Produção, purificação e caracterização bioquímica funcional de uma α-L-arabinofuranosidase secretada pelo fungo Coriolopsis byrsina
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2023-04-14
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Universidade Estadual Paulista (Unesp)
Resumo
As α-L-arabinofuranosidases podem ser produzidas por fungos, bactérias e plantas e são responsáveis pela clivagem de ligações glicosídicas α-1,2, α-1,3 e α-1,5 envolvendo arabinose. Nos últimos anos, muitos estudos têm demonstrado suas aplicações no processamento de sucos, visando diminuir a viscosidade e promoção da clarificação, e hidrólise de biomassa vegetal. Outro interesse por estas enzimas decorre de sua capacidade para melhorar a liberação de compostos aromatizantes presentes em bebidas como vinho, cerveja e sucos. Em vista das diferentes aplicações, o presente trabalho focou na prospecção de α-L-arabinofuranosidases dos fungos filamentosos Thermothelomyces heterothallica, Rasamsonia emersoni e Coriolopsis byrsina SXS16. Em seguida, conforme a capacidade de produção de α-L-arabinofuranosidases, foi purificada uma enzima secretada pelo fungo C. byrsina SXS16 e investigada suas propriedades bioquímicas funcionais. A enzima purificada, com massa molecular estimada em 55 kDa, exibiu máxima atividade a pH 3,5-4,0 e 50 ºC, e estabilidade em faixa de pH 3-8 a 4 °C por 24 horas, e temperatura de 10 a 60 ºC por 1 hora, cujas atividades remanescentes foram superiores a 70%. A enzima também se mostrou estável a etanol 5-10% após 24 horas de incubação a 28 °C, retendo atividade enzimática superior a 75% após incubação com etanol 5%, e para etanol 10%, superior a 60% de atividade. Ao avaliar a atividade enzimática sob a influência de íons metálicos, foi observado uma redução da catálise para para todos os íons, especialmente Fe3+ e Hg2+, cujas atividades enzimáticas residuais foram 25% e 33%, respectivamente. Em presença de D e L arabinose, a inibição enzimática foi notada apenas com L-arabinose, exibindo 50% de atividade residual a 0,2 mol L-1 e 30% a 0,9 mol L-1 deste açúcar. A enzima foi ativa sobre p-Nitrofenil-α-L-arabinofuranosideo (pNPA) e p-Nitrofenil-β-D-xilopiranosideo (pNPX), com 6,71 ± 0,23 e 0,38 ± 0,07 U mg-1, respectivamente, e para cinética enzimática com o substrato pNPA, notamos um KM de 3,45 ± 0,9 mmol L-1 e Vmax de 198,2 ± 24 μmol min-1 mg-1. A enzima também foi capaz de hidrolisar α-1,5-L-arabinana linear, mas nenhuma atividade foi notada sobre os substratos p-Nitrofenil-β-D-glucopiranosídeo e p-Nitrofenil-N-acetil-β-D-glicosaminídeo.
α-L-arabinofuranosidases can be produced by fungi, bacteria and plants and are responsible for the cleavage of α-1,2, α-1,3 and α-1,5 glycosidic bonds involving arabinose. In recent years, many studies have demonstrated its applications in juice processing, aiming to reduce viscosity and promote clarification and hydrolysis of plant biomass. Another interest in these enzymes is related to their ability to improve the release of flavoring compounds present in beverages such as wine, beer and juices. In view of the different applications, the present work focused on the prospection of α-Larabinofuranosidases from the filamentous fungi Thermothelomyces heterothallica, Rasamsonia emersoni and Coriolopsis byrsina SXS16. Then, according to the production capacity of α-L-arabinofuranosidase, it was purify the enzyme secreted by the fungus C. byrsina SXS16 and investigated its functional biochemical properties. The purified enzyme, with an estimated molecular mass of 55 kDa, exhibited maximum activity at pH 3.5-4.0 and 50 ºC, and stability in a range of pH 3-8 at 4 °C for 24 hours, and temperature from 10 to 60 ºC for 1 hour, whose remaining activities were greater than 70%. The enzyme was also stable for 5-10% ethanol after 24 hours of incubation at 28 °C, retaining enzyme activity greater than 75% after incubation with 5% ethanol, and for 10% ethanol, greater than 60% activity. When evaluating enzymatic activity under the influence of metal ions, a reduction in catalysis was observed for all ions, especially Fe3+ and Hg2+ , whose residual enzymatic activities were 25% and 33%, respectively. In the presence of D and L arabinose, enzyme inhibition was noted only with L-arabinose, exhibiting 50% residual activity at 0.2 mol L-1 and 30% at 0.9 mol L-1 of this sugar. The enzyme was active on p-Nitrophenyl-α-L-arabinofuranoside (pNPA) and p-Nitrophenyl-β-Dxylopyranoside (pNPX), with 6.71 ± 0.23 and 0.38 ± 0.07 U mg-1 , respectively, and for enzyme kinetics under pNPA substrate, we noted a KM of 3.45 ± 0.9 mmol L-1 and Vmax of 198.2 ± 24 µmol min-1 mg-1 . The enzyme was also able to hydrolyze linear 1,5 α-Larabinan, but no activity was noted on the substrates p-Nitrophenyl-β-D-glucopyranoside and p-Nitrophenyl-N-acetyl-β-D-glucosaminide.
α-L-arabinofuranosidases can be produced by fungi, bacteria and plants and are responsible for the cleavage of α-1,2, α-1,3 and α-1,5 glycosidic bonds involving arabinose. In recent years, many studies have demonstrated its applications in juice processing, aiming to reduce viscosity and promote clarification and hydrolysis of plant biomass. Another interest in these enzymes is related to their ability to improve the release of flavoring compounds present in beverages such as wine, beer and juices. In view of the different applications, the present work focused on the prospection of α-Larabinofuranosidases from the filamentous fungi Thermothelomyces heterothallica, Rasamsonia emersoni and Coriolopsis byrsina SXS16. Then, according to the production capacity of α-L-arabinofuranosidase, it was purify the enzyme secreted by the fungus C. byrsina SXS16 and investigated its functional biochemical properties. The purified enzyme, with an estimated molecular mass of 55 kDa, exhibited maximum activity at pH 3.5-4.0 and 50 ºC, and stability in a range of pH 3-8 at 4 °C for 24 hours, and temperature from 10 to 60 ºC for 1 hour, whose remaining activities were greater than 70%. The enzyme was also stable for 5-10% ethanol after 24 hours of incubation at 28 °C, retaining enzyme activity greater than 75% after incubation with 5% ethanol, and for 10% ethanol, greater than 60% activity. When evaluating enzymatic activity under the influence of metal ions, a reduction in catalysis was observed for all ions, especially Fe3+ and Hg2+ , whose residual enzymatic activities were 25% and 33%, respectively. In the presence of D and L arabinose, enzyme inhibition was noted only with L-arabinose, exhibiting 50% residual activity at 0.2 mol L-1 and 30% at 0.9 mol L-1 of this sugar. The enzyme was active on p-Nitrophenyl-α-L-arabinofuranoside (pNPA) and p-Nitrophenyl-β-Dxylopyranoside (pNPX), with 6.71 ± 0.23 and 0.38 ± 0.07 U mg-1 , respectively, and for enzyme kinetics under pNPA substrate, we noted a KM of 3.45 ± 0.9 mmol L-1 and Vmax of 198.2 ± 24 µmol min-1 mg-1 . The enzyme was also able to hydrolyze linear 1,5 α-Larabinan, but no activity was noted on the substrates p-Nitrophenyl-β-D-glucopyranoside and p-Nitrophenyl-N-acetyl-β-D-glucosaminide.
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α-L-arabinofuranosidases, Agliconas terpénicas, Cultivo em estado sólido, Fungos, Microrganismos, Terpenic aglicone, Solid state contivatin, Fungi, Microorganisms