactions of several analogs such as d,l-homocysteine and l-homoserine in addition to l-SeMet. This enzyme also catalyzed the α,β-elimination reaction of Se-methylseleno-l-cysteine. However, l-methionine was inerts. Therefore, the purified enzyme was different from the bacterial l-methionine γ-lyase that metabolizes l-SeMet to CH₃SeH, in terms of the substrate specificity. These results were the first identification of a mammalian enzyme that specifically catalyzes the α,γ-elimination reaction of l-SeMet and immediately converts it to CH₃SeH, an important metabolite of Se.     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60

Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M _r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K _m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co²⁺, Mn²⁺ and Fe²⁺ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH₂-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-l-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.Abbreviations BMT S-adenosyl-l-methionine: bergaptol O-methyltransferase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Detection and characterization of anS-adenosyl-l-methionine: Flavonoid 7-O-methyltransferase from leaves ofPrunus x yedoensis matsum. with special reference to prunetrin formation

An enzyme,S-adenosyl-l-methionine: flavonoid 7-O-methyltransferase (F7OMT), catalyzing the transfer of the methyl group fromS-adenosyl-l-methionine (SAM) to the 7 position of sophoricoside (5, 7, 4′-trihydroxyisoflavone 4′-O-glucoside) and some of the other flavonoids, was detected in extracts from leaves ofPrunus x yedoensis, and it was partially purified (about 203-fold) by a combination of gel filtration and ion-exchange column chromatographies. F7OMT was isolated as a soluble enzyme with a pH optimun of 7.5 in K-phosphate buffer. The molecular mass of F7OMT, which had an isoelectric point at pH 4.1, was estimated by elution from a column of Sephadex G-100 to be about 36 kDa. The activity of F7OMT was stimulated by 14 mM 2-Co²⁺ and reagents that react with sulfhydryl groups. The apparentKm values for sophoricoside, its aglycone genistein (5, 7, 4′-trihydroxyisoflavone) and quercetin were 1.49, 2.19 and 1.89 μM, respectively. The apparentKm value for SAM as methyl donor was 2.08 mM. The specificity of F7OMT for methyl acceptors was not strict; flavonols, flavanones and flavanonols in addition to isoflavones served as methyl acceptor. An examination ofP. x yedoensis leaves during spring and autumn showed variations in the activities of F7OMT and UDP-glucose: isoflavone 4′-O-glucosyltransferase (I4′ GT). The activities of F7OMT and I4′GT increased in enlarging leaf tissues and then markedly declined when the leaves approached maturation. In autumn leaves F7OMT activity was scarcely detected, but a small peak of I4′GT activity was observed during autumnal reddening.