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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering

Full text
Author(s):
Alponti, Juliana Sanchez [1] ; Maldonado, Raquel Fonseca [2] ; Ward, Richard J. [3, 1]
Total Authors: 3
Affiliation:
[1] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ave Bandeirantes 3900, BR-14040901 Sao Paulo - Brazil
[2] Univ Sao Paulo, FMRP USP, Dept Bioquim & Imunol, BR-14040901 Sao Paulo - Brazil
[3] CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol CTBE, Sao Paulo - Brazil
Total Affiliations: 3
Document type: Journal article
Source: International Journal of Biological Macromolecules; v. 87, p. 522-528, JUN 2016.
Web of Science Citations: 10
Abstract

Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis (XynA), five single mutants (522E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed in Escherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (T-opt) of 55 degrees C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55 degrees C (t(50)) decreased in the order S22E/N32D >N181R > S22E > Wild-type > S27E= N32D,--IN54E. The values of the van't Hoff denaturation enthalpy change (AHND), melting temperature (Tm) and heat capacity at constant pressure (ACp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328 K (AG328) S22E/N32D > N181R > S22E> Wild-type >S275E=N32D approximate to N54E. correlates well with the thermotolerance results, and is dominated by changes in AHND which is consistent with increased in hydrogen bonding in the thermostable mutants. (C) 2016 Elsevier B.V. All rights reserved. (AU)

FAPESP's process: 10/18850-2 - Identification, characterization and engineering of plant cell wall degrading enzymes
Grantee:Richard John Ward
Support Opportunities: Research Projects - Thematic Grants