Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Microbial and fungal protease inhibitors—current and potential applications

Proteolytic enzymes play essential metabolic and regulatory functions in many biological processes and also offer a wide range of biotechnological applications. Because of their essential roles, their proteolytic activity needs to be tightly regulated. Therefore, small molecules and proteins that inhibit proteases can be versatile tools in the fields of medicine, agriculture and biotechnology. In medicine, protease inhibitors can be used as diagnostic or therapeutic agents for viral, bacterial, fungal and parasitic diseases as well as for treating cancer and immunological, neurodegenerative and cardiovascular diseases. They can be involved in crop protection against plant pathogens and herbivorous pests as well as against abiotic stress such as drought. Furthermore, protease inhibitors are indispensable in protein purification procedures to prevent undesired proteolysis during heterologous expression or protein extraction. They are also valuable tools for simple and effective purification of proteases, using affinity chromatography. Because there are such a large number and diversity of proteases in prokaryotes, yeasts, filamentous fungi and mushrooms, we can expect them to be a rich source of protease inhibitors as well.     

Conservative ecological and evolutionary patterns in liverwort–fungal symbioses

Liverworts, the most ancient group of land plants, form a range of intimate associations with fungi that may be analogous to the mycorrhizas of vascular plants. Most thalloid liverworts contain arbuscular mycorrhizal glomeromycete fungi similar to most vascular plants. In contrast, a range of leafy liverwort genera and one simple thalloid liverwort family (the Aneuraceae) have switched to basidiomycete fungi. These liverwort switches away from glomeromycete fungi may be expected to parallel switches undergone by vascular plants that target diverse lineages of basidiomycete fungi to form ectomycorrhizas. To test this hypothesis, we used a cultivation-independent approach to examine the basidiomycete fungi associated with liverworts in varied worldwide locations by generating fungal DNA sequence data from over 200 field collections of over 30 species. Here we show that eight leafy liverwort genera predominantly and consistently associate with members of the Sebacina vermifera species complex and that Aneuraceae thalloid liverworts associate nearly exclusively with Tulasnella species. Furthermore, within sites where multiple liverwort species co-occur, they almost never share the same fungi. Our analyses reveal a strikingly conservative ecological and evolutionary pattern of liverwort symbioses with basidiomycete fungi that is unlike that of vascular plant mycorrhizas.     

Do bacterial and fungal communities assemble differently during primary succession?

High‐throughput sequencing technologies are now allowing us to study patterns of community assembly for diverse microbial assemblages across environmental gradients and during succession. Here we discuss potential explanations for similarities and differences in bacterial and fungal community assembly patterns along a soil chronosequence in the foreland of a receding glacier. Although the data are not entirely conclusive, they do indicate that successional trajectories for bacteria and fungi may be quite different. Recent empirical and theoretical studies indicate that smaller microbes (like most bacteria) are less likely to be dispersal limited than are larger microbes – which could result in a more deterministic community assembly pattern for bacteria during primary succession. Many bacteria are also better adapted (than are fungi) to life in barren, early‐successional sediments in that some can fix nitrogen and carbon from the atmosphere – traits not possessed by any fungi. Other differences between bacteria and fungi are discussed, but it is apparent from this and other recent studies of microbial succession that we are a long way from understanding the mechanistic underpinnings of microbial community assembly during ecosystem succession. We especially need a better understanding of global and regional patterns of microbial dispersal and what environmental factors control the development of microbial communities in complex natural systems.     

Bacterial and fungal communities associated with Tuber magnatum‐productive niches

Abstract

Truffles are hypogeous ectomycorrhizal fungi of ecological interest for forestry in soils of the northern hemisphere, and of economical relevance for food markets worldwide. The molecular mechanisms that control truffle body formation are largely unknown, as well as the environmental factors that are likely involved. Among the latter, it has been hypothesized that soil‐borne communities may have an impact on truffle production. To address this question, we investigated bacterial and fungal communities resident in productive versus adjacent non‐productive grounds of the white truffle Tuber magnatum by using PCR‐DGGE. Although bacterial communities were generally highly similar across all samples within the grounds, profiles did cluster according to the productivity of circumscribed niches, and a Moraxella osloensis population appeared to be associated with productive sites. Fungal communities revealed several populations, yet showed no obvious patterns in relation to productivity, although Mortierella and Fusarium oxysporum appeared to be more abundant in the productive area. Our results offer a first glimpse into microbial communities thriving in truffle productive niches, and open the question as to whether microbe‐mediated mechanisms may facilitate/inhibit truffle fruiting‐body production or, vice versa, i.e. whether truffle sporocarps have an impact on the microbes living in the rhizosphere.     

Inhibition of fungal growth by plant chitinases andβ-1,3-glucanases

Summary Plant chitinases and -1,3-glucanases have been demonstrated to inhibit fungal growth in model experiments, both on agar plates or in liquid media. Here,Trichoderma longibrachiatum was taken as a model to study the morphological changes caused by chitinase and glucanase treatments, using cytochemical techniques in combination with fluorescence and electron microscopy. Chitinase, alone or in the presence of glucanase, arrested growth of the hypha: it affected the extreme tip of the fungus producing a thinning of the wall, a balloon-like swelling and a rupture of the plasma membrane. Chitin and glucans were present in the wall, as shown by lectinand enzyme-binding experiments, but they had a different susceptibility to chitinase and -1,3-glucanase. Chitin was present at the apex and in the inner parts of the lateral walls; it was more susceptible to chitinase at the tip than in the subapical part. Glucans mostly occurred on the outer layer where they were degraded by glucanase. The latter did not affect the inner hyphal skeleton. It is suggested that the growth inhibition ofTrichoderma by hydrolytic enzymes is the consequence of a thinning of the cell wall in the hyphal apex, leading to an imbalance of turgor pressure and wall tension which causes the tip to swell and to burst.Abbreviations WGA-FITC wheat germ agglutinin labelled with fluorescein isothiocyanate - ConA-FITC concanavalin A labelled with fluorescein isothiocyanate - PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy     

The Spitzenkörper: a choreographer of fungal growth and morphogenesis

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A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.     

The fungal–mineral interface: challenges and considerations of micro-analytical developments

Over recent years, the role of fungi, especially mycorrhizal fungi, in the weathering of rock-forming minerals has been increasingly recognised. Much of our understanding of the effects of fungi on mineral weathering is based on macroscopic studies. However, the ability of fungi to translocate materials, including organic acids and siderophores, to specific areas of a mineral surface leads to significant spatial heterogeneity in the weathering process. Thus, geomycologists are confronted with unique challenges of how to comprehend and quantify such a high degree of diversity and complicated arrays of interactions. Recent advances in experimental and analytical techniques have increased our ability to probe the fungal–mineral interface at the resolution necessary to decouple significant biogeochemical processes. Modern microscopy, spectroscopy, mass spectrometry, wet chemistry, and scattering techniques allow for the selective extraction of physical, chemical, and structural data at the micro- to nano-scale. These techniques offer exciting possibilities to study fungal–mineral interactions at the scale of individual hyphae. In this review, we give an overview of some of these techniques with their characteristics, advantages and limitations, and how they can be used to further our understanding of biotic mineral weathering.     

Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β-N-acetylhexosaminidase

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.     

ITS, OTUs and beyond—fungal hyperdiversity calls for supplementary solutions

Molecular species recognition of fungi emerged years before DNA barcoding ( Seifert 2009 ). While the ideal fungal DNA barcode seems Utopian, two research decades nevertheless highlight the internal transcribed spacer (ITS) as the best available choice ( Seifert 2009 ). Databases providing reliable ITS sequences of known fungi require enormous efforts, but are urgently needed ( Abarenkov et al. 2010a,b ; Begerow et al. 2010 ). Any criticism of such a commitment seems unjustified. However, exclusive focus on the development of ITS reference libraries will delay the progress towards a deeper ecological insight. It is widely acknowledged that ITS fails to recognize species, particularly in some ascomycete lineages ( Balajee et al. 2009 ; Seifert 2009 ). It also appears paradoxical to solely rely on ITS for ecological recognition of fungal species when modern fungal systematics rely on phylogenetic species recognition with concordance of multiple gene genealogies (see Blackwell 2011 ). Considering that at least 98% of the predicted ～5 million fungal species remain undescribed ( Blackwell 2011 ), how will reliance on ITS alone influence the biodiversity estimates and ecological understanding? In this issue, Gazis et al. (2011) elegantly demonstrate through multi‐locus sequence phylogeny analyses that ITS largely underestimates the species diversity of tropical fungal endophytes and even more importantly obscures fundamental ecological and biogeographical patterns. This thorough reflection on species delimitation criteria and their implications for ecological and biogeographical inferences underline that ITS, particularly in hyperdiverse habitats, provides no shortcut to deeper knowledge of fungal ecology.     

Design and synthesis of marine fungal phthalide derivatives as PPAR-γ agonists

On the basis of a marine fungal phthalide (paecilocin A) skeleton, we synthesized 20 analogs and evaluated them for peroxisome proliferator-activated receptor gamma (PPAR-γ) binding and activation. Among these analogs, 6 and 7 had significant PPAR-γ binding activity, and 7 showed further PPAR-γ activation in rat liver Ac2F cells. In docking simulation, 7 formed H bonds with key amino acid residues of the PPAR-γ binding domain, and the overall positioning was similar to rosiglitazone. This new phthalide derivative is considered an interesting new molecular class of PPAR-γ ligands.     

A new procedure for the measurement of fungal and bacterial α-amylase

Summary A procedure for the measurement of fungal and bacterial -amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end)p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and -glucosidase, and is absolutely specific for -amylase. The assay procedure is simple, reliable and accurate.     

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized β-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.     

Cordyceps: a traditional Chinese medicine and another fungal therapeutic biofactory?

Traditional Chinese medicines (TCM) are growing in popularity. However, are they effective? Cordyceps is not studied as systematically for bioactivity as another TCM, Ganoderma. Cordyceps is fascinating per se, especially because of the pathogenic lifestyle on Lepidopteron insects. The combination of the fungus and dead insect has been used as a TCM for centuries. However, the natural fungus has been harvested to the extent that it is an endangered species. The effectiveness has been attributed to the Chinese philosophical concept of Yin and Yang and can this be compatible with scientific philosophy? A vast literature exists, some of which is scientific, although others are popular myth, and even hype. Cordyceps sinensis is the most explored species followed by Cordyceps militaris. However, taxonomic concepts were confused until a recent revision, with undefined material being used that cannot be verified. Holomorphism is relevant and contamination might account for some of the activity. The role of the insect has been ignored. Some of the analytical methodologies are poor. Data on the "old" compound cordycepin are still being published: ergosterol and related compounds are reported despite being universal to fungi. There is too much work on crude extracts rather than pure compounds with water and methanol solvents being over-represented in this respect (although methanol is an effective solvent). Excessive speculation exists as to the curative properties. However, there are some excellent pharmacological data and relating to apoptosis. For example, some preparations are active against cancers or diabetes which should be fully investigated. Polysaccharides and secondary metabolites are of particular interest. The use of genuine anamorphic forms in bioreactors is encouraged.     

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.     

Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

A nitrilase from Aspergillus niger and an amidase from Rhodococcus erythropolis co-immobilized on a 1-mL Butyl Sepharose column were used for the hydrolysis of 4-cyanopyridine into isonicotinic acid. The former enzyme converted the nitrile into the acid:amide mixture (molar ratio ca. 3:1), while the latter enzyme hydrolyzed the amide by-product. Therefore, the ratio of amide in the total product decreased to about 5%. Sodium sulfate was used as a component of the elution buffer, as the commonly used ammonium sulfate (0.8 M) acted as an amidase inhibitor. The hydrolysis of 4-cyanopyridine by a nitrilase from F. solani gave isonicotinic acid and isonicotinamide at a molar ratio of about 98:2. When using this enzyme and the amidase immobilized on two columns operated in tandem, the percentage of isonicotinamide in total product decreased to <0.2%.