Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate.

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.

Characterisation of the extracellular polysaccharides produced by isolates of the fungus Acremonium.

Solid state (13)C NMR studies of the extracellular glucans from the fungi Acremonium persicinum C38 (QM107a) and Acremonium sp. strain C106 indicated a backbone of (1-->3)-beta-linked glucosyl residues with single (1-->6)-beta-linked glucosyl side branches for both glucans. Analyses of enzymatic digestion products suggested that the average branching frequency for the A. persicinum glucan (66.7% branched) was much higher than that of the Acremonium sp. strain C106 glucan (28.6% branched). The solid state (13)C NMR spectra also indicated that both glucans are amorphous polymers with no crystalline regions, and the individual chains are probably arranged as triple helices.

Biochemistry and molecular biology of exocellular fungal beta-(1,3)- and beta-(1,6)-glucanases.

Many fungi produce exocellular beta-glucan-degrading enzymes, the beta-glucanases including the noncellulolytic beta-(1,3)- and beta-(1,6)-glucanases, degrading beta-(1,3)- and beta-(1,6)-glucans. An ability to purify several exocellular beta-glucanases attacking the same linkage type from a single fungus is common, although unlike the beta-1,3-glucanases, production of multiple beta-1,6-glucanases is quite rare in fungi. Reasons for this multiplicity remain unclear and the multiple forms may not be genetically different but arise by posttranslational glycosylation or proteolytic degradation of the single enzyme. How their synthesis is regulated, and whether each form is regulated differentially also needs clarifying. Their industrial potential will only be realized when the genes encoding them are cloned and expressed in large quantities. This review considers what is known in molecular terms about their multiplicity of occurrence, regulation of synthesis and phylogenetic diversity. It discusses how this information assists in understanding their functions in the fungi producing them. It deals largely with exocellular beta-glucanases which here refers to those recoverable after the cells are removed, since those associated with fungal cell walls have been reviewed recently by Adams (2004). It also updates the earlier review by Pitson et al. (1993).

The three beta-1,3-glucanases from Acremonium blochii strain C59 appear to be encoded by separate genes.

Three exocellular beta-1,3-glucanases from Acremonium blochii strain C59, BGN3.2, BGN3.3 and BGN3.4, were purified. Two, BGN3.2 and BGN3.4 appeared to act as exo-enzymes against laminarin from Laminaria digitata, while BGN3.3 displayed an endo-mode of action. The N-terminal amino acid sequence data for BGN3.2 and BGN3.4 suggested these two enzymes may be encoded by different genes. The gene encoding the BGN3.2 glucanase was fully sequenced, and its deduced amino acid sequence was similar to those for all other sequenced fungal exo-beta-1,3-glucanases. This BGN3.2 gene consists of an uninterrupted ORF of 2349 bp encoding 783 amino acids possibly with two cleavage sites for the potential removal of a pre- and pro-protein, respectively. A DNA fragment encoding a portion of the BGN3.4 gene was amplified by PCR, and the nucleotide sequence of this fragment confirmed that BGN3.2 and BGN3.4 are encoded by different genes. The internal peptide sequences of BGN3.3 were not present in the amino acid sequence deduced from the BGN3.2 gene, reinforcing the view that BGN3.3 is also genetically different to BGN3.2. Genetic differences between multiple forms of fungal beta-1,3-glucanases from a single fungus have not been reported previously.

Structure and assembly of epiglucan, the extracellular (1-->3;1-->6)-beta-glucan produced by the fungus Epicoccum nigrum strain F19.

In a previous article [Carbohydr. Res.2001, 331, 163-171] two different structures for the possible modular repeating unit of the extracellular beta-glucan, epiglucan produced by the fungus Epicoccum nigrum strain F19 were proposed. Clarifying which was the more likely one was considered essential before attempts were made to understand how epiglucan was assembled by this fungus. Data from Smith degradation analyses of epiglucan were consistent with the repeating unit of structure I, where single glucosyl residues are attached by (1-->6)-beta-linkages to two out of every three glucosyl residues in the (1-->3)-beta-linked glucan backbone. Repeated Smith degradations of 14C-glucose labelled epiglucan showed that chain elongation occurred from its non-reducing end. Side chain insertion into the growing glucan was followed by analysis of real time incorporation of 13C-glucose into epiglucan by 13C NMR, and 14C-glucose by enzymic digestion of the synthesised 14C-epiglucan. All data obtained were consistent with the view that single (1-->6)-beta-linked glucosyl side residues are inserted simultaneously as the glucan backbone elongates.

Which morphological forms of the fungus Aureobasidium pullulans are responsible for pullulan production?

Attempts were made to clarify the precise location and possible site of production of the alpha-glucan pullulan in different morphological forms of the fungus Aureobasidium pullulans. Gold-conjugated pullulanase was used as the specific probe for this purpose. No cell wall pullulan-like material was detected by transmission electron microscopy (TEM) in any morphological form of this fungus, although intracellular electron transparent material bound this probe. When silver enhancement of this gold-conjugated pullulanase probe was used, the data strongly suggested that only swollen cells and chlamydospores, and neither hyphae nor unicellular blastospores, often held responsible for pullulan formation, appeared to produce pullulan-like material.

Purification and characterization of the (1-->3)-beta-glucanases from Acremonium sp. IMI 383068.

Three extracellular (1-->3)-beta-glucanases were purified from the fungus Acremonium sp. IMI 383068. Higher activities were unexpectedly obtained with pustulan, a (1-->6)-beta-glucan as carbon source, than when grown with laminarin, a (1-->3)-beta-glucan. Preliminary evidence suggests that these enzymes are not constitutive, but are inducible, and that their synthesis is repressed by glucose. All three had the same molecular masses, similar pH and temperature optima and none were glycosylated. They all appeared to have an exo-hydrolytic mode of substrate attack. N-terminal amino acid sequence data indicate that substantial post-translational modification of these had occurred, and that while two may be encoded by the same gene, the third may be genetically different.