caa a22 4500 605500711 CHVBK 20210128100557.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-014-6324-0 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6324-0 Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera [Elektronische Daten] [Gilvan Furtado, Camila Santos, Rosa Cordeiro, Lucas Ribeiro, Luiz de Moraes, André Damásio, Maria Polizeli, Marcos Lourenzoni, Mário Murakami, Richard Ward] Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03s−1; XegA, 144.31s−1) and reduced KM (chimera, 1.30mgmL−1; XegA, 1.50mgmL−1) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60°C, the thermostabilitiy of the chimera at 60°C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera. Springer-Verlag Berlin Heidelberg, 2015 GH12 glycosylhydrolase nationallicence X-ray crystallography nationallicence Small-angle X-ray scattering nationallicence Furtado Gilvan Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut Santos Camila Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut Cordeiro Rosa Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut Ribeiro Lucas Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut de Moraes Luiz Computational Modelling Group: Simulation and Evolution of Biomolecules, Fundação Oswaldo Cruz—Fiocruz-CE, Fortaleza, CE, Brazil aut Damásio André Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisas em Energia e Materiais, Campinas, SP, Brazil aut Polizeli Maria Departamento de Biologia, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut Lourenzoni Marcos Computational Modelling Group: Simulation and Evolution of Biomolecules, Fundação Oswaldo Cruz—Fiocruz-CE, Fortaleza, CE, Brazil aut Murakami Mário Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut Ward Richard Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisas em Energia e Materiais, Campinas, SP, Brazil aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5095-5107 0175-7598 99:12<5095 2015 99 253 https://doi.org/10.1007/s00253-014-6324-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6324-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Furtado Gilvan Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut NATIONALLICENCE 700 1- Santos Camila Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut NATIONALLICENCE 700 1- Cordeiro Rosa Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut NATIONALLICENCE 700 1- Ribeiro Lucas Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut NATIONALLICENCE 700 1- de Moraes Luiz Computational Modelling Group: Simulation and Evolution of Biomolecules, Fundação Oswaldo Cruz—Fiocruz-CE, Fortaleza, CE, Brazil aut NATIONALLICENCE 700 1- Damásio André Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisas em Energia e Materiais, Campinas, SP, Brazil aut NATIONALLICENCE 700 1- Polizeli Maria Departamento de Biologia, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil aut NATIONALLICENCE 700 1- Lourenzoni Marcos Computational Modelling Group: Simulation and Evolution of Biomolecules, Fundação Oswaldo Cruz—Fiocruz-CE, Fortaleza, CE, Brazil aut NATIONALLICENCE 700 1- Murakami Mário Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil aut NATIONALLICENCE 700 1- Ward Richard Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisas em Energia e Materiais, Campinas, SP, Brazil aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5095-5107 0175-7598 99:12<5095 2015 99 253