Agroindustrial biomass for xylanase production by Penicillium chrysogenum: purification, biochemical properties and hydrolysis of hemicelluloses

Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.

Yeast HXK2 gene reverts glucose regulation mutation of penicillin biosynthesis in P. chrysogenum

The mutant Penicillium chrysogenum strain dogR5, derived from strain AS-P-78, does not respond to glucose regulation of penicillin biosynthesis and β-galactosidase, and is partially deficient in D-glucose phosphorilating activity. We have transformed strain dogR5 with the (hexokinase) hxk2 gene from Saccharomyces cerevisiae. Transformants recovered glucose control of penicillin biosynthesis in different degrees, and acquired a hexokinase (fructose phosphorylating) activity absent in strains AS- P-78 and dogR5. Interestingly, they also recovered glucose regulation of β-galactosidase. On the other hand, glucokinase activity was affected in different ways in the transformants; one of which showed a lower activity than the parental dogR5, but normal glucose regulation of penicillin biosynthesis. Our results show that Penicillium chrysogenum AS-P-78 and dogR5 strains lack hexokinase, and suggest that an enzyme with glucokinase activity is involved in glucose regulation of penicillin biosynthesis and β-galactosidase, thus signaling glucose in both primary and secondary metabolism; however, catalytic and signaling activities seem to be independent.

Biodegradation of phenol in static cultures by Penicillium chrysogenum ERK1: catalytic abilities and residual phototoxicity / Biodegradación de fenol en cultivos estáticos por Penicillium chrysogenum ERK1: habilidades catalíticas y fitotoxicidad residual

A phenol-degrading fungus was isolated from crop soils. Molecular characterization (using internal transcribed spacer, translation elongation factor and beta-tubulin gene sequences) and biochemical characterization allowed to identify the fungal strain as Penicillium chrysogenum Thorn ERK1. Phenol degradation was tested at 25 °C under resting mycelium conditions at 6, 30, 60, 200, 350 and 400 mg/l of phenol as the only source of carbon and energy. The time required for complete phenol degradation increased at different initial phenol concentrations. Maximum specific degradation rate (0.89978 mg of phenol/day/mg of dry weight) was obtained at 200 mg/l. Biomass yield decreased at initial phenol concentrations above 60 mg/l. Catechol was identified as an intermediate metabolite by HPLC analysis and catechol dioxygenase activity was detected in plate assays, suggesting that phenol metabolism could occur via ortho fission of catechol. Wheat seeds were used as phototoxicity indicators of phenol degradation products. It was found that these products were not phytotoxic for wheat but highly phytotoxic for phenol. The high specific degradation rates obtained under resting mycelium conditions are considered relevant for practical applications of this fungus in soil decontamination processes.

Un aislamiento fúngico capaz de degradar fenol como única fuente de carbono y energía fue aislado de suelos agrícolas. La caracterización molecular (basada en el empleo de secuencias de espaciadores de transcriptos internos, de factores de la elongación de la traducción y del gen de la beta-tubulina) y la caracterización bioquímica permitieron identificar a esta cepa como Penicillium chrysogenum Thom ERK1. Se estudió la degradación de fenol a 25 °C en cultivos estáticos con 6, 30, 60, 200, 350 y 400 mg/l de fenol inicial. El tiempo requerido para completar la degradación de fenol aumentó al elevarse las concentraciones iniciales de dicho compuesto. La máxima tasa de degradación específica (0,89978 mg de fenol/día/mg de peso seco) se obtuvo con 200 mg/l. El rendimiento en biomasa disminuyó con concentraciones Iniciales de fenol mayores de 60 mg/l. Se identificó al catecol como intermediarlo metabolico por HPLC y se observó actividad de catecol dioxigenasa en placa, lo que sugiere que el metabolismo de degradación del fenol ocurre vía orto fisión del catecol. Se utilizaron semillas de trigo como indicadores de fitotoxicidad de los productos de degradación. Estos productos no fueron fitotóxicos para trigo, mientras que el fenol mostró una alta fitotoxicidad. La alta tasa de degradación específica obtenida en condiciones estáticas resulta de gran interés para la aplicación de este hongo en procesos de descontaminación de suelos.