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Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach.
Akbarzadeh, Ali; Pourzardosht, Navid; Dehnavi, Ehsan; Ranaei Siadat, Seyed Omid; Zamani, Mohammad Reza; Motallebi, Mostafa; Nikzad Jamnani, Farnaz; Aghaeepoor, Mojtaba; Barshan Tashnizi, Mohammad.
Afiliación
  • Akbarzadeh A; Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran; Gene Transfer Pioneers Research Group, Shahid Beheshti University of Medical Science, Tehran, Iran; Biotechnology Protein Research Center (PRC), Shahid Beheshti University, GC, Tehra
  • Pourzardosht N; Biochemistry Department, Guilan University of Medical Sciences, Rasht, Iran.
  • Dehnavi E; Gene Transfer Pioneers Research Group, Shahid Beheshti University of Medical Science, Tehran, Iran.
  • Ranaei Siadat SO; Biotechnology Protein Research Center (PRC), Shahid Beheshti University, GC, Tehran, Iran. Electronic address: o_ranaei@sbu.ac.ir.
  • Zamani MR; Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Electronic address: zamani@nigeb.ac.ir.
  • Motallebi M; Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
  • Nikzad Jamnani F; Biotechnology Protein Research Center (PRC), Shahid Beheshti University, GC, Tehran, Iran.
  • Aghaeepoor M; Gene Transfer Pioneers Research Group, Shahid Beheshti University of Medical Science, Tehran, Iran.
  • Barshan Tashnizi M; Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, Tehran, Iran.
Int J Biol Macromol ; 120(Pt B): 1572-1580, 2018 Dec.
Article en En | MEDLINE | ID: mdl-30267817
EndoglucanaseII (Cel5A) of Trichoderma reesei is widely used industrially with the high catalytic efficiency, but it is not stable high temperatures. Structural comparison with the closest thermophilic endoglucanase homolog, Cel5A from Thermoascus aurantiacus, demonstrates disulfide bond differences. Replacement of Cysteine99 with Valine and Cysteine323 with Histidine by site directed mutagenesis caused elimination of two disulfide bonds. Recombinant expression in Pichia pastoris showed the catalytic efficiency (kcat/Km) increment toward CMC for single mutant enzymes, C99V and C323H, about 1.87 and 1.3 folded respectively. This indicates that the elimination of disulfide bond in substrate binding cleft around the catalytic domain of mutant EndoglucanaseII may be increased the flexibility of protein, to form a suitable E-S complex. In direct contrast with previous studies suggesting the existence of disulfide bonds increase the protein stability, the results showed mutant endoglucanase enzymes with disulfide bond reduction have higher thermal stability. The thermal stability of C99V and C323H in 80 °C were increased 2.4 and 2.34 folded, respectively. In this project, theoretical data had a good agreement with the experimental results. Because of high enzyme activity and thermal stability, both of C99V and C323H mutant have high potential suitable for different industrial applications.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Temperatura / Trichoderma / Celulasa / Mutagénesis Sitio-Dirigida / Disulfuros Idioma: En Revista: Int J Biol Macromol Año: 2018 Tipo del documento: Article Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Temperatura / Trichoderma / Celulasa / Mutagénesis Sitio-Dirigida / Disulfuros Idioma: En Revista: Int J Biol Macromol Año: 2018 Tipo del documento: Article Pais de publicación: Países Bajos