Purification, characterization and synthetic application of a thermally stable laccase from Hexagonia tenuis MTCC-1119.

A thermally stable laccase was purified from the culture filtrate of Hexagonia tenuis MTCC-1119. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion-exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native-PAGE) both gave single protein bands, indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 100 kDa. The purification fold and percentage recovery of the enzyme activity were 12.75 and 30.12%, respectively. The pH and the temperature optima were 3.5 and 45○C, respectively. The enzyme was most stable at pH 4.0 when exposed for 1 h. Using 2,6-dimethoxyphenol (DMP), 2,2’ [azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 3,5-dimethoxy-4-hydroxybenzaldehyde azine (syringaldazine) as the substrates, the Km, kcat and kcat/Km values of the laccase were 80 μM, 2.54 s-1, 3.17 × 104 M-1s-1, 36 μM, 2.54 s-1, 7.05 × 104 M-1s-1 and 87 μM, 2.54 s-1, 2.92 × 104 M-1s-1, respectively. The purified laccase was finally used for the selective biotransformation of aromatic methyl group to aldehyde group in presence of diammonium salt of ABTS as the mediator and products were characterized by HPLC, IR and 1H NMR. The percentage yields of these transformed products were >91%.

Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust

Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc) and manganese peroxidase (MnP). New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm-1 of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm-1, respectively). The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes.

Mycoremediation of Congo red dye by filamentous fungi

Azo, anthroquinone and triphenylmethane dyes are the major classes of synthetic colourants, which are difficult to degrade and have received considerable attention. Congo red, a diazo dye, is considered as a xenobiotic compound, and is recalcitrant to biodegradative processes. Nevertheless, during the last few years it has been demonstrated that several fungi, under certain environmental conditions, are able to transfer azo dyes to non toxic products using laccases. The aim of this work was to study the factors influencing mycoremediation of Congo red. Several basidiomycetes and deuteromycetes species were tested for the decolourisation of Congo red (0.05 g/l) in a semi synthetic broth at static and shaking conditions. Poor decolourisation was observed when the dye acted as the sole source of nitrogen, whereas semi synthetic broth supplemented with fertilizer resulted in better decolourisation. Decolourisation of Congo red was checked in the presence of salts of heavy metals such as mercuric chloride, lead acetate and zinc sulphate. Decolourisation parameters such as temperature, pH, and rpm were optimized and the decolourisation obtained at optimized conditions varied between 29.25- 97.28 percent at static condition and 82.1- 100 percent at shaking condition. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis revealed bands with molecular weights ranging between 66.5 to 71 kDa, a characteristic of the fungal laccases. High efficiency decolourisation of Congo red makes these fungal forms a promising choice in biological treatment of waste water containing Congo red.

Lignin degradation, ligninolytic enzymes activities and exopolysaccharide production by Grifola frondosa strains cultivated on oak sawdust

Fourteen strains of Grifola frondosa (Dicks.) S. F. Gray, originating from different regions (Asia, Europe and North America) were tested for lignin degradation, ligninolytic enzyme activities, protein accumulation and exopolysaccharide production during 55 days of cultivation on oak sawdust. Lignin degradation varied from 2.6 to7.1 percent of dry weight of the oak sawdust substrate among tested strains. The loss of dry matter in all screened fungi varied between 11.7 and 33.0 percent, and the amount of crude protein in the dry substrate varied between 0.94 to 2.55 percent. The strain, MBFBL 596, had the highest laccase activity (703.3 U/l), and the maximum peroxidase activity of 22.6 U/l was shown by the strain MBFBL 684. Several tested strains (MBFBL 21, 638 and 662) appeared to be good producers of exopolysaccharides (3.5, 3.5 and 3.2 mg/ml respectively).

Production of laccase by Pynoporus sanguineus using 2, 5 - Xylidine and ethanol

Enzyme application in biotechnological and environmental processes has had increasing interest due to its efficiency, selectivity and mainly for being environmentally healthful, but these applications require a great volume of enzymes. In this work the effect of different concentrations of ethanol and 2,5 - xylidine on growth and production of laccase by Pycnoporus sanguineus was investigated. In a medium containing 200 mg.L-1 of 2,5 - xylidine or 50 g.L-1 of ethanol, the maximum activity of laccase was 2019 U.L-1 and 1035 U.L-1, respectively. No direct correlation between biomass and activity of laccase was observed for any of the inducers used during the tests. Ethanol concentrations, larger than or equal to 20 g.L-1, inhibited the radial growth of P. sanguineus. This study showed that ethanol, which has less toxicity and cost than the majority of the studied inducers, presents promising perspectives for laccase production by P. sanguineus.

Xenobiotics enhance laccase activity in alkali-tolerant gama-proteobacterium JB / Xenobióticos aumentam a atividade de lacase em gama-Proteobacterium JB alcali-tolerante

Various genotoxic textile dyes, xenobiotics, substrates (10 µM) and agrochemicals (100 µg/ml) were tested for enhancement of alkalophilic laccase activity inã-proteobacterium JB. Neutral Red, Indigo Carmine, Naphthol Base Bordears and Sulphast Ruby dyes increased the activity by 3.7, 2.7, 2.6 and 2.3 fold respectively. Xenobiotics/substrates like p-toluidine, 8-hydroxyquinoline and anthracine increased it by 3.4, 2.8 and 2.3 fold respectively. Atrazine and trycyclozole pesticides enhanced the activityby 1.95 and 1.5 fold respectively.

Vários corantes têxteis genotóxicos, xenobióticos, substratos (10 mM) e agroquímicos (100 mM/mL) foram testados quanto ao aumento da atividade de lacase em ã-Proteobacterium JB. Os corantes Neutral Red, Indigo Carmine, Naphtol Base Bordears e Sulphast Ruby aumentaram a atividade em 3,7, 2,7, 2,6 e 2,3 vezes, respectivamente. Xenobióticos/substratos como p-toluidina, 8-hidroxiquinolina e antracina aumentaram a atividade em 3,4, 2,8 e 2,3 vezes, respectivamente. Atrazina e pesticidas triciclozol aumentaram a atividade em 1,95 e 1,5 vezes, respectivamente.

Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes / Produção de ligninases por linhagens de fungos Basidiomicetos usando resíduos agrícolas lignocelulósicos e aplicação das enzimas na descoloração de corantes sintéticos

Wood rotting Basidiomycetes collected in the ôEstação Ecológica do Noroeste Paulistaõ, São José do Rio Preto, São Paulo State, Brazil, concerning Aphyllophorales order and identified as Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp SXS48, Picnoporus sanguineus SXS 43 and Phellinus rimosus SXS47 were tested for ligninases production by solid state fermentation (SSF) using wheat branor rice straw as culture media. C. byrsina produced the highest laccase (200 U mL-1) and Lentinus sp produced the highest activities of manganese peroxidase (MnP) and lignin peroxidase (LiP) (7 and 8 U mL-1, respectively), when cultivated on wheat bran. The effect of N addition on enzyme production was studied in medium containing rice straw and the data showed an increase of 3 up to 4-fold in the laccase production compared to that obtained in SSF on wheat bran. The laccases presented optimum pH at 3.0-3.5 and were stable at neutral pH values. Optimum pH for MnP and LiP activities was at 3.5 and between 4.5 and 6.0, respectively. All the strains produced laccase with optimum activities between 55-60ºC while the peroxidases presented maximum activity at temperatures of 30 to 55ºC. The crude enzymes promoted decolorization of chemically different dyes with around 70% of decolorization of RBBR and cybacron blue 3GA in 6h oftreatment. The data indicated that enzymes from these basidiomycetes strains are able to decolorize synthetic dyes.

Fungos decompositores de madeira, do grupo Basidiomicetes, coletados na ôEstação Ecológica do NoroestePaulistaõ, São José do Rio Preto, São Paulo, Brasil, pertencentes a ordem Aphyllophorales e identificados como Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp. SXS48,Picnoporus sanguineus SXS 43 e Phellinus rimosus SXS47 foram estudados para a produção de ligninases por FES (fermentação em estado sólido) usando farelo de trigo ou palha de arroz como meio de cultura. A espécie C. byrsina produziu a maior quantidade de lacase (200 U mL-1) enquanto que Lentinus sp. foi o melhor produtor de manganês peroxidase (MnP) e lignina peroxidase (LiP) (7 e 8 U mL-1, respectivamente), quando cultivados em meio composto por farelo de trigo. A avaliação do efeito da suplementação de nitrogênio do substrato sólido lignocelulósico (palha de arroz) indicou um aumento de 3 a 4 vezes na produção de lacase. A caracterização das enzimasmostrou que as lacases apresentaram atividade ótima em pH 3,0-3,5 e foram estáveis em pH de neutro a alcalino. O pH ótimo para atividade de MnP e LiP foi de 3,5 e entre 4,5 e 6,0, respectivamente. Todas as linhagens produziram lacase com atividade ótima a 55-60ºC, enquanto as peroxidases apresentaram atividades máximas entre temperaturas de 30 e 55ºC. A aplicaçãodas soluções enzimáticas brutas, obtidas pelo cultivo das linhagens em meio de farelo de trigo, em testes de descoloração de corantes sintéticos de diferentes grupos químicos levou amais 70% de perda de cor dos corantes RBBR e de cybacron blue 3GA, em 6h de tratamento. Os dados obtidos indicaramque as soluções enzimáticas contendo ligninases produzidas pelas linhagens de basidiomicetos estudadas promoveram adescoloração de corantes sintéticos.

Lignolytic enzymes produced by Trametes villosa ccb176 under different culture conditions

The expression of the enzymatic system produced by basidiomycetous fungi, which is involved in the degradation of xenobiotics, mainly depends on culture conditions, especially of the culture medium composition. Trametes villosa is a strain with a proven biotechnological potential for the degradation of organochlorine compounds and for the decolorization of textile dyes. The influence of glucose concentration, addition of a vegetable oil-surfactant emulsion, nature of the surfactant and the presence of manganese and copper on the growth, pH and production of laccase, total peroxidase and manganese-dependent peroxidase activities were evaluated. In general, acidification of the medium was observed, with the pH reaching a value close to 3.5 within the first days of growth. Laccase was the main activity detected under the different conditions and was produced throughout the culture period of the fungus, irrespective of the growth phase. Supplementation of the medium with vegetable oil emulsified with a surfactant induced manganese-dependent peroxidase activity in T. villosa. Higher specific yields of laccase activity were obtained with the addition of copper.

A expressão do sistema enzimático produzido por fungos basidiomicetos envolvido na degradação de xenobióticos é bastante dependente das condições de cultivo, principalmente da composição do meio de cultivo. Trametes villosa CCB176 é uma linhagem com comprovado potencial biotecnológico para degradação de compostos organoclorados e descoloração de corantes têxteis. Foi avaliada a influência da concentração de glicose, adição de emulsão de óleo vegetal e surfactante, natureza do surfactante e os metais manganês e cobre no crescimento, pH e na produção das atividades de lacase, de peroxidases totais e de peroxidase dependente de manganês. No geral, ocorreu acidificação do meio com pH atingindo valor próximo a 3,5, nos primeiros dias de crescimento. Lacase foi a principal atividade detectada nas diferentes condições e sua produção se deu durante todo o período de cultivo do fungo, independente da fase de crescimento. Suplementação do meio com óleo vegetal emulsificado com surfactante resultou em indução da atividade de peroxidase dependente de manganês produzida por T. villosa. Maiores valores de rendimento específico da atividade de lacase foram propiciados pelo cobre.