Robert F.H. Dekker

Optimization of laccase production by Botryosphaeria sp. in the presence of veratryl alcohol by the response-surface method.

Abstract Botryosphaeria sp. produced two laccases (PPO-I and PPO-II) constitutively, whose titers were enhanced by veratryl alcohol. The effect of veratryl alcohol and yeast extract concentration, time of cultivation and agitation speed were evaluated by factorial analysis to select variables for optimizing the production of laccases. Maximal laccase production was determined using a second-order central-composite design and analyzed by the response-surface method. Veratryl alcohol concentration and time of cultivation were the main factors increasing laccase production, while yeast extract had no influence within the range 0.2–2.0% w/v. Response-surface analysis showed that 30.4 mM veratryl alcohol, for 4.5 days at 28°C and 180 rpm, were the optimal conditions to maximize PPO-I production, while conditions for maximal PPO-II production occurred within a range of 28–35 mM veratryl alcohol over a growth period of 4–5.5 days. The model predicted 5.6 U ml −1 for PPO-I, and 0.6–1.0 U ml −1 for PPO-II, which agreed with the experimentally observed results.

Structural characterization of Botryosphaeran: a (1→3;1→6)-β-d-glucan produced by the ascomyceteous fungus, Botryosphaeria sp.

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomyceteous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and 13C NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1-->3)-beta-D-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1-->6)-beta-linked glucosyl, and (1-->6)-beta-linked gentiobiosyl residues.

A new role for veratryl alcohol: Regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.

Genetic diversity among Botryosphaeria isolates and their correlation with cell wall-lytic enzyme production

Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and β-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and β-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Comparison of the laccases, molecular marker proteins, and induction of pycnidia by three species of botryosphaeriaceous fungi

Three species of botryosphaeriaceous fungi,Botryosphaeria sp. isolate MAMB-5,Botryosphaeria ribis andLasiodiplodia theobromae, were compared for the production of pycnidia and laccases. Laccases were produced both intra- and extra-cellularly when the fungi were cultivated on basal medium in the presence and absence of veratryl alcohol, withBotryosphaeria sp. MAMB-5 showing the highest enzyme titres. Electrophoretic examination of intracellular marker proteins (esterases and phosphatases) and laccases indicated that the three species were genetically distinctly different, although the laccase zymograms for the three fungi showed similarity. The production of pycnidia occurred under continuous lighting at 28°C, but conditions differed among the three fungal species. Production could be induced on artificial media (potato-dextrose and oat agar) under stress-induced conditions where the mycelium was stimulated by physical abrasion, and in the case ofBotryosphaeria sp. isolate MAMB-5 on eucalypt woodchips. Evidence is presented that veratryl alcohol facillitated the secretion of intracellular-localised laccases into the extracellular medium.

Phytotoxin produced by Bipolaris euphorbiae in-vitro is effective against the weed Euphorbia heterophylla

Four virulent strain isolates of the fungus, Bipolaris euphorbiae (previously identified as a Helminthosporium sp.), isolated from host plants in four states within Brazil were screened for the production of phytotoxins that promoted wilting and defoliation of the Brazilian weed, Euphorbia heterophylla, commonly found growing among soyabean crops. Only one isolate, B. euphorbiae Strain I (EUPH petropar from Mato Grosso state), produced phytotoxin in-vitro when grown in stationary culture for 7 d at 28 ° C on minimum salts medium supplemented with 1.5 % glucose as the sole carbon source. Phytotoxin was also produced when the fungal strain was grown on fructose, galactose, mannose, xylose and sucrose. The addition of nitrogen source (yeast extract, peptone or malt extract) to the culture medium did not influence phytotoxin production. The phytotoxin produced by Strain I was most active at pH 6.0, stable between pH 3-9, and was highly thermostable, remaining fully active when heated at 90 ° C for 1 h.

Structural characterization of the bglH gene encoding a beta-glucosidase-like enzyme in an endophytic Bacillus pumilus strain

A beta-glucosidase-like enzyme-encoding gene (bglH) of an endophytic Bacillus pumilus strain (CL16) was cloned using a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned fragment (2484 bp) was determined and characterized. An incomplete open reading frame (ORF) of 534 bp (ORF1) designated bglP and a complete ORF of 1419 bp (ORF2) designated bglH, located in the fragment, are organized in an operon. The protein deduced from 1419 bp (ORF2) had 472 amino acid residues without a characteristic signal peptide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced from bglH gene had high similarity with b-glucosidases from the glycosyl hydrolase family 1. Over-expression of the B. pumilus bglH gene in E. coli showed a 54 kDa protein whose identity was confirmed by mass spectrometry (MALDI-TOF).

Structure and Biological Functions of d-Glucans and Their Applications

Abstract Glucans are natural macromolecules obtained from the cell wall of yeast and cereals or as an extracellular metabolite from filamentous fungi and bacteria. Glucans have wide commercial applications in the chemical and pharmaceutical sectors because of their biomedical and technological properties. They constitute the group of biomacromolecules with demonstrated activity in stimulating the immune system and have potential in treating human disease conditions. Such biomacromolecules display anticoagulant, antithrombotic, antioxidant, and antiinflammatory activities, and they have been effective in lowering blood cholesterol levels, in reducing the risk of cardiovascular problems, and in treating various illnesses such as cancer, diabetes, and microbial infections. The chemical and conformational structure of glucans has a close relationship with biological and biomedical activities. The biological response depends on the number of units, degree of polymerization, frequency of branching, type of connections, and chain length, which corresponds to primary structure. Furthermore, there exist a considerable number of studies that described the importance of the three-dimensional conformation of the molecule on its biological properties. In this review, we show the latest advances and citations regarding the relationship between the chemical and conformational structure of glucans on antioxidant activity, immune response, antiproliferative, and hypocholesterolemic activities, as well as in the control of obesity and other benefits in human health.