| Resumo: |
Non-productive adsorption of cellulase and beta-glucosidase enzymes onto lignin is shown to be a limiting factor to enzymatic hydrolysis of lignocellulosic. This study evaluated the interaction of cellulases and beta-glucosidase from Trichoderma reesei (Cellic Ctec2) and Aspergillus niger (Novo 188) commercial cocktail with lignin preparations isolated from liquid hot water sugarcane bagasse by enzymatic (LDHE) and acid hydrolysis (LDHA). Furthermore, a glucose tolerant β-glucosidase was selected from the collection of working fungi of Laboratório de Bioquímica e Microbiologia (Ibilce/Unesp), effect of LDHE and liquid hot water pretreatment-derived phenolic compounds were evaluated. LDHA had higher adsorption capacity on enzymes than LDHE. At 45°C T. reesei enzymes bound to both LDHE and LDHA. Endoglucanase, beta-glucosidase and exoglucanase remained activities in the supernatant after incubation with LDHE were 28, 45 e 63%. Exoglucanase and betaglucosidase were completely adsorbed onto LDHA while endoglucanase activity remained in the supernatant was 14%. A. niger beta-glucosidase was less adsorption than enzymes from T. reesei, it maintained 94 and 48% of the activity in the presence of LDHA and LDHE. Purified beta-glucosidase from A. niger showed 89 and only 2% activity in the supernatant after incubation with LDHE LDHA, while beta-glucosidase from enzymatic cocktail showed 100 and 95% of the enzyme activity. The decrease of temperature to 30°C decreased non-productive adsorption of the enzymes onto LDHE. Endoglucanases, exoglucanases and beta-glucosidase from T. reesei showed 93%, 70% and 83% of the activity in the supernatant, while A. niger beta-glucosidase exhibited 98% of activity. Purified beta-glucosidase from A. niger and the enzyme from enzymatic cocktail weren’t adsorbed at 30°C. In the search for a beta-glucosidase with desirable properties to hydrolysis, T. indicae-seudaticae N31, A. fumigatus M.7.1, R. miehei GF3.3, L. ramosa GF1.5, L. corymbifera GF1.2 e R. pusillus fungi were evaluated for beta-glucosidase production and glucose tolerance. The fungal beta-glucosidase from L. ramosa GF1.5 showed the best results with 44.4% residual activity at 80 mM glucose and 6.4 IU/mL of beta-glucosidase. The fungus only produces a betaglucosidase with estimated molecular weight in 57 kDa, which was characterized with regard to optimum pH and temperature and pH and thermal stability, and then partially purified. The enzyme was strongly inhibited by phenolic compounds and inhibitors from liquid fraction of the liquid hot water pretreatment of sugarcane bagasse. The remaining activity was only 8.5% after 6h of incubation. However, the enzyme wasn’t absorbed onto LDHE, showing increase of up to 14% of the activity in the supernatant. |
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