Engineering the GH1 beta-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose

Autor(es)

Meleiro, Luana Parras ^[1] ; Santos Salgado, Jose Carlos ^[2] ; Maldonado, Raquel Fonseca ^[3] ; Carli, Sibeli ^[1] ; Beraldo Moraes, Luiz Alberto ^[1] ; Ward, Richard John ^[1] ; Jorge, Joao Atilio ^[4] ; Melo Furriel, Rosa Prazeres ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, Ribeirao Preto, SP - Brazil

^[3] Inst Fed Educ Ciencia & Tecnol Sao Paulo, Sao Jose Dos Campos, SP - Brazil

^[4] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Biol, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PLoS One; v. 12, n. 11 NOV 16 2017.
• Citações Web of Science: 9

Resumo

The activity of the GH1 beta-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-beta-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 16/17582-0 - Modulação da termoestabilidade de uma ß-glucosidase estimulada por glicose e xilose empregando técnicas de glicosilação sítio dirigida
• Beneficiário: Luana Parras Meleiro Garcia
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 14/14415-0 - Enzimas lignocelulolíticas de fungos filamentosos: identificação, purificação, caracterização, relação estrutura-função e potencial de aplicação biotecnológica
• Beneficiário: Rosa dos Prazeres Melo Furriel
• Modalidade de apoio: Auxílio à Pesquisa - Regular