Thermophilic fungi are known to produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we report the characterization of a protease produced in state-solid (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus My-celiophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a media composed by a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. ...view middle of the document...
, 1998), and among them alkaline proteases have great biotechnological potential for industrial sectors like laundry detergents, leather process-ing, brewing, food and pharmaceutical industries (Kembhavi et al., 1993).
Thermophilic fungi are known to produce thermostable enzymes. The use of these enzymes may present many advantages due to the high processing temperatures that could be applied, which are related to an increase in reaction rates, improved solubility of reagents, and a decrease in mesophilic contamination. Besides thermal stability, these enzymes also exhibit higher stability towards other protein denaturating conditions when compared to similar mesophilic enzymes (Gusek and Kinsella, 1988).
The available literature shows that a large amount of work has been devoted to the immobilization of enzymes in polymeric carriers (Kennedy et al., 1990). The main advantage of using immobilized enzymes as biocatalysts is that it is possible to reuse them since they can be easily recovered, thereby making the process economically feasible (da Silva et al., 2009). Therefore, for industrial applications, the immobilization of enzymes can offer several advantages and may contribute to increase their stability, making feasible their widespread use in industry.
For immobilization purposes, alginates are one of the most used polymers due to their mild gelling properties and non-toxicity (Won et al., 2005). Alginate is a water-soluble ani-onic linear polysaccharide composed of 1,4-linked -D-mannuronic acid and -L-guluronic acid in different proportions in sequential arrangements, which can be precipitated by the addition of Ca2+ ions (Ertesvag and Valla, 1998; Smidsrod and Skajak-Braek, 1990) allowing to obtain microspheres with good strength and flexibility (De Queiroz et al., 2006).
In this work we report the isolation of a thermophilic fungi and protease production through state-solid and submerged fermentations, and is presented a comparative study on the properties of the protease produced in both fermentative systems. Additionally, we show data on the immobilization of the enzyme from SSF on beads of calcium alginate and also compare the properties of the free and immobilized proteases.
Materials and Methods
Casein, glycine, agar, CaCl2 were obtained from Vetec. Yeast extract was obtained from Oxoid. NaCl, Na2CO3, MnCl2.4H2O, FeSO4.7H2O, sodium phosphate were obtained from Synth. K2SO4, MgCl2 were obtained from Nuclear. Glycerol, ZnSO4.7H2O, CuSO4.5H2O, EDTA, H3BO3, (NH4)2HPO4, CoCl2.5H2O, (NH4)Mo7O24.4H2O were obtained from Merck. (NH4)2SO4, trichloroacetic acid, Hepes, Taps, Caps and BSA were obtained from Sigma. MgSO4 .7H2O and NH4NO3 was obtained from Dinamica. Sodium citrate dehydrate, sodium acetate were obtained from Mallinckrodt Baker. Sodium alginate was obtained from Fluka.
Isolation and Identification
Several thermophilic fungal strains were isolated from box fat, poultry, poultry litter and a composting plant....