Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H₂O₂ produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β- d -glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H₂O₂ was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H₂O₂ will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO^·. Mycelial extracts from each of the mycorrhizal fungi produced HO^· in the presence of cellobiose and Fe(II), presumably mediated by H₂O₂ produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO^· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.     

Aryl alcohols in the physiology of ligninolytic fungi

Abstract: White-rot fungi have a versatile machinery of enzymes which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. This review will focus on the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H₂0₂. The aryl alcohol/aldehyde couple is well protected against degradation by the fungi's extracellular ligninolytic enzymes and their concentrations in the extracellular fluid are highly regulated by intracellular enzymes.     

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.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

Novel heme-containing enzyme possibly involved in lignin degradation by the white-rot fungus Junghuhnia separabilima

Abstract The white-rot fungus Junghuhnia separabilima (Pouz.)Ryv, showed high levels of laccase production in cultures supplemented with veratric acid. Laccase, lignin peroxidase and an unknown peroxidase were separated from the extracellular culture fluid using anion-exchange FPLC. Three laccase species, three lignin peroxidases and a novel heme-containing protein were characterized by gel electrophoresis and isoelectric focusing. The new hemoprotein has a molecular mass of 44 kDa, isoelectric point of 3,4 and pH optimum of 5.5 for oxidation of o -dianisidine in the presence of H₂O₂. However it oxidised diaminobenzidine and guaiacol in the absence of H₂O₂. Veratryl alcohol and phenol red were not substratesfor this enzyme with or without addition of H₂O₂ and Mn(II). In addition the enzyme did not produce H₂O₂.     

Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium

Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K _m values for activity showed a difference between the cellular fractions with a K _m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V _max values observed were 0.83 nmol min⁻ (nmol microsomal P-450) ⁻¹ and 0.4 nmol min⁻¹ (nmol cytosolic P-450)⁻¹. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.     

De-novo biosynthesis of chlorinated aromatics by the white-rot fungus Bjerkandera sp. BOS55. Formation of 3-chloro-anisaldehyde from glucose.

The white-rot fungus Bjerkandera sp. BOS55 produced de-novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi, other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde and a yet unknown compound containing two chlorine atoms. Additionally GC/MS analysis revealed the production of small amounts of anisyl alcohol and 3-chloro-anisyl alcohol. After 14 days, the extracellular fluid of Bjerkandera BOS55 contained 100 microM veratraldehyde and 50 microM 3-chloro-anisaldehyde. This is the first report of de-novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. Anisaldehyde and 3-chloro-anisaldehyde were also produced by Bjerkandera adusta but not by Phanerochaete chrysosporium.     

Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency

Abstract A lignin-degrading enzyme has been detected in culture supernatants of Phanerochaete chrysosporium strain INA-12 grown under non-limiting nitrogen conditions. Highest levels of enzyme activity were observed when glycerol served as carbon source. Veratryl alcohol, a known secondary metabolite of P. chrysosporium , was also produced in high nitrogen/glycerol cultures of strain INA-12 and closely followed the development of the 'ligninase' activity. Evolution of ¹⁴CO₂ from ¹⁴C-ring-DHP was readily observed when a hydrogen peroxide-generating system was added to 5-day-old high nitrogen/glycerol cultures which contained high amounts of enzyme.     

Molecular subtyping within a single Salmonella typhimurium phage type, DT204c, with a PCR-generated probe for IS200

Abstract Pullulan is an industrial biopolymer produced by the yeast-like fungus Aureobasidium , usually by direct fermentation of starch. Despite evidence that autogenous amylases produced during these fermentations are detrimental to the final molecular mass of the product, fundamental studies of these enzymes have not been reported. Total extracellular amylases were studied from the promising production strain NRRL Y-12,974. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml⁻¹ in glucose-grown cultures to 0.03 IU ml⁻¹ in soluble-starch-grown cultures. All cultures showed both α-amylase activity and activity against pullulan. Synthetic oligosaccharide substrates were apparently attacked by an α-glucosidase, produced in highest levels by maltose-grown cultures.     

A novel type of oxygenolytic ring cleavage: 2,4-oxygenation and decarbonylation of 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline

Abstract The utilization of quinaldine (2-methylquinoline) by Arthrobacter sp. Rü61a proceeds via 1 H -4-oxoquinaldine, 1 H -3-hydroxy-4-oxoquinaldine, and N -acetyl-anthranilic acid. By analogy, 1 H -4-oxoquinoline is degraded by Pseudomonas putida 33/1 via 1 H -3-hydroxy-4-oxoquinoline and N -formylanthranilic acid. Using the purified enzymes from both organisms, the mode of N -heterocyclic ring cleavage was investigated. The conversions of 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinoline to N -acetyl- and N -formylanthranilic acid, respectively, were both accompanied by the release of carbon monoxide. The enzyme-catalysed transformations were performed in an [¹⁸O]O₂ atmosphere and resulted in the incorporation of two oxygen atoms into the respective products, N -acetyl- and N -formylanthranilic acid, indicating an oxygenolytic attack at C-2 and C-4 of both 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinolone.     

Phosphodiesterase and phosphotriesterase in Rhizobium and Bradyrhizobium strains and their roles in the degradation of organophosphorus pesticides

Of 13 Rhizobium and Bradyrhizobium strains investigated for the production of cellular and extracellular phosphodiesterase and phosphotriesterase, all were found to produce both enzymes. Phosphodiesterase was produced at a much higher level than phosphotriesterase. Rhizobium meliloti TAL 1373 was the most productive. The extracellular enzymes were activated by inclusion in the assay mixture of Ca²⁺ or Mg²⁺. The enzymes were inhibited by Zn²⁺ but not significantly affected by Cu²⁺, Co²⁺ and Mn²⁺. Both hydrolases were inhibited by dithiothreitol but not by thiol-directed inhibitors, suggesting that sulphydryl groups are not directly involved in catalysis. The enzymes have the ability to hydrolyse some organophosphorus compounds, suggesting that Rhizobium and Bradyrhizobium strains play an important role in the degradation of organophosphorus pesticides.     

Mode of interference of chlorosis-inducing herbicides with peroxisomal enzyme activities

In rye leaves ( Secale cereale L. cv. Petkus "Kustro") bleached in the presence of the chlorosis-inducing herbicides aminotriazole, haloxidine, San 6706 or difunone in white light of 54.2 W m^-2 (5000 lx), catalase activity was very low. In addition, the activities of glycolate oxidase and hydroxypyruvate reductase were strongly diminished in treatments with San 6706 and difunone. The lowering of the peroxisomal enzyme activities was observed in red, but not in blue light and did not occur after treatment with the non-bleaching pyridazinone derivative San 9785. The deficiencies of the peroxisomal enzymes did not appear to be involved in the initiation of the chlorosis. Instead they are probably produced as secondary consequences of the bleaching. Low peroxisomal enzyme activities were also obtained without herbicide treatment by growing the leaves in an atmosphere of 2% O₂ and 3% CO₂, but in this case were not accompanied by an increased sensitivity of the Chl to photooxidative bleaching. The peroxisomal enzymes reached as high activities as in untreated controls when the herbicide-treated leaves were grown at a low light intensity of 0.106 W m^-2 (10 lx). After transfer of herbicide-treated leaves grown under 0.106 W m^-2 to 306 W m^-2 (30 000 lx), catalase was strongly inactivated, even at 0°C. In treatments with San 6706 and difunone the increase of the activities of glycolate oxidase and hydroxypyruvate reductase was either stopped, remaining unchanged, or the enzymes were slightly inactivated after exposure to 306 W m^-2 (30 000 lx). The observations suggest that the inactivation of peroxisomal enzymes results from photooxidative events in the chloroplasts.     

Formation of 2-hydroxy-4-methylpentanoic acid from l-leucine by Clostridium butyricum

Abstract Five Clostridium butyricum strains of different origin were grown in trypticase-yeast extract-hemin medium with or without d-glucose (TGYH or TYH medium, respectively) and in a synthetic basal medium with d-glucose (BMG medium). 2-Hydroxy-4-methylpentanoic acid was detected by gas chromatography-mass spectrometry (GC-MS) for the five strains whether grown in TGYH or TYH medium (270 or 170 μM, respectively). In BMG medium supplemented with l-leucine (10 mM), the concentration of this metabolite was strongly increased (2.8 mM versus 10 μM in the control). After culture in TGYH or TYH medium supplemented with l-( methyl -²H₃)leucine, 2-hydroxy-4-([²H₃]methyl)pentanoic acid was detected by GC-MS. This observation demonstrates that C. butyricum is able to convert l-leucine into the corresponding 2-hydroxy acid and opens a new aspect in the study of C. butyricum metabolism.     

Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: Proposal of a novel glycolytic pathway based on 13C labelling data and enzyme activities

Abstract Suspensions of maltose-grown cells of the hyperthermophilic archaeon Pyrococcus furiosus , when incubated at 90°C with 35 mM [1-¹³C]glucose or [3-¹³C]glucose, consumed glucose at a rate of about 10 nmol min⁻¹ (mg protein)⁻¹. Acetate (10 mM), alanine (3 mM), CO₂ and H₂ were the fermentation products. The ¹³C-labelling pattern in alamine and acetate were analyzed. With [1-¹³C]glucose the methyl group of both alanine and acetate was labelled; with [3-¹³C]glucose only the carboxyl group of alanine was labelled whereas acetate was unlabelled. Extracts of maltose-grown cells contained glucose isomerase (12.8 U mg⁻¹, 100°C), ketohexokinase (0.23 U mg⁻¹, 100°C), and fructose 1-phosphate aldolase (0.06 U mg^{−1, 100°C). Enzymes catalyzing the formation of fructose 1,6-bisphosphate from fructose 1-phosphate or fructose 6-phosphate could not be detected. As publihed previously by our group and other authors P. furiosus also contains enzymes of glyceraldehyde conversion to 2-phosphoglycerate according to a non-phosphorylated Entner-Doudoroff pathway, of dihydroxyacetone phosphate conversion to 2-phosphoglycerate according to the Embden-Meyerhof pathway, and of 2-phosphoglycerate conversion - via pyruvate - to acetate and alanine. Based on the enzyme activities in P. furiosus , the following pathway for glucose degradation to alanine and acetate in cell suspensions is proposed which can explain the [13}C]glucose labelling data: glucose→ fructose → fructose 1- phosphate → dihydroxyacetone phosphate + glyceraldehyde and further conversion of both trioses to alanine and acetate via pyruvate.     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Ligninase production in agitated conditions by Phanerochaete chrysosporium

Abstract Extracellular H₂O₂-dependent ligninase activity of Phanerochaete chrysosporium was produced in agitated culture conditions when veratryl alcohol or veratraldehyde were added to the cultures. The enzyme production was suppressed by cycloheximide indicating that true protein synthesis occurred. The activated cultures were also able to degrade synthetic lignin. Reduction of veratraldehyde to corresponding alcohol during secondary metabolism was a good indicator of the effect of agitation on cell metabolism. Too high agitation speed led to complete inhibition of both the reduction reaction and the ligninolytic activity.     

Extracellular pectin lyase produced by Neocallimastix sp. LM1, a rumen anaerobic fungus

A viscometric assay was used to assess the extracellular pectinolytic enzyme activity produced by Neocallimastix sp. LM1 during growth in a medium containing grass leaves as substrate. The highest activity was measured at pH 8.0, in the presence of CaCl₂. This anaerobic fungus apparently produced an endo-acting pectin lyase (EC 4.2.2.10), which was induced in the presence of pectin.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.