Jamile Queiroz Pereira

Pigment Production by Filamentous Fungi on Agro-Industrial Byproducts: an Eco-Friendly Alternative

The search for new sources of natural pigments has increased, mainly because of the toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic industries. Fungi provide a readily available alternative source of natural pigments. In this context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3, Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments producers. The fungal identification was performed using ITS and part of the β-tubulin gene sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-industrial residues, with the exception of P. vasconiae that produced pigments only on potato dextrose broth. The production of yellow pigments was predominant and the two strains of P. chrysogenum were the largest producers. In addition, the production of pigments and mycotoxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract, the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude filtrates have potential to be used in the textile industry; nevertheless, additional pigment purification is required for food and pharmaceutical applications.

Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

Bacillus spp. Isolated from Puba as a Source of Biosurfactants and Antimicrobial Lipopeptides

Several products of industrial interest are produced by Bacillus, including enzymes, antibiotics, amino acids, insecticides, biosurfactants and bacteriocins. This study aimed to investigate the potential of two bacterial isolates (P5 and C3) from puba, a regional fermentation product from cassava, to produce multiple substances with antimicrobial and surface active properties. Phylogenetic analyses showed close relation of isolates P5 and C3 with Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. Notably, Bacillus sp. P5 showed antimicrobial activity against pathogens such as Listeria monocytogenes and Bacillus cereus, in addition to antifungal activity. The presence of genes encoding pre-subtilosin (sboA), malonyl CoA transacylase (ituD), and the putative transcriptional terminator of surfactin (sfp) were detected in Bacillus sp. P5, suggesting the production of the bacteriocin subtilosin A and the lipopeptides iturin A and surfactin by this strain. For Bacillus sp. C3 the presence of sboA and spas (subtilin) genes was observed by the first time in members of B. cereus cluster. Bacillus sp. P5 showed emulsifying capability on mineral oil, soybean biodiesel and toluene, while Bacillus sp. C3 showed emulsifying capability only on mineral oil. The reduction of the surface tension in culture medium was also observed for strain P5, confirming the production of surface-active compounds by this bacterium. Monoprotonated molecular species and adducts of sodium and potassium ions of surfactin, iturin, and fengycin were detected in the P5 culture medium. Comparative MS/MS spectra of the peak m/z 1030 (C14 surfactin A or C15 surfactin B [M+Na]+) and peak m/z 1079 (C15 iturin [M+Na]+) showed the same fragmentation profile of standards, confirming the molecular identification. In conclusion, Bacillus sp. P5 showed the best potential for the production of antifungal, antibacterial, and biosurfactant substances.

Proteolytic system of Bacillus sp. CL18 is capable of extensive feather degradation and hydrolysis of diverse protein substrates

ABSTRACT 1. Feathers are recalcitrant protein-rich wastes produced in huge amounts by poultry processing for meat production. Hence, feather bioconversion and protease production by Bacillus sp. CL18 were investigated. 2. Bacillus sp. CL18 demonstrated a remarkable feather-degrading potential. Through cultivations on feather broth (10 g l−1 feathers), 94.5% ± 3% of whole feathers were degraded after 4 d. Increases in soluble protein contents were observed and protease production was maximal also at d 4. This strain produced diverse proteolytic enzymes during growth. 3. Crude protease displayed optimal activity at 55°C (50–62°C), pH 8.0 (7.0–9.0) and a low thermal stability. Proteolytic activity increased in the presence of Ca2+, Mg2+, Triton X-100, Tween 20 and dimethyl sulphoxide. Inhibition profile indicated that crude protease contains, mainly, serine proteases. Enzyme preparation hydrolysed mainly casein and soy protein isolate. 4. The keratinolytic capacity of Bacillus sp. CL18 at moderate temperatures (30°C) might be appropriate for feather conversion, resulting in protein hydrolysates and proteolytic enzymes. Proteases are postulated to be added-value products that can be obtained from such a bioprocess.

Prospecting soil bacteria from subtropical Brazil for hydrolases production

Abstract Eighteen bacterial strains were isolated from soil of an urban area located in a transition zone between the Atlantic Forest and Pampa biomes, in southern Brazil. These strains were screened for cellulolytic, lipolytic and proteolytic potentials. Eleven isolates (61%) were able to produce cellulolytic enzymes on carboxymethylcellulose (CMC) agar plates, 14 isolates (78%) were proteolytic on skim milk agar plates, and all isolates demonstrated lipolytic/esterolytic potential on tributyrin agar (TBA) plates. From the 18 bacteria, nine (50%) were shown to produce the three investigated enzyme activities. Selected isolates were then evaluated for growth and enzyme production at different conditions of temperature and pH on CMC agar, TBA, and feather meal agar plates. As a general trend, growth and hydrolysis zones were observed at pH 6.0–9.0 and 30–37∘C. Sequencing of 16S rRNA gene fragments indicated that 10 isolates belonged to the genus Bacillus, three to Lysinibacillus genus, and the remaining isolates were representatives of Serratia, Phyllobacterium, Paenibacillus, Acinetobacter, and Curtobacterium. The isolate Bacillus sp. CL18 displayed competence for feather degradation when cultured in mineral medium (30∘C, pH 7.0) containing a single feather as the only organic substrate. Results from bioprospection indicate the functional versatility of the bacterial isolates, which might be of significance from both ecological and biotechnological perspectives.

Whole-Genome Shotgun Sequence of the Keratinolytic Bacterium Lysobacter sp. A03, Isolated from the Antarctic Environment

ABSTRACT Lysobacter sp. strain A03 is a protease-producing bacterium isolated from decomposing-penguin feathers collected in the Antarctic environment. This strain has the ability to degrade keratin at low temperatures. The A03 genome sequence provides the possibility of finding new genes with biotechnological potential to better understand its cold-adaptation mechanism and survival in cold environments.

A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: Insights about enzyme activity at low temperatures

Currently, there is a great interest for customized biocatalysts that can supply the ongoing demand of industrial processes, but also deal with the growing concern about the environment. In this scenario, cold-adapted enzymes have features that make them very attractive for industrial and biotechnological purposes. Here, we describe A03Pep1, a new cold-adapted serine peptidase isolated from Lysobacter sp. A03 by screening a genomic library. The enzyme is synthesized as a large inactive prepropeptidase that, after intramolecular processing, gives rise to the active form, of 35kDa. The heterologous expression of A03Pep1 was carried out in E. coli cells harboring the vector pGEX-4T-2-a0301. Its activity was optimal at pH 9.0 and 40°C, in the presence of 25mM Ca2+, which may contribute to the thermal stability of the enzyme. The 3D structure modelling predicted a less deep and more open binding pocket in A03Pep1 than that observed in the crystal structure of its mesophilic homologous AprV2, presumably as a way to enhance the probability of substrate binding at low temperatures. These results provide possible approaches in developing new biotechnologically relevant peptidases active at low to moderate temperatures.