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.
The endo-1,4-b-xylanases (EC 22.214.171.124) are responsible for the hydrolysis of b-1,4 connections present in the internal chain of xylan molecule, producing fragments as xilotriose and xilobiose. Therefore, this doctoral project has as an object of study a xylanase present in the thermophilic fungus genome, Termoascus aurantiacus (xynA). This theme is linked to the process 2010/12624-0, which theme is "Application of physico-chemical methods and the enzymatic saccharification of sugarcane bagasse: Studies of microorganisms, fermentation processes and related hydrolysis methods." The xylanase in study is a thermophilic enzyme, with potential application in industrial sectors, as in pulp and paper industry. Its native form is stable in wide pH range (3.5 - 10.5) and temperature (35 - 75° C) after 24 hours and 1 hour of incubation respectively, with optimal activity in pH 5.0 at 75°C. Thus, this project aims to improve the thermostability of the T. aurantiacus xylanase, through the technique of random mutation by PCR mutagenic (error-prone PCR). For this purpose, the following steps are performed: (1) cloning vector Y1PGK1, mutation and expression in heterologous system (S. cerevisiae), (2) purification of xylanase (native and mutated), (3) studies of biochemical and physical-chemistry characterization of xylanases pure and wild enzyme, for data comparison and measurement of increased thermostability; and (4) study of structural modeling from the mutated protein by homology, since the exchange can modify the folding of the protein, as well as its specificity, and others. This project is interesting and innovative, because it uses tools of genetic engineering to change the protein properties and increase the range of industrial applications, with no reported study.
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