Study of the production of alkalophilic keratinases by fungal strains isolated from Buenos Aires forests

CAVELLO, IVANA; GALARZA, BETINA; GORTARI, CECILIA; CANTERA, CARLOS; HOURS, ROQUE ALBERTO; CAVALITTO, SEBASTIÁN FERNANDO

Curitiba, Brasil

Congreso; 4th International Congress on Bioprocess in Food Industries – X Southern Regional meeting on Food Science and Technology; 2010

Federal University of Parana

Keratinases are a particular class of proteolytic enzymes that has the capability to hydrolyze insoluble keratin. Poultry waste and leather industries wastes are mainly keratin protein. Feather can be used for animal feeding but hair waste has to be properly disposed and represent a serious environmental problem. Due to it is knwon that keratin degradation is facilitated at high pH values and most of the microbial keratinases are alkaline or neutral proteases the search for novel alkalophilic microorganims producer of keratinases are of continuous interest. In order to select a an alkalophilic fungal strain with good productivity of keratinolytic activity, a comparative study was done from the colection of Spegazinni institute. Six non pathogenic fungal strains locally isolated from forest alkaline soils with keratinolic activity were primary selected: Acremonium murorum (Corda) Gams var. murorum LPS # 57 , Aspergillus sidowii LPS # 931, Cladosporium cladosporoides LPS # 953, Neurospora tetrasperma LPS #837, Paecilomyces. lilacinus (Thom) Samson LPS # 876 and Westerdikella dispersa LSP # 834. All of them were incubated on agar feather meal for 15 days at 28°C to detect the production of keratinolityc enzymes (detected as a clear zone around the colony). Fungal strains showing the highest activity on agar feather meal were tested further in a solid state fermentation and in submerged shaken cultures using “hair waste” as a sole energy, carbon and nitrogen source. Solid state fermentation was performed in Petri dishes containing 3 gr of “hair waste”, inoculated with 106 spores/ml suspended in mineral medium (MM) and incubated at 28° C in humid atmosphere for 28 days. The same methodology was performed for the submerged shaken cultures at 28°C and 200 rpm All the samples were tested for proteolityc activity using azocasein and azokeratin as susbtrates. After selected the one who has the highest keratinolityc activity, the study on the effect of reducing agents on the submerged culture using L-Cysteine Hydrocholoride monohydrate  5 mM, Sodium sulfite  5mM and Thioglycolate  5 mM was done.. All the fungus were capable to growth on agar feather meal but only three of them, A. murorum, C. cladosporoides and P. lilacinus, clarified the keratin agar, probably those who did not was due the lack of extracellular enzymes. Altought the three of them grew on solid state “hair waste”, P.lilacinus had the biggest growth level and enzyme production with a maximum of azocaseinolytic and azokeratinolityc activity on 10th day. On the other hand, P.lilacinus grown on submerged cultures showed to have the maximum activity on the 5th day and was higher when the basal medium has the addition of Thioglycolate 5 mM. Change in the pH of the medium towards alkalinity was also noted day after day on the submerged culture. It is proposed that the basis of keratinolysis is the high level of deamination which renders the medium alkaline. Considering the results of this research, the optimization of the culture conditions to increase the yield of the pool enzymes will be the target of the future work.