T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus / Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.

Cellulose and xylan degrading enzymes in Thecotheus pelletieri

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.