Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha-D-mannopyranosyl-(1-->2)-D-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha-D-mannopyranosyl-(1-->3)-D-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as 'decoys' of receptors of type-1 fimbriae carried by enterobacteria.

Imobilização do fungo Penicillium citrinum CBMAI 1186 e lipase de Pseudomonas fluorescens em biopolímeros para aplicações em biocatálise

Diversos biomateriais podem ser aplicados como suportes na imobilização de células totais de fungos filamentosos ou enzimas isoladas, visando a manutenção e o prolongamento da atividade enzimática em processos biocatalíticos. Exemplos promissores de biomateriais são a fibroína da seda e o alginato de sódio. A fibroína é um material protéico com alta estabilidade térmica, elasticidade, resistência à tensão, não sofre ataque microbiano, baixo custo de purificação e alta tenacidade, o alginato é um biopolímero versátil, devido a suas propriedades gelificantes em soluções aquosas. Assim, neste trabalho empregou-se micélios do fungo derivado de ambiente marinho, Penicillium citrinum CBMAI 1186, livres e imobilizados em biopolímeros (fibra de algodão, fibra de fibroína da seda e fibra de paina) na biorredução quimiosseletiva, regiosseletiva e enantiosseletiva da ligação α,β-C=C de enonas α,β-, α,β,γ,δ- e di-α,β-insaturadas previamente sintetizados pela a reação de condensação aldólica. Foi possível a utilização do fungo P. citrinum CBMAI 1186 na redução quimiosseletiva, regiosseletiva e enantiosseletiva da ligação dupla carbono-carbono de sistemas α,β-insaturados. A imobilização do fungo P. citrinum CBMAI 1186 em biopolímeros (algodão, fibroína da seda, paina e quitosana) permitiu a prolongamento da atividade celular do fungo. O protocolo desenvolvido foi capaz de obter compostos até então descritos apenas por síntese clássica. Também foi realizado reações de resolução enzimática de derivados de haloidrinas por diferentes lipases microbianas de: Pseudomonas fluorescens, Candida cylindracea, Rhizopus niveus e Aspergillus niger. A lipase de P. fluorescens foi imobilizada em esferas de fibroína do bicho da seda (método 1, via adsorção) e em blenda com alginato de cálcio (método 2, via encapsulação) em diferentes condições, tais como, variação de solvente, variação da quantidade de enzima imobilizada e tempo de reação. As condições otimizadas foram empregadas em diferentes haloidrinas, rendendo elevados excessos enantioméricos (ee > 99%) e alta razão enanantiomérica (E > 200) para os produtos acetilados. Foi possível desenvolver um protocolo simples, barato e prático para a síntese enantiosseletiva de haloidrina reforçando a versatilidade da fibroína e do alginato como suportes de imobilização para catalisadores heterogêneos. Também foi possível utilizar a lipase imobilizada (método 2) na reação de transesterificação para obtenção do biodiesel etílico. As melhores condições para o bom funcionamento do biocatalisador foram: 30% do biocatalisador, 20% de n-hexano, relação óleo e etanol de 1:4 a 32 ºC por 48 h em agitação magnética (400 rpm). Essas condições permitiram a formação de 42% de rendimento do biodiesel etílico. O biocatalisador apresentou algumas limitações reacionais, tais como, fragilidade frente a elevadas temperaturas (> 32 ºC) e prolongado tempo de agitação magnética. Porém, permaneceu apto no meio por 4 ciclos consecutivas. Conclui-se que os biomateriais (fibroína, alginato e quitosana) podem ser utilizados como alternativas versáteis na imobilização de micélios de fungos filamentoso e de enzimas isoladas para aplicações em biocatalíticas.

Use of Experimental Design for the Optimization of the Production of New Secondary Metabolites by Two Penicillium Species

A fractional factorial design approach has been used to enhance secondary metabolite production by two Penicillium strains. The method was initially used to improve the production of bioactive extracts as a whole and subsequently to optimize the production of particular bioactive metabolites. Enhancements of over 500% in secondary metabolite production were observed for both P. oxalicum and P. citrinum. Two new alkaloids, citrinalins A (5) and B (6), were isolated and identified from P. citrinum cultures optimized for production of minor metabolites.

Investigação química e biológica dos metabólitos secundários derivados de macroalgas marinhas e microrganismos associados

Pós-graduação em Química - IQ

Perfil enzimático e potencial biotecnológico de fungos isolados de jardins das formigas cortadeiras

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immobilization of marine fungi on silica gel, silica xerogel and chitosan for biocatalytic reduction of ketones

The scanning electron microscopy (SEM) analysis showed that whole living hyphal of marine fungi Aspergillus sclerotiorum CBMAI 849 and Penicillium citrinum CBMAI 1186 were immobilized on support matrices of silica gel, silica xerogel and/or chitosan. P. citrinum immobilized on chitosan catalyzed the quantitative reduction of 1-(4-methoxyphenyl)-ethanone (1) to the enantiomer (S)-1-(4-methoxyphenyl)-ethanol (3b), with excellent enantioselectivity (ee > 99%, yield = 95%). Interestingly, ketone 1 was reduced with moderate selectivity and conversion to alcohol 3b (ee = 69%, c 40%) by the free mycelium of P. citrinum. This free mycelium of P. citrinum catalyzed the production of the (R)-alcohol 3a, the antipode of the alcohol produced by the immobilized cells. P. citrinum immobilized on chitosan also catalyzed the bioreduction of 2-chloro-1-phenylethanone (2) to 2-chloro-1-phenylethanol (4a,b), but in this case without optical selectivity. These results showed that biocatalytic reduction of ketones by immobilization hyphal of marine fungi depends on the xenobiotic substrate and the support matrix used. (c) 2012 Elsevier B.V. All rights reserved.

Stereoselective Bioreduction of 1-(4-Methoxyphenyl)ethanone by Whole Cells of Marine-Derived Fungi

Nine strains of marine-derived fungi (Aspergillus sydowii Ce15, A. sydowii Ce19, Aspergillus sclerotiorum CBMAI 849, Bionectria sp. Ce5, Beauveria felina CBMAI 738, Cladosporium cladosporioides CBMAI 857, Mucor racemosus CBMAI 847, Penicillium citrinum CBMAI 1186, and Penicillium miczynskii Gc5) were screened, catalyzing the asymmetric bioreduction of 1-(4-methoxyphenyl) ethanone 1 to its corresponding 1-(4-methoxyphenyl) ethanol 2. A. sydowii Ce15 and Bionectria sp. Ce5 produced the enantiopure (R)-alcohol 2 (>99% ee) in accordance with the anti-Prelog rule and, the fungi B. felina CBMAI 738 (>99% ee) and P. citrinum CBMAI 1186 (69% ee) in accordance with the Prelog rule. Stereoselective bioreduction by whole cells of marine-derived fungi described by us is important for the production of new reductases from marine-derived fungi.

Occurrence of molds on laminated paperboard for aseptic packaging, selection of the most hydrogen peroxide- and heat-resistant isolates and determination of their thermal death kinetics in sterile distilled water

This study aimed at enumerating molds (heat-labile and heat-resistant) on the surface of paperboard material to be filled with tomato pulps through an aseptic system and at determining the most heat-and hydrogen peroxide-resistant strains. A total of 118 samples of laminated paperboard before filling were collected, being 68 before and 50 after the hydrogen peroxide bath. Seven molds, including heat-resistant strains (Penicillium variotii and Talaromyces flavus) with counts ranging between 0.71 and 1.02 CFU/cm(2) were isolated. P. variotii was more resistant to hydrogen peroxide than T. flavus and was inactivated after heating at 85 degrees C/15 min. When exposed to 35 % hydrogen peroxide at 25 degrees C, T. flavus (F5E2) and N. fischeri (control) were less resistant than P. variotti (F1A1). P. citrinum (F7E2) was shown to be as resistant as P. variotti. The D values (the time to cause one logarithmic cycle reduction in a microbial population at a determined temperature) for spores of P. variotii (F1A1) and N. fischeri (control) with 4 months of age at 85 and 90 degrees C were 3.9 and 4.5 min, respectively. Although the contamination of packages was low, the presence of heat-and chemical-resistant molds may be of concern for package sterility and product stability during shelf-life. To our knowledge, this is the first report that focuses on the isolation of molds, including heat-resistant ones, contaminating paperboard packaging material and on estimating their resistance to the chemical and physical processes used for packaging sterilization.

Synthesis and antimicrobial activity of some 2- Piperidinomethylamino-4-(7-H/substituted coumarin-3-yl)- 6-chlorosubstitutedphenyl pyrimidines

Purpose: To prepare and evaluate some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6- chlorosubstitutedphenyl pyrimidines as antimicrobial agents. Methods: Some 2-piperidinomethylamino-4-(7-H/substitutedcoumarin-3-yl)-6-chlorosubstitutedphenyl pyrimidines were prepared by reacting 2-amino-4-(7-H/substitutedcoumarin-3-yl)-6- (chlorosubstitutedphenyl) pyrimidines with piperidine and formaldehyde. The chemical structures of the synthesized compounds were elucidated by Fourier transform infrared (FTIR), 1H-nuclear magnetic resonance (1H-NMR), mass spectrometry and elemental analysis. These compounds were investigated for their antimicrobial activity against ten bacteria and five fungi by serial plate dilution method using standard drugs, namely, ofloxacin and ketoconazole, respectively, and their minimum inhibitory concentrations (MICs) were also determined. Results: A total of eighteen new compounds (1a-18a) were synthesized. Compound 6a (MIC = 50 μg/mL; p < 0.05 or less) displayed the highest activity against S. aureus , E. faecalis , Staphylococcus epidermidis , B. subtilis , and B. cereus . Compound 6a further showed good activity (MIC = 25 μg/mL; p < 0.05 or less) against E. coli ; P. aeruginosa K. pneumonia , B. bronchiseptica , and P. vulgaris . Compounds 6a (MIC = 25 μg/mL; p < 0.0001) and 17a (MIC = 25 μg/mL; p < 0.0001) displayed very good activity against C. albicans , A. niger , A. flavus , M. purpureous , and P. citrinum , respectively. Analysis of structure-activity relationship revealed that the presence of bromo group at 7-postion of the coumarin moiety along with the 4-chlorophenyl group at position-6 of the pyrimidine ring is critical for antimicrobial activity against Gram-positive bacteria, Gram negative bacteria and fungi. Conclusion: The synthesized 2-piperidino derivatives are better antifungal and antibacterial agents than the earlier reported 2-morpholino derivatives, but require further investigations against other microbial strains to ascertain their broad spectrum antimicrobial activity.

Utilização do resíduo líquido de indústria de processamento de suco de laranja como meio de cultura de Penicillium citrinum: depuração biológica do resíduo e produção de enzima

Penicillium citrinum grown in orange juice processing wastes medium under continuous agitation was studied in order to establish optimal conditions (incubation period, incubation temperature, initial pH and nitrogen addition) for biomass and ribonuclease production, as well as, biological depuration of the wastes. Nitrogen addition to wastes medium increased the biomass and ribonuclease production and provides COD reduction. The soy meal shows to be the best nitrogen source. The conditions for a more favorable enzyme and biomass production and COD reduction were initial pH 6.0 and temperature of 27°C. The maximum value obtained for these parameters on optimal conditions of cultivation was 11 U/mL of enzyme, 4 mg/mL of biomass (dry matter) and 55% of COD reduction, in 96 hours of incubation.

Biosynthesis of two dihydropyrrole-polyketides from a marine-derived Penicillium citrinum

Feeding experiments with 13C-labeled precursors were performed in order to establish the biosynthesis of two N-acylated dihydropyrroles, (8E)-1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldec8-ene-1,3-dione (1) and 1-(2,3-dihydro-1H-pyrrol-1-yl)-2-methyldecane-1,3-dione (2), isolated from the cultures of a marine-derived Penicillium citrinum. The biosynthesis of both, 1 and 2, involves the incorporation of acetate, methionine and ornithine.