Title
Thermostability enhancement of xylanase Aspergillus fumigatus RT-1
Author
Mohd Khairul Hakimi bin Abdul Wahab, Mohd Anuar bin Jonet, Rosli Md Illias
Year
2016
Journal
Journal of Molecular Catalysis B: Enzymatic
Abstract
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 5000 afxynG1 mutants was generated and thermally screened at 60° C for 30 min. 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 60 min of incubation while the wild type had lost its activity completely after 50 min 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 42 min at 70° C, which is a 3.5-fold increase compared to the wild type enzyme which only showed a t1/2 of 12 min at 70 °C. This is followed by mutant A60D, t1/2 of 31 min (2.7-fold), S68R, t1/2 of 29 min (2.4-fold) and Q47P/S159R, t1/2 of 27 min (2.25-fold). Based on homology modelling conducted to analyze the mutants’ structures, it showed that hydrophobicity and hydrogen bonds were the driving forces that lead to the improvement of the thermal stability of these xylanase mutants.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Thermal stability, Biochemistry