Barbara M. McDougall

Noncellulolytic fungal β-glucanases: Their physiology and regulation

The occurrence, regulation, and action of fungal enzymes capable of degrading noncellulosic β-glucans, especially 1,3-β- and 1,6-β-glucans, are reviewed. Special consideration is given to their roles in both metabolic and morphogenetic events in the fungal cell, including cell wall extension, hyphal branching, sporulation, budding, and autolysis. Also examined are the protocols currently available for their purification, with some of the properties of purified β-glucanases discussed in terms of their potential applications in industrial, agricultural, and medical fields.

Structure of epiglucan, a highly side-chain/branched (1→3;1→6)-β-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht

The extracellular fungal polysaccharide, epiglucan, synthesised by Epicoccum nigrum is a side-chain/branched (1 --> 3;1 --> 6)-D-beta-glucan. Methylation analysis, 13C DEPT NMR and specific enzymic digestion data show slight variation in branching frequency among the epiglucans from the three strains examined. The (1 --> 3)-beta-linked backbone has (1 --> 6)-beta-linked branches at frequencies greater than the homologous glucans, scleroglucan and schizophyllan, from Sclerotium spp. and Schizophyllum commune, respectively. The structural analyses do not allow a distinction to be made between structures I and II. [structures: see text] Epiglucan displays non-Newtonian shear thinning rheological properties, typical of these glucans.

Purification and characterization of an extracellular β-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in β-glucan degradation

A beta-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH4)2SO4 precipitation followed by anion-exchange and gel filtration chromatography. SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa. The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5. Comparison of the N-terminal amino acid sequence revealed similarities between the A. persicinum enzyme and several other extracellular fungal beta-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala. In addition to the hydrolysis of p-nitrophenyl-beta-glucoside, the enzyme was also active against several other aryl-beta-glucosides as well as a range of beta-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose. D-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by N-bromosuccinimide, N-acetylimidazole, dicyclohexyl carbodiimide, Woodwards Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4, and some metal ions. Possible roles for this enzyme in the noncellulolytic fungus A. persicinum are discussed in light of the increase in the rate of reducing sugar release from beta-glucans by (1-->3)- and (1-->6)-beta-glucanases when the beta-glucosidase is also present in the reaction mixtures.

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.

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans.

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans.

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.

Production of β-glucan degrading enzymes by Acremonium and Cephalosporium species

Thirty-one isolates of the form genera Acremonium and Cephalosoporium were screened for their ability to excrete enzymes capable of degrading b-glucans. Most produced both (1 → 3)- and (1 → 6)-b-glucanases together, although the yields varied with carbon source used. Surprisingly, higher yields of (1 → 3)-b-glucanases were often seen from isolates grown on pustulan, a (1 → 6)-b-glucan which is not hydrolysed by these enzymes. Lower enzyme yields were generally obtained with glucose than with either laminarin, a (1 U 3)- b-glucan or pustulan as carbon sources, suggesting regulation of synthesis by either catabolite repression and}or induction. However, a few isolates, most notably Cephalosporium sp. OXF C13 and Acremonium strictum appeared to have some constitutive- b-glucanase activity. Most of the isolates screened were only very weakly cellulolytic against carboxymethyl cellulose or filter paper as substrates.

Purification and characterization of the (1→3)-β-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.

Factors affecting the synthesis of (1 → 3) and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 grown in batch culture

Abstract Batch fermentations in continuously stirred tank reactors (CSTR) and low shear airlift vessels were operated under a range of conditions, in attempts to optimize the specific activities (units of activity per unit of biomass) of the extracellular (1→3)- and (1→6)-β-glucanases synthesized by the fungus Acremonium sp. IMI 383068. A marked change in the hyphal growth unit (HGU) in this organism was observed in the CSTR with both pustulan and scleroglucan as sole carbon source, in response to changes in agitation speeds. HGU did not change when culture pH or aeration rate was varied in the CSTR with pustulan, even though (1→3)-β-glucanase and to a lesser extent, (1→6)-β-glucanase specific activities were affected by these changes. Higher specific activities for both were obtained under these conditions when scleroglucan was used as sole carbon source in the CSTR at the same concentration. In direct contrast to data from experiments using pustulan, with scleroglucan grown cultures, specific activities of both enzymes could be increased further by increasing aeration rates, an increase not corresponding to any change in HGU of the fungus. FPLC analysis of filtrates showed the presence of an extra (1→3)-β-glucanase in scleroglucan grown cultures that was not present when pustulan was used as sole carbon source, and which accounted for the additional (1→3)-β-glucanase activity measured in media containing scleroglucan. The stability of these enzymes appeared to be insensitive to changes in the fermenter conditions examined. As differences in HGU in response to varying culture conditions did not always correspond to changes in their specific activities, the influence of shear rate on extracellular β-glucanase production in this fungus is considered more likely to be mediated by its effect on oxygen mass transfer rates.

Purification and properties of a (1→6)-β-glucanase from Acremonium sp. IMI 383068

Abstract Several isolates of Acremonium sp. grew well on pustulan, a (1→6)-β-glucan, as sole carbon source and produced extracellular pustulan degrading enzymic activity. The extracellular enzymic activity of Acremonium sp. IMI 383068 against pustulan was due to the synthesis of a single (1→6)-β-glucanase, which was purified to homogeneity by FPLC. The molecular weight of this enzyme was estimated by SDS-PAGE to be 41.2 kDa. The enzyme is non-glycosylated with a pI of 4.5. It hydrolysed pustulan and lutean, both (1→6)-β-glucans, with a lower activity against lutean, and Eisenia bicyclis laminarin, a (1→3)(1→6)-β-glucan. Other substrates examined gave little or no detectable activity. TLC analyses of degradation products from enzymic digests of pustulan were consistent with the (1→6)-β-glucanase having an endo-hydrolytic mode of attack and it may be classified as glucan (1→6)-β-D-glucan glucanohydrolase [EC 3.2.1.75]. N-terminal sequence [SWIS-PROT P82288] analysis and BLAST searching revealed only a low level (ca 50%) of amino acid homology with the (1→6)-β-glucanase from Trichoderma harzianum , the only other fungal (1→6)-β-glucanase sequence contained in the data base. Furthermore, this homology was only revealed after alignment of the Acremonium sp. IMI 383068 sequence starting at amino acid 45 of the T. harzianum sequence, suggesting that the first 44 amino acids were missing from the Acremonium (1→6)-β-glucanase. The possible reasons for this are discussed. Homology was also seen with N-terminal amino acid sequences of several fungal (1→3)-β-glucanases.