Molecular properties of extracellular Botrytis cinerea laccase

Two molecular forms of extracellular laccase induced by different phenolics were studied in Botrytis cinerea. The enzyme induced by grapejuice had a MW of 38 000 and contained 80 % sugar while that induced by gallic acid had a MW of 36 000 and contained 70 % sugar. Both forms contained arabinose, xylose, mannose, galactose and glucose but differed markedly in the relative content of these sugars. Tunicamycin, which inhibits glycosylation of peptide chains, considerably reduced the level of laccase in both hyphae and medium. The two enzyme forms differed also in their isoelectric focusing pattern and amino acid composition, the grape juice enzyme being richer in basic amino acids and poorer in acidic ones. A third form, induced by p-coumaric acid, resembled the laccase induced by gallic acid in many of its properties but was apparently not identical to it. The possible significance of the various forms in relation to the infection process by the fungus is discussed.     

Purification and properties of laccase from Botrytis cinerea

The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its K_m for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.     

Properties of gallic acid-induced extracellular laccase of Botrytis cinerea

The properties of the extracellular laccase produced by Botrytis cinerea induced by garlic acid are compared with the laccase produced by Botrytis in the presence of grape juice. The extra- and intracellular laccases are compared and found to be very similar.     

Pectin,a second inducer for laccase production by Botrytis cinerea

Pectin acts as a second inducer of extracellular laccase formation by Botrytis cinerea, in the presence of a phenolic substance as a first inducer, but pectin alone fails to induce enzyme formation. The possible advantages of this mechanism for the fungus during the process of infection and overcoming host resistance are discussed.     

Induction of laccase formation in Botrytis

Optimal conditions for laccase excretion by Botrytis cinerea were determined. Addition of gallic acid to the culture medium induced maximal enzyme production and excretion. Other inducers previously reported for other organisms were ineffective. The relationships between internal and external laccase formation were determined.     

毛栓菌胞外漆酶的纯化及部分性质研究

毛栓菌胞外漆酶经盐析、透析、sephadexG75和G2 5四步纯化 ,粗酶液被纯化了 39 1倍 ,比活力 12 152 ,回收率 4 5 3%。漆酶最适pH值为 4 0 ,最适反应温度为 30℃。K Cu 2 、Zn2 离子可激活漆酶 ,而Ag 、Fe3 离子可抑制漆酶的活性。漆酶的Km值为 1 81× 10 3mol/L。     

Properties of laccase in Schinus molle

The properties of laccase isolated from Schinus molle, including its MW, amino acid and carbohydrate composition, are described. The enzyme is distinct from Rhus laccase both in K_m and in carbohydrate composition.     

Extracellular laccase from Monilinia fructicola: isolation,primary characterization and application

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5–5.0 and 3.0–5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.     

Inhibitors of polygalacturonase and laccase of Botrytis cinerea and their application to the control of this fungus

EDTA, calcium chloride, and two siderophores (rhodotorulic acid produced by Rhodotorula glutinis BNM 0524, and enterochelin from the bacterium Rahnella aquatilis BNM 0523) were evaluated as possible inhibitors of polygalacturonase (PG) and laccase (LC) from Botrytis cinerea. The aim was to apply them to the control of this pathogen, taking into account the fact that these enzymes are related to the invasion and installation of the fungus in the host. Two B. cinerea Pers.:Fr strains (BNM 0527 and BNM 0528) were used. Enzyme activities were measured in the supernatant of 7-day-old cultures. EDTA, calcium chloride, rhodotorulic acid, or enterochelin were added in the reaction mixture. Laccase activity from two strains was more affected by enterochelin (70-80% inhibition) than by the other compounds, while polygalacturonase was more inhibited (45% inhibition) by calcium chloride. The inhibitors were added to the growth medium and after 7 days of culture, the activities of the enzymes were measured in the supernatants. The production of PG and LC in both strains was lower when enterochelin or calcium chloride was added. In the third step, when the inhibitors were tested on apple, all them provided both effects, preventive and curative, against infections caused by B. cinerea, with EDTA and rhodotorulic acid exhibiting more preventive effects while calcium chloride and enterochelin provided more control of pre-existing infections (curative effect), coinciding with their ability to inhibit the production of polygalacturonase and laccase.     

Conversion of wyerone to wyerol by Botrytis cinerea and B. fabae in vitro

The phytoalexin wyerone was induced to accumulate in cotyledons of Vicia faba infected with Botrytis cinerea or B. fabae. The acetylenic keto ester, wyerone, was converted to the less antifungal corresponding hydroxy ester, wyerol, by both species of Botrytis in vitro.     

Antifungal activity of zinc oxide nanoparticles against Botrytis cinerea and Penicillium expansum

Antifungal activities of zinc oxide nanoparticles (ZnO NPs) and their mode of action against two postharvest pathogenic fungi (Botrytis cinerea and Penicillium expansum) were investigated in this study. ZnO NPs with sizes of 70 ± 15 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ were used. Traditional microbiological plating, scanning electron microscopy (SEM), and Raman spectroscopy were used to study antifungal activities of ZnO NPs and to characterize the changes in morphology and cellular compositions of fungal hyphae treated with ZnO NPs. Results show that ZnO NPs at concentrations greater than 3 mmol l⁻¹ can significantly inhibit the growth of B. cinerea and P. expansum. P. expansum was more sensitive to the treatment with ZnO NPs than B. cinerea. SEM images and Raman spectra indicate two different antifungal activities of ZnO NPs against B. cinerea and P. expansum. ZnO NPs inhibited the growth of B. cinerea by affecting cellular functions, which caused deformation in fungal hyphae. In comparison, ZnO NPs prevented the development of conidiophores and conidia of P. expansum, which eventually led to the death of fungal hyphae. These results suggest that ZnO NPs could be used as an effective fungicide in agricultural and food safety applications.     

白灵侧耳漆酶分离纯化及其酶学性质研究

为了探索白灵侧耳Pleurotus eryngii var.tuoliensis漆酶性质,以白灵侧耳菌株00485为试验材料,从发酵液中分离纯化得到胞外漆酶并对其酶学性质进行测定。纯化流程依次为DEAE-Cellulose阴离子交换层析,CM-Cellulose阳离子交换层析,SP-Sepharose强阳离子交换层析以及Superdex 75凝胶过滤层析,获得胞外白灵侧耳漆酶(Pn Lac)。SDS-PAGE检测结果表明Pn Lac为65k Da的单一蛋白。Pn Lac经过胰蛋白酶水解得到3种肽段,经过NBCI-BLAST后发现它们与糙皮侧耳、环柄韧伞、刺芹侧耳等的漆酶具有同源性。底物为2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)时该种漆酶的最适反应温度和p H分别为50℃和3.0,Ca2+和Hg2+能够抑制它的活性,相反地Cu2+和Mn2+能够提高它的活性,米氏常数Km和Vmax分别是0.17mmol/L和1.76OD/min/U。     

Cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase in wheat leaves infected with Botrytis cinerea

Increases in cinnamate 4-hydroxylase and hydroxycinnamate:CoA ligase activities preceded the deposition of lignin around wounds in wheat leaves infecte     

灵芝EIM-40漆酶的分离纯化及酶学性质

采用冻干浓缩、（NH4）2S04盐析、HiTrapphenyl（FF）疏水层析和QSepharose FastFlow离子交换层析对灵芝EIM-40发酵液进行分离纯化,获得纯化漆酶,纯化倍数为14．6,回收率为5．3％。SDS-PAGE银染的结果为单一条带,相对分子质量约为6．53×104。以愈创木酚和2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）为催化底物进行酶学性质研究,最适pH分别为4．8和4．5,最适温度分别为55和50℃,2种底物在pH4．0。5．0范围内,温度低于50℃时,酶的稳定性都很好。以愈创木酚为底物,Km=645．0umol／L;以ABTS为底物,Km=22．2txmol／L。Cu2＋对该酶起激活作用,Fe2＋、Ca2＋、Ba2＋则完全抑制酶的活性。     

Isolation of a natural sterol and an aliphatic acid from Vernonia cinerea

From the roots of Vernonia cinerea a new natural sterol and a new aliphatic acid characterized as stigmast-5,17(20)-dien-3β-ol and 26-methylheptacosanoic acid, respectively, have been isolated together with stigmasterol and sitosterol.     

Induction of tannin acyl hydrolase (EC 3.1.1.20) activity in some members of fungi imperfecti

The potential of gallotannin, methyl gallate, gallic acid, and pyrogallol to induce tannin acyl hydrolase (EC 3.1.1.20) activity in Aspergillus niger, Aspergillus fischerii, Fusarium solani, and Trichoderma viride has been investigated. The maximum induction ratios recorded were A. fischerii (26.7), F. solani (26.1), and T. viride (40.7) when the fungi were induced with gallotannin, gallic acid, and methyl gallate, respectively. A. niger was devoid of basal enzyme activity. The gallotannin tolerance limits for A. niger, A. fischerii, F. solani, and T. viride were determined by progressively increasing the gallotannin concentration and were found to be 20, 4, 3, and 3%, respectively. F. solanii induced with 3% gallotannin has been recommended for the production of tannase enzyme.     

A galactoglucomannan from extracellular polysaccharides of suspension-cultured cells of Nicotiana tabacum

A polysaccharide containing d-xylose, l-arabinose, d-mannose, d-galactose and d-glucose residues in the molar ratio of 0.07:0.16:1.     

Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption

An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method. The support immobilized 95% of the total laccase units and 35% of the total tyrosinase units. Different mixtures of phenols were passed through the column with co-immobilized laccase and tyrosinase. This method removed 42–90% of different phenolic substances by a single passage through the bioreactor. The second step employed Polyclar for additional removal of phenolic substances from mixtures. The degree of removal depends on the nature of the phenols. Complete removal was achieved for a-naphthol, 2,4-dichlorophenol, 4-methoxyphenol, b-naphthol, 4-chloro-3-methylphenol and catehin. The operational stability of the immobilized system was 10–90 h depending on the substrate. The biocatalyst was capable of continuous transformation of different phenols in mixtures. Journal of Industrial Microbiology & Biotechnology (2000) 24, 383–388. Received 12 August 1999/ Accepted in revised form 18 February 2000     

Biotransformation of geranylated- and acetylated-phloroglucinols by Gibberella fujikuroi into molecules with increased antifungal activity against Botrytis cinerea

Terpenylated phenols possess interesting biological activities. These properties vary mainly according to the type of terpene associated and the degree of oxidation of the molecule. The search for new active molecules for application in different areas of knowledge includes the structural modification of these through ecological methodologies, such as biotransformation. The aims of this study were the biotransformation of geranylated- and acetylated-phloroglucinol by the fungus Gibberella fujikuroi and the evaluation of the antifungal activity of the derivatives. Five major derivatives were identified after biotransformation, highlighting the formation of specific monoacetylated products. In vitro antifungal activity assays against the phytopathogenic fungus Botrytis cinerea indicated that deacetylated derivatives possess higher activity compared to the precursor molecule. In other biotransformation reactions, a relationship between the release of the alkyl chain from the aromatic ring with a decrease of the antifungal activity, was observed. The in vivo tests in infected tomato plants with B. cinerea confirmed the antifungal activity of the derivatives observed in in vitro experiments.