Thermostability enhancement of xylanase Aspergillus fumigatus RT-1

This study aimed to improve the thermostability of endo-1,4-β-xylanase (afxynG1) from Aspergillus fumigatus RT-1 using error-prone PCR. Since the wild type enzyme has an optimum temperature stability at 50°C, the improvement of its stability will widen its application in industries with operating processes at higher temperatures. A library containing approximately 5,000 afxynG1 mutants was generated and thermally screened at 60°C for 30min. Four mutants (T16A/T39I/L176Q, S68R, A60D and Q47P/S159R) were selected for enzymatic characterization because of their higher catalytic activity compared to the wild type. Among these mutants, the mutant T16A/T39I/L176Q showed highest stability at 70°C and retained 45.9% of its activity after 60min of incubation while the wild type had lost its activity completely after 50min of incubation. The other mutants, A60D, S68R and Q47P/S159R also showed improvement in thermostability by retaining 33.2%, 25.8% and 23.8% of their activity respectively. The optimum temperature for mutants also significantly increased. The optimum temperature for T16A/T39I/L176Q increased up to 70°C, followed by A60D increased up to 60°C while the rest remained the same, similar to the wild type enzyme. The mutant T16A/T39I/L176Q had the highest half-life time (t1/2) of 42min at 70°C, which is a 3.5-fold increase compared to the wild type enzyme which only showed a t1/2 of 12min at 70°C. This is followed by mutant A60D, t1/2 of 31min (2.7-fold), S68R, t1/2 o...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research