Improvement of bilirubin oxidase productivity of Myrothecium verrucaria and studies on the enzyme overproduced by the mutant strain in the solid-state fermentation.

Bilirubin oxidase has applications in the health and environmental sectors. Hence, several attempts have been made to increase enzyme yields. However, improvements were not very high. We report here the development of a mutant strain of Myrothecium verrucaria by using UV-rays, which produced 28.8 times more enzyme compared with the parent and was higher than the yields reported in earlier submerged cultures. The mutant strain produced 35.6 times more enzyme than the parent in solid-state fermentation, which is better than that previously reported for a solid-state fermentation process. The specific activity of the enzyme produced by the mutant was higher than that of the parental enzyme. Bilirubin oxidase from both strains showed an optimum activity at pH 7 and 40°C. However, the time required to inactivate half of the initial enzyme activity at 60°C was much higher in the case of the enzyme obtained from the mutant compared with the parental enzyme. The improved thermostability of the enzyme from the mutant strain could be due to the point mutations induced during the UV irradiation, since there was no change in the mass of the enzyme compared with the parental enzyme. The bilirubin oxidase of the mutant strain degraded the bilirubin faster than the enzyme obtained from the parent under similar conditions. Faster activity of the enzyme obtained from the mutant strain could be due to its lower Km (79.4 µM) compared with that of the parental enzyme (184 µM). Hence, the mutant enzyme showed a better functionality and thermostability, which will be beneficial for industrial applications.

The unique characteristics of HOG pathway MAPKs in the extremely halotolerant Hortaea werneckii.

HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte concentrations, and regulates expression of a similar set of genes independent of osmolyte type. To understand how HwHog1 kinases activity is regulated in H. werneckii, we studied HwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activities of recombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and soluble recombinant Hog1 homologues were isolated from insect cells. Our results demonstrate that HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl and KCl concentrations. Importantly, phosphorylation profiles differ depending on the osmolyte type. Additionally, phosphorylated recombinant HwHog1A/B show lower specific kinase activities compared to Hog1ΔC. In summary, HOG pathway MAPKs in the extremely halotolerant H. werneckii show unique characteristics compared to S. cerevisiae homologues. The reported findings contribute to defining the key determinants of H. werneckii osmotolerance, which is important for its potential transfer to economically relevant microorganisms and crops.

Isolation of a chitinolytic Bacillus licheniformis S213 strain exerting a biological control against Phoma medicaginis infection.

Among nine chitinase-producing strains isolated from Tunisian soil, one isolate called S213 exhibited a potent chitinolytic activity. S213 strain was identified as Bacillus licheniformis by API 50CH system and sequence analysis of its partial 16S ribosomal DNA. Chitinolytic activity was induced either by colloidal chitin or fungal cell walls, and the highest chitinase activity reached at the late stationary phase exhibiting optimal temperature and pH of 50-60 °C and pH 6.0, respectively. SDS-PAGE analysis of the secreted colloidal chitin-induced proteins showed a major protein of about 65 kDa. This protein was identified as chitinase on the basis of its peptide sequences which displayed high homology with chitinase sequence of B. licheniformis ATCC 14580. Moreover, chitinolytic activity containing supernatant inhibited the growth of several phytopathogenic fungi including Phoma medicaginis. Interestingly, S213 strain reduced efficiently the damping-off disease caused by P. medicaginis in Medicago truncatula and should be envisaged in enzyme-based biopesticides against phytopathogen application.

The nematophagous fungus Monacrosporium thaumasium and its nematicidal activity on Angiostrongylus vasorum.

BACKGROUND: The dog acts as a reservoir and environmental disseminator of potentially zoonotic parasites. AIMS: The objective of this work was to study the fungus Monacrosporium thaumasium regarding its nematicidal potential in laboratory trials and its proteolytic profile. METHODS: The in vitro test was carried out through two assays (A and B). In assay A, conidia of the fungus N34a were added in positive coprocultures for Angiostrongylus vasorum. In assay B, crude extract (treated group) and distilled water (control group) were added to coprocultures. Next, the proteolytic profile of crude extract of the nematophagous fungus M. thaumasium (NF34a) was revealed by performing a zymogram. RESULTS: There was a reduction (p<0.01) in the averages of larvae recovered from the treated groups (conidia and crude extract) in relation to control groups. The zymogram suggested that the nematophagous fungus M. thaumasium produces a protease of approximately 40 kDa. CONCLUSIONS: The results of this work confirm that the conidia as well as the crude extract of the fungus M. thaumasium may be used to control A. vasorum L1. The proteolytic profile suggested the presence of one protease (Mt1) of approximately 40 kDa that in the future may be used in biological control of L1 of this nematode.

[Solid-State and membrane-surface liquid cultures of micromycetes: specific features of their development and enzyme production (a review)].

Specific features in the development of micromycetes, typical mechanisms of their enzyme production, and conditions providing for an increase in enzyme secretion by the microscopic fungi in solid-state (on natural substrates and inert carriers) and membrane-surface liquid cultures are considered. The prospects and advantages of these fermentation methods for the production of extracellular enzymes are discussed and compared with submerged cultures.

Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

Expression of neutral ß-glucosidase from Scytalidium thermophilum in Candida glabrata for ethanol production from alkaline-pretreated rice straw.

We successfully expressed the neutral ß-glucosidase (BGL4) from Scytalidium thermophilum in the thermotolerant yeast Candida glabrata. Compared to the strain expressing Aspergillus acidic ß-glucosidase (BGL1), the BGL4-expressing strain showed a higher cellobiose fermentation ability at pH 6.0 and 40°C, leading to a higher ethanol production from alkaline-pretreated rice straw.

An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

The enzymes of ß-lactam biosynthesis.

The ß-lactam antibiotics and related ß-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, ß-lactams including penicillins, cephalosporins, and clavulanic acid are produced via fermentation or via modification of fermented intermediates, with important exceptions being the carbapenems and aztreonam. The desire for more efficient routes to existing antibiotics and for access to new and synthetically challenging ones stimulates continued interest in ß-lactam biosynthesis. We review knowledge of the pathways leading to ß-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation

Twenty-seven thermophilic and thermotolerant fungal strains were isolated from soil, decaying organic matter and sugarcane piles based on their ability to grow at 45ºC on medium containing corn straw and cardboard as carbon sources. These fungi were identified in the genera Aspergillus, Thermomyces, Myceliophthora, Thermomucor and Candida. The majority of the isolated strains produced xylanase and cellulases under solid state fermentation (SSF). The highest cellulase and xylanase productions were obtained by the cultivation of the strains identified as Aspergillus fumigatus M.7.1 and Myceliophthora thermophila M.7.7. The enzymes from these strains exhibited maximum activity at pH 5.0 and at 60 and 70ºC. The endo-glucanase from A. fumigatus was stable from 40ºC to 65ºC and both endo-glucanase and xylanase from M. thermophila were stable in this temperature range when in absence of substrate. The enzymes were stable from pH 4.0 to 9.0.

Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes.

A novel 'white' laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml(-1) on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na(+), Mn(2+), Cu(2+) and Zn(2+) while inhibited by DTT, NaN(3) and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications.

Laccase activity in soils: considerations for the measurement of enzyme activity.

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K(M)) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL(-1) or 0.199×10(-12) mol mL(-1) of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed.

Enantiomeric scaffolding of α-tetralone and related scaffolds by EKR (enzymatic kinetic resolution) and stereoselective ketoreduction with ketoreductases.

Stereochemically pure compounds containing an all carbon quaternary stereocenter based on 1-tetralone, 1-indanone and 4-chromanone scaffolds have been synthesized by employing Lipase PS (Burkholderia cepacia) catalyzed kinetic resolution. These scaffolds are further functionalized by microbial ketoreductase enzymes (Geotrichum candidum, Candida parapsilosis and Aspergillus niger) to access stereochemically pure diols which, on further synthetic manipulation, yield novel cyclic compounds.

Alternatives to Trichoderma reesei in biofuel production.

Mutant strains of Trichoderma reesei are considered indisputable champions in cellulase production among biomass-degrading fungi. So, it is not surprising that most R&D projects on bioethanol production from lignocellulosics have been based on using T. reesei cellulases. The present review focuses on whether any serious alternatives to T. reesei enzymes in cellulose hydrolysis exist. Although not widely accepted, more and more data have been accumulated that demonstrate that fungi belonging to the genera Penicillium, Acremonium and Chrysosporium might represent such alternatives because they are competitive to T. reesei on some important parameters, such as protein production level, cellulase hydrolytic performance per unit of activity or milligram of protein.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts.

Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Δgdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Δgdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast.

BEAUVERIA BASSIANA: quercetinase production and genetic diversity

Beauveria bassiana genetic diversity and ability to synthesize quercetin 2,3-dioxygenase (quercetinase) were analyzed. B. bassiana isolates, obtained from Brazilian soil samples, produced quercetinase after induction using 0.5 g/L quercetin. B. bassiana ATCC 7159 (29.6 nmol/mL/min) and isolate IP 11 (27.5 nmol/ml/min) showed the best performances and IP 3a (9.5 nmol/mL/min) presented the lowest level of quercetinase activity in the culture supernatant. A high level of polymorphism was detected by random amplified polymorphic DNA (RAPD) analysis. The use of internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP) did not reveal characteristic markers to differentiate isolates. However, the ITS1-5.8S-ITS2 region sequence analysis provided more information on polymorphism among the isolates, allowing them to be clustered by relative similarity into three large groups. Correlation was tested according to the Person's correlation. Data of our studies showed, that lower associations among groups, level of quercetinase production, or geographical origin could be observed. This study presents the production of a novel biocatalyst by B. bassiana and suggests the possible industrial application of this fungal species in large-scale biotechnological manufacture of quercetinase.

Culturable fungal diversity of shrimp Litopenaeus vannamei boone from breeding farms in Brazil

Litopenaeus vannamei, which is the most common shrimp species cultivated in the northeast of Brazil, is very susceptible to microbial diseases, and this consequently affects productivity. There are reports of bacteria, viruses and protozoa in these shrimp, but not fungi. This study aims to isolate and identify fungi present in shrimp Litopenaeus vannamei, and in their nursery waters, at two breeding farms in Brazil. The pathogenic potential of the isolates was assessed through the qualitative detection of proteases and aflatoxin B production. The 146 isolated fungi comprised 46 species. Aspergillus, Penicillium and Furarium were the three most relevant genera and Aspergillus flavus was the predominant species with a total of 33 isolates. Most of the isolated species are known as potentially pathogenic to humans and other animals. Eighteen isolates of A. flavus and two of A. parasiticus were able to produce aflatoxin B and 33 out of the 46 species produced protease, indicating that these fungi may also become pathogenic to shrimp and their consumers.