Recombinant GH3β-glucosidase stimulated by xylose and tolerant to furfural and 5-hydroxymethylfurfural obtained from Aspergillus nidulans  

作　　者：Diandra de Andrades Robson C.Alnoch Gabriela S.Alves Jose C.S.Salgado Paula Z.Almeida Gabriela Leila Berto Fernando Segato Richard J.Ward Marcos S.Buckeridge Maria de Lourdes T.M.Polizeli 

机构地区：[1]Department of Biology,Faculty of Philosophy,Sciences and Letters of Ribeirão Preto,University of São Paulo,Ribeirão Preto,SP 14040-901,Brazil [2]Department of Biochemistry and Immunology,Faculty of Medicine of Ribeirão Preto,University of São Paulo,Ribeirão Preto,SP 14049-900,Brazil [3]Laboratory of Enzymology and Molecular Biology of Microorganisms,Institute of Biology,Campinas State University(UNICAMP),Campinas 13083-970,SP,Brazil [4]Department of Chemistry,Faculty of Philosophy,Sciences and Letters of Ribeirão Preto,University of São Paulo,Ribeirão Preto,SP 14040-901,Brazil [5]Department of Biotechnology,Lorena School of Engineering,University of São Paulo,Lorena 12602-810,Brazil [6]Bioscience Institute,University of São Paulo,São Paulo 05508-090,Brazil

出　　处：《Bioresources and Bioprocessing》2024年第1期1026-1040,共15页生物资源与生物加工(英文)

基　　金：gratefully acknowledge the São Paulo Research Foundation(FAPESP)(Grant no:2018/07522-6,2021/06679-1,2022/00539-6,and 2023/01547-5;FCT(POCI-01-0145-FEDER-032206)-transnational cooperation project EcoTech,Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq)(310340/2021-7);National Institute of Science and Technology of Bioethanol(INCT)(CNPq 465319/2014-9/FAPESP no:2014/50884-5)for financial support;Research scholarships were granted to RCA by CNPq(Grant no:151187/2023-1);by FAPESP(Grant no:2023/09627-8);to DA by FAPESP(Grant no:2020/15510-8 and 2023/01338-7);to JCSS by CNPq(Grant no:384465/2023-4);FAPESP(Grant no:2019/21989-7);GSA by CAPES(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,Finance Code 001).

摘　　要：Theβ-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A.nidulans A773.The resulting purifiedβ-glucosidase,named AnGH3,is a monomeric enzyme with a molecular weight of approximately 80 kDa,as confirmed by SDS-PAGE.Circular dichroism further validated its unique canonical barrel fold(β/α),a feature also observed in the 3D homology model of AnGH3.The most striking aspect of this recombinant enzyme is its robustness,as it retained 100%activity after 24 h of incubation at 45 and 50ºC and pH 6.0.Even at 55°C,it maintained 72%of its enzymatic activity after 6 h of incubation at the same pH.The kinetic parameters Vmax,KM,and Kcat/KM forρ-nitrophenyl-β-D-glucopyranoside(ρNPG)and cellobiose were also determined.UsingρNPG,the enzyme demonstrated a Vmax of 212 U mg−1,KM of 0.0607 mmol L−1,and Kcat/KM of 4521 mmol L−1 s−1 when incubated at pH 6.0 and 65°C.The KM,Vmax,and Kcat/KM using cellobiose were 2.7 mmol L−1,57 U mg−1,and 27 mmol-1 s−1,respectively.AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L−1 and 25%,respectively.Even in challenging conditions,at 65°C and pH 6.0,the enzyme maintained its activity,retaining 100%and 70%of its initial activity in the presence of 200 mmol L−1 furfural and 5-hydroxymethylfurfural(HMF),respectively.The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum,where it led to a 48%increase in glucose release after 24 h.These unique characteristics,including high catalytic performance,good thermal stability in hydrolysis temperature,and tolerance to elevated concentrations of ethanol,D-xylose,furfural,and HMF,position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail,thereby opening new avenues in the field of biotechnology and enzymology.

关 键 词：Enzymatic hydrolysis Xylose-stimulated Tolerant to biomass products Homologous expression 

分 类 号：O64[理学—物理化学]