Improved thermal stability of Thermoascus aurantiacus GH10 xylanase, expressed in E. coli, by directed evolution and rational design: Production, purification, biochemical and physico-chemical characterization of the enzyme.

Author(s)

Angélica Rodrigues de Souza

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2018-05-25.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2016-03-04
• Advisor: Roberto da Silva; Jeffrey Mertens; Fernando Araripe Gonçalves Torres

Abstract

The initial purpose of this work was the improvement of xylanase thermal stability of fungus T. aurantiacus (XynA_Ta), expressed in S. cerevisiae (XynA_Sc) from the building previously obtained in P. pastoris (xynA_Pp). However, the reduced expression of XynA_Sc, combined with two mutations of amino acids, led to the recloning xynA_Ta gene in E. coli (XynA_Ec). Chapter 1 describes the study of the production and characterization of XynA_Sc, XynA_Pp and XynA_Ec, previous mutation corrections. These showed great expression with 650, 5.8 and 815 U.mL-1, after 96 h and activity at pH 5-5.5 and 65-70 °C. XynA_Ec displayed wide stability range from 40 to 85 °C, after 1h, and XynA_Pp showed 100% activity at pH 6-8, after 24 h. XynA_Sc and XynA_Pp exhibited stronger activation in organic solvents. The XynA_Sc was inhibited by concentrations greater than 20 mg.mL-1 of substrate. Chapter 2 describes the native correction (XynAc_Ec) and construction of eleven mutant obtained by the MSD. All strains were expressed, purified and biochemical factors helped in the selection of the three most heat resistant. These were characterized, along with XynAc_Ec, by biophysical (CD) and structural factors (homology - 1TUX-PDB). H209N mutant displayed higher thermal stability, catalytic activity and Tm of 71.3 °C. The increase in thermal stability was related to short helices content, salt bridges, and the positive charge in the catalytic core (published work). Chapter 3 presents results of in silico combination of the three thermostable mutants, based on structural data (homology - 1TUX-PDB). Q158R / H209N / N257D showed higher content of bridges interactions and lower RMSF. Therefore, XynA_Pp, XynA_Ec and H209N are potential enzymes for applied bioprocesses; and studies in silico suggest that mutant Q158R / H209N / N257D should be obtained in situ. (AU)

• FAPESP's process: 11/22461-4 - Improved thermal stability of Thermoascus aurantiacus GH10 xylanase, expressed in E. coli, by directed evolution and rational design: Production, purification, biochemical and physico-chemical characterization of the enzyme.
• Grantee: Angelica Rodrigues de Souza
• Support Opportunities: Scholarships in Brazil - Doctorate