Biotechnological application of endophytic filamentous bipolaris and curvularia: a review on bioeconomy impact.

The filamentous Bipolaris and Curvularia genera consist of species known to cause severe diseases in plants and animals amounting to an estimated annual loss of USD $10 billion worldwide. Despite the harmful effect of Bipolaris and Curvularia species, scarce attention is paid on beneficial areas where the fungi are used in industrial processes to generate biotechnological products. Catalytic potential of Bipolaris and Curvularia species in the production of biodiesel, bioflucculant, biosorbent, and mycoherbicide are promising for the bioeconomy. It is herein demonstrated that knowledge-based application of some endophytic Bipolaris and Curvularia species are indispensable vectors of sustainable economic development. In the twenty-first century, India, China, and the USA have taken progress in the biotechnological application of these fungi to generate wealth. As such, some Bipolaris and Curvularia species significantly impact on global crop improvement, act as catalyst in batch-reactors for biosynthesis of industrial enzymes and medicines, bioengineer of green-nanoparticle, agent of biofertilizer, bioremediation and bio-hydrometallurgy. For the first time, this study discusses the current advances in biotechnological application of Bipolaris and Curvularia species and provide new insights into the prospects of optimizing their bioengineering potential for developing bioeconomy.

Bilirubin Oxidase Adsorption onto Charged Self-Assembled Monolayers: Insights from Multiscale Simulations.

The efficient immobilization and orientation of bilirubin oxidase (BOx) on different solid substrates are essential for its application in biotechnology. The T1 copper site within BOx is responsible for the electron transfer. In order to obtain quick direct electron transfer (DET), it is important to keep the distance between the T1 copper site and electrode surface small and to maintain the natural structure of BOx at the same time. In this work, the combined parallel tempering Monte Carlo simulation with the all-atom molecular dynamics simulation approach was adopted to reveal the adsorption mechanism, orientation, and conformational changes of BOx from Myrothecium verrucaria (MvBOx) adsorbed on charged self-assembled monolayers (SAMs), including COOH-SAM and NH2-SAM with different surface charge densities (±0.05 and ±0.19 C·m-2). The results show that MvBOx adsorbs on negatively charged surfaces with a "back-on" orientation, whereas on positively charged surfaces, MvBOx binds with a "lying-on" orientation. The locations of the T1 copper site are closer to negatively charged surfaces. Furthermore, for negatively charged surfaces, the T1 copper site prefers to orient closer to the surface with lower surface charge density. Therefore, the negatively charged surface with low surface charge density is more suitable for the DET of MvBOx on electrodes. Besides, the structural changes primarily take place on the relatively flexible turns, coils, and α-helix. The native structure of MvBOx is well preserved when it adsorbs on both charged surfaces. This work sheds light on the controlling orientation and conformational information on MvBOx on charged surfaces at the atomistic level. This understanding would certainly promote our understanding of the mechanism of MvBOx immobilization and provide theoretical support for BOx-based bioelectrode design.

A Chrysoporthe cubensis enzyme cocktail produced from a low-cost carbon source with high biomass hydrolysis efficiency.

Low cost and high efficiency cellulolytic cocktails can consolidate lignocellulosic ethanol technologies. Sugarcane bagasse (SCB) is a low cost agro-industrial residue, and its use as a carbon source can reduce the costs of fungi cultivation for enzyme production. Chrysoporthe cubensis grown under solid state fermentation (SSF) with wheat bran has potential to produce efficient enzymatic extracts for SCB saccharification. This fungus was grown under submersed fermentation (SmF) and SSF with in natura SCB, pretreated with acid or alkali and with others carbon sources. In natura SCB induced the highest carboxymethylcellulase (CMCase), xylanase, ß-xylosidase, α-galactosidase and mannanase activities by C. cubensis under SSF. In natura and washed SCB, inducers of enzyme production under SSF, did not induce high cellulases and hemicellulases production by C. cubensis in SmF. The C. cubensis enzymatic extract produced under SSF with in natura SCB as a carbon source was more efficient for lignocelulolic biomass hydrolysis than extracts produced under SSF with wheat bran and commercial cellulolytic extract. Chrysoporthe cubensis showed high potential for cellulases and hemicellulases production, especially when grown under SSF with in natura SCB as carbon source.

The discovery of novel LPMO families with a new Hidden Markov model.

BACKGROUND: Renewable biopolymers, such as cellulose, starch and chitin are highly resistance to enzymatic degradation. Therefore, there is a need to upgrade current degradation processes by including novel enzymes. Lytic polysaccharide mono-oxygenases (LPMOs) can disrupt recalcitrant biopolymers, thereby enhancing hydrolysis by conventional enzymes. However, novel LPMO families are difficult to identify using existing methods. Therefore, we developed a novel profile Hidden Markov model (HMM) and used it to mine genomes of ascomycetous fungi for novel LPMOs. RESULTS: We constructed a structural alignment and verified that the alignment was correct. In the alignment we identified several known conserved features, such as the histidine brace and the N/Q/E-X-F/Y motif and previously unidentified conserved proline and glycine residues. These residues are distal from the active site, suggesting a role in structure rather than activity. The multiple protein alignment was subsequently used to build a profile Hidden Markov model. This model was initially tested on manually curated datasets and proved to be both sensitive (no false negatives) and specific (no false positives). In some of the genomes analyzed we identified a yet unknown LPMO family. This new family is mostly confined to the phyla of Ascomycota and Basidiomycota and the class of Oomycota. Genomic clustering indicated that at least some members might be involved in the degradation of ß-glucans, while transcriptomic data suggested that others are possibly involved in the degradation of pectin. CONCLUSIONS: The newly developed profile hidden Markov Model was successfully used to mine fungal genomes for a novel family of LPMOs. However, the model is not limited to bacterial and fungal genomes. This is illustrated by the fact that the model was also able to identify another new LPMO family in Drosophila melanogaster. Furthermore, the Hidden Markov model was used to verify the more distant blast hits from the new fungal family of LPMOs, which belong to the Bivalves, Stony corals and Sea anemones. So this Hidden Markov model (Additional file 3) will help the broader scientific community in identifying other yet unknown LPMOs.

Lateral Protein-Protein Interactions at Hydrophobic and Charged Surfaces as a Function of pH and Salt Concentration.

Surface adsorption of Thermomyces lanuginosus lipase (TLL)-a widely used industrial biocatalyst-is studied experimentally and theoretically at different pH and salt concentrations. The maximum achievable surface coverage on a hydrophobic surface occurs around the protein isoelectric point and adsorption is reduced when either increasing or decreasing pH, indicating that electrostatic protein-protein interactions in the adsorbed layer play an important role. Using Metropolis Monte Carlo (MC) simulations, where proteins are coarse grained to the amino acid level, we estimate the protein isoelectric point in the vicinity of charged surfaces as well as the lateral osmotic pressure in the adsorbed monolayer. Good agreement with available experimental data is achieved and we further make predictions of the protein orientation at hydrophobic and charged surfaces. Finally, we present a perturbation theory for predicting shifts in the protein isoelectric point due to close proximity to charged surfaces. Although this approximate model requires only single protein properties (mean charge and its variance), excellent agreement is found with MC simulations.

Efficient production of pullulan using rice hull hydrolysate by adaptive laboratory evolution of Aureobasidium pullulans.

Pullulan production by Aureobasidium pullulans CCTCC M 2012259 using rice hull hydrolysate as the carbon source was conducted. The acetic acid in the hydrolysate was demonstrated to exert a negative effect on pullulan biosynthesis. Instead of employing expensive methods to remove acetic acid from the hydrolysate, a mutant A. pullulans ARH-1 was isolated following 20 cycles of adaptive laboratory evolution of the parental strain on medium containing acetic acid. The maximum pullulan production achieved by the adapted mutant at 48 h using the hydrolysate of untreated rice hull was 22.2 g L(-1), while that obtained by the parental strain at 60 h was 15.6 g L(-1). The assay of key enzymes associated with pullulan biosynthesis revealed that acetic acid inhibited enzyme activity rather than suppressing enzyme synthesis. These results demonstrated that adaptive evolution highly improved the efficiency of pullulan production by A. pullulans using the hydrolysate of untreated rice hull.

Ultrasonic pilot-scale reactor for enzymatic bleaching of cotton fabrics.

The potential of ultrasound-assisted technology has been demonstrated by several laboratory scale studies. However, their successful industrial scaling-up is still a challenge due to the limited pilot and commercial sonochemical reactors. In this work, a pilot reactor for laccase-hydrogen peroxide cotton bleaching assisted by ultrasound was scaled-up. For this purpose, an existing dyeing machine was transformed and adapted by including piezoelectric ultrasonic devices. Laboratory experiments demonstrated that both low frequency, high power (22 kHz, 2100 W) and high frequency, low power ultrasounds (850 kHz, 400 W) were required to achieve satisfactory results. Standard half (4 g/L H2O2 at 90 °C for 60 min) and optical (8 g/L H2O2 at 103 °C for 40 min) cotton bleaching processes were used as references. Two sequential stages were established for cotton bleaching: (1) laccase pretreatment assisted by high frequency ultrasound (850 kHz, 400 W) and (2) bleaching using high power ultrasound (22 kHz, 2100 W). When compared with conventional methods, combined laccase-hydrogen peroxide cotton bleaching with ultrasound energy improved the whitening effectiveness. Subsequently, less energy (temperature) and chemicals (hydrogen peroxide) were needed for cotton bleaching thus resulting in costs reduction. This technology allowed the combination of enzyme and hydrogen peroxide treatment in a continuous process. The developed pilot-scale reactor offers an enhancement of the cotton bleaching process with lower environmental impact as well as a better performance of further finishing operations.

Production of multi-fiber modifying enzyme from Mamillisphaeria sp. for refining of recycled paper pulp.

Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.

Transcriptional monitoring of steady state and effects of anaerobic phases in chemostat cultures of the filamentous fungus Trichoderma reesei.

BACKGROUND: Chemostat cultures are commonly used in production of cellular material for systems-wide biological studies. We have used the novel TRAC (transcript analysis with aid of affinity capture) method to study expression stability of approximately 30 process relevant marker genes in chemostat cultures of the filamentous fungus Trichoderma reesei and its transformant expressing laccase from Melanocarpus albomyces. Transcriptional responses caused by transient oxygen deprivations and production of foreign protein were also studied in T. reesei by TRAC. RESULTS: In cultures with good steady states, the expression of the marker genes varied less than 20% on average between sequential samples for at least 5 or 6 residence times. However, in a number of T. reesei cultures continuous flow did not result in a good steady state. Perturbations to the steady state were always evident at the transcriptional level, even when they were not measurable as changes in biomass or product concentrations. Both unintentional and intentional perturbations of the steady state demonstrated that a number of genes involved in growth, protein production and secretion are sensitive markers for culture disturbances. Exposure to anaerobic conditions caused strong responses at the level of gene expression, but surprisingly the cultures could regain their previous steady state quickly, even after 3 h O2 depletion. The main effect of producing M. albomyces laccase was down-regulation of the native cellulases compared with the host strain. CONCLUSION: This study demonstrates the usefulness of transcriptional analysis by TRAC in ensuring the quality of chemostat cultures prior to costly and laborious genome-wide analysis. In addition TRAC was shown to be an efficient tool in studying gene expression dynamics in transient conditions.