Purificação e caracterização bioquímica de β-glicosidases de Penicillium chrysogenum e sua aplicação na suplementação de coquetéis enzimáticos para a hidrólise de biomassas

• Autor(a) principal: Costa, Samara Graciane da
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2459
• topic: Beta-glicosidases; Penicillium chrysogenum; Biomassas; Beta-glucosidase; Penicillium chrysogenum; Biomass; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

β-glucosidases have received attention in many fields, mainly because of their practical applications. The objectives of this work were to stablish the best conditions for P. chrysogenum cultivation in submerged culture containing wheat bran as carbon source and the purification and characterization of β-glucosidases found. The culture supernatant was subjected to ammonium sulphate precipitation and hydrophobic interaction chromatography. Two β-glucosidases were found, PcβGlu1 and PcβGlu2. For further purification, PcβGlu2 was subjected to gel filtration chromatography. PcβGlu1 was more stable than PcβGlu2 when subjected to high temperatures and both have optimum activity at pH 5.0. PcβGlu1 and PcβGlu2 have respective native molecular masses of 241 kDa and 95 kDa (gel filtration). The determination of anomeric carbon configuration demonstrates that enzymes hydrolyze their substrates with retention of configuration. The PcβGlu2 activity was more inhibited for Cu 2+ , Mg 2+ , Zn 2+ e Hg + at 10 mM than PcβGLu1. PcβGlu1 showed Michaelian-Menten kinetics for all substrates tested with K M values ranging from 0.09 ± 0.01 (laminarin) to 1.7 ± 0.1 mM (C2). PcβGlu2 showed substrate inhibition for MUβGlu, pNPβGlu, cellodextrins (C3, C4 and C5), octilβGlu and Lb, with K M values ranging from 0.014 ± 0.001 (MUβGlu) to 0.64 ± 0.06 mM (Cb). Glucose is competitive inhibitor and celobiose is a mixed-type inhibitor of PcβGlu1 and PcβGlu2 when pNPβGlu is used as substrate. PcβGlu2 was more inhibited than PcβGlu1for both inhibitors. PcβGlu1 was identified with 15% of coverage as the product coded by the gene Pc18g01940 and PcβGlu2 was identified as as the product coded by the gene Pc20g10170 with 3,5 % of coverage. The two enzymes bind glucosyl residues with more affinity at -1 and +1 subsites. Comparing the two enzymes PcβGlu2 has more pontetial to be applied on biomass hydrolysis because it was able to hydrolyse several sources of biomass without addition of supplementary enzymes.