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Produção de [beta]-1,3 glucanases, proteases liticas e quitinases por microrganismos e aplicação na lise de leveduras
Luciana Francisco Fleuri
DATA DE PUBLICAÇÃO
The aim of this work was the study of b-1,3 glucanases, proteases and chitinases production by B1, B22, B26, FXX, Oerskovia sp. nO4 and Cellulomonas cartae nº191 strains in culture media containing differents inductors as well as their application on yeast cell lysis. The strains B26 and Cellulomonas cartae nO191 showed highest protease production using the culture medium II containing 8% of instant yeast. The strains Bl and C. cartae nº191 showed highest b-1,3 glucanase production using the culture medium III containing 1% of yeast cell wall obtained by Dyno-Mill. The strain C. cartae nº191 showed highest chitinase production using the culture medium IV containing 1.5% of chitin neutralized. The culture medium III supernatants obtained by fermentation of strains B1 and C. cartae nº191 demonstrated the biggest yeast cell lysis activity. The enzymes precipitation studies revealed that 60% ammonium sulphate was the best concentration for protease, b-1,3 glucanase and chitinase separation. The b-1,3 glucanase extract obtained by Bl and C. cartae nº191 strains demonstrated lysis activity against Kluyveromyces lodderi, Saccharomyces cerevisiae (yeast Fleischmann), Saccharomyces cerevisiae (yeast Itaiquara), Saccharomyces cerevisiae KL-88, Saccharomyces diastaticus NCYC 713, Saccharomyces cerevisiae NCYC 1001, Candida glabrata NCYC 388, Kluyveromyces marxianus NCYC 587 and Hansenula mrakii NCYC 500. K. marxianus NCYC 587 and H mrakii NCYC 500 were more sensitive to b-1,3 glucanases action, whereas Itaiquara yeast and C. glabrata NCYC 388 were more resistant when compared with S. cerevisiae KL-88 susceptibility. The chitinase extract obtained by C. cartae nO191, in some cases increased the susceptibility of yeast to cellular lysis. The previous treatment of the yeast suspensions with protease from C. cartae nº191, decreased the yeasts cell lysis, mainly when used at high concentrations. The maximum b-1,3 glucanase production by C. cartae nº191, using culture medium III, occurred after 48 hours of fermentation at 35°C and 200 rpm. However, when the fermentation was perform after 24 hours of fermentation at 30ºC and 200 rpm, the b-1,3 glucanase activity obtained was almost the same. The maximum protease production by C. cartae nº191, using culture medium II, occurred after 30 hours of fermentation at 35ºC and 150 rpm, while the highest chitinase production by C. cartae nº191, using culture medium IV, occurred after 72 hours of fermentation at 35°C and 150 rpm. The experimental design study showed that the best conditions to S. cerevisiae KL-88 lysis by b-1,3 glucanase extract were pH 6,5 and 35°C. This study also demonstrated that the yeast cells were more susceptible to lysis after 10 hours cultivation in flasks without agitation. Lysis activity was increased when S. cerevisiae KL-88 cell suspension was treated b-1,3 glucanase and cystein 1mM. The enzyme invertase of S. cerevisiae KL-88 and K marxianus NCYC 587 was extracted after treatment of cell suspension with b-1,3 glucanase obtained from C. cartae nº191. The previous treatment of S. cerevisiae KL-88 and K marxianus NCYC 587 with b-1,3 glucanase, increased the susceptibility to lysis when ultrasonic treatment was used