The effects of aeration and veratryl alcohol on the production of two laccases by the ascomycete Botryosphaeria sp

The ascomycete, Botryosphaeria sp, produced two extracellular constitutive laccases (PPO-I and PPO-II) active toward the substrates: 2, 21-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) [ABTS], and 2,6-dimethoxyphenol (DMP), respectively. The production of both laccases increased when the fungal isolate was grown in the presence of veratryl alcohol, and resulted in optimal laccase production (100- and 25- fold, respectively) at 40 mM. The effect of aeration on growth and laccase production was studied in baffled flasks, and showed that aeration of the cultures increased the production of both enzymes 4-5 fold in the presence of veratryl alcohol. Both laccases were susceptible to inhibition by azide, acetate and chloride anions. Veratryl alcohol inhibited the laccase-catalyzed polymerization of DMP. Growing cultures of Botryosphaeria sp. produced an exopolysaccharide of the β-glucan type whose synthesis was depressed when grown in the presence of veratryl alcohol. (C) 2001 Elsevier Science Inc. The ascomycete, Botryosphaeria sp, produced two extracellular constitutive laccases (PPO-I and PPO-II) active toward the substrates: 2,2-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) [ABTS], and 2,6-dimethoxyphenol (DMP), respectively. The production of both laccases increased when the fungal isolate was grown in the presence of veratryl alcohol, and resulted in optimal laccase production (100- and 25-fold, respectively) at 40 mM. The effect of aeration on growth and laccase production was studied in baffled flasks, and showed that aeration of the cultures increased the production of both enzymes 4-5 fold in the presence of veratryl alcohol. Both laccases were susceptible to inhibition by azide, acetate and chloride anions. Veratryl alcohol inhibited the laccase-catalyzed polymerization of DMP. Growing cultures of Botryosphaeria sp. produced an exopolysaccharide of the β-glucan type whose synthesis was depressed when grown in the presence of veratryl alcohol

A simple method for monitoring chromatography column eluates for laccase activity during enzyme purification

A simple and economical method is described that allows rapid detection of laccase activity in chromatography column fractions during enzyme purification. Aliquots of column eluants are applied to filter paper coated with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing a numbered grid, and incubated at ambient temperature for 20 min. Indications of enzyme activity are simply observed by a colour change. This method avoids having to manually assay each fraction of a chromatographic run for enzyme activity

Veratryl alcohol as an inducer of laccase by an ascomycete, Botryosphaeria sp., when screened on the polymeric dye Poly R-478

Forty fungi isolated from diverse environments in Western Australia were screened for ligninolytic activity based on in-vivo decolorization of the polymeric dye Poly R-478. Three isolates identified as Aspergillus, Botryosphaeria and Coniochaeta species were selected for further studies. The Botryosphaeria and Coniochaeta isolates were found to produce laccase constitutively in submerged culture when grown on glucose, or on ryegrass seed by solid state fermentation. A comparison of the three isolates grown on glucose in the presence of 3,4-dimethoxybenzyl (veratryl) alcohol (40 mmol 1−1) showed that only the Botryosphaeria isolate produced laccase, with laccase activities 115-fold higher than when grown on glucose alone

The effect of lignin-related compounds on the growth and production of laccases by the ascomycete, Botryosphaeria sp

The concentration at which 50% inhibition (I50) of fungal growth occurred for various lignin-related compounds was evaluated on Botryosphaeria sp. MAMB-5 cultivated on agar medium. A trend for inhibition of growth on lignin-related aromatic acids and phenolic compounds was observed and was based upon chemical structural features: benzoic acids containing di-methoxyl substituents on the aromatic ring were less inhibitory than the parent unsubstituted compound; p-phenolic benzoates containing a single methoxyl group were less inhibitory than those containing a di-methoxyl group; methoxylated derivatives of p-phenolic cinnamic acids were less inhibitory than the unsubstituted parent compound; but un-methylated hydroxyls on di-phenols allowed growth compared to the mono- and di-O-methylated derivatives, which were inhibitory. Botryosphaeria sp. grew on basal media containing up to 10% (w/v) lignosulfonate and kraft lignin in stationary liquid culture. In submerged liquid cultivation, Botryosphaeria sp. grew on basal medium containing abietic acid, catechol, 4-chlorophenol, guaiacol, vanillyl alcohol, veratraldehyde and lignosulfonate, and in each case produced laccases. The production of laccases implicated a possible role for their involvement in the biodegradation of aromatic compounds by Botryosphaeria sp. 4-Chlorophenol was the most toxic of the compounds examined

Synergistic effects of a cellulase-producing Micromonospora carbonacea and an antibiotic-producing Streptomyces violascens on the suppression of Phytophthora cinnamomi root rot of Banksia grandis

Three polyvalent Streptomyces phages were used to isolate four Micromonospora species (M. carbonacea, M. chalcea, M. purpureochromogenes, and M. inositola) from mine-site rhizosphere soils in Western Australia. Streptomyces violascens was isolated using selective isolation techniques from the same soils. The Micromonspora spp. were examined for their ability to produce cellulases. Micromonospora carbonacea, M. chalcea, and M. purpureochromogenes, which were found to produce the enzyme, caused lysis of Phytophthora cinnamomi hyphae. Glasshouse trials showed that the use of the cellulase-producing M. carbonacea isolate, in conjunction with the antibiotic-producing S. violascens isolate, had a synergistic effect on the suppression of the Phytophthora root rot and in promoting growth of Banksia grandis. The importance of using a number of antagonists with different antagonistic abilities to control plant pathogenic fungi is discussed

Screening for microbial trehalases: extracellular trehalases produced by Fusarium species

Two hundred microorganisms comprising actinomycetes, bacteria, fungi and yeasts were screened for extracellular trehalases by their growth on trehalose in solid and liquid culture.Candida albicans, Gelasinospora retispora and four isolates belonging to the genusFusarium produced extracellular trehalases. The production of trehalase by theFusarium spp. was influenced by the nutrient composition of the medium and the carbon source; of the substrates examined starch produced the highest enzyme titre. Trehalase was an inducible enzyme and was repressed when theFusarium spp. were grown on glucose. The properties of the trehalases from two of theFusarium spp. (isolates MU-105 and TR-8) were typical of non-regulatory trehalases. Activity of several α-glucosidases, an amylase, an invertase and a cellobiase was also demonstrated when the two isolates were grown on trehalose

The combined synergistic effects of a cellulase-producing Micromonospora carbonacea and an antibiotic-producing Streptomyces violascens on the suppression of root-rot disease of Banksia grandis caused by Phytophthora cinnamomi

Three polyvalent Streptomyces phages were used to isolate four Micromonospora species (M. carbonacea, M. chalcea, M. purpureochromogenes and M. inositola) from rhizosphere soils from a rehabilitated bauxite mine in Western Australia. Streptomyces violascens was isolated using the selective dry heat technique. M. carbonacea, M. chalcea and M. purpureochromogenes were found to produce cellulases in vitro and cause lysis of Phytophthora cinnamomi hyphae. Glasshouse trials showed that a cellulase-producing M. carbonacea isolate used in conjunction with an antibiotic-producing isolate of S. violascens, had a synergistic effect on the suppression of Phytophthora root-rot, and significantly promoted the growth of Banksia grandis. This study illustrates the importance of combining potential biological control agents with different antagonistic abilities, to control soil-borne plant pathogens