Thermostability enhancement of xylanase Aspergillus fumigatus RT-1

This study aimed to improve the thermostability of endo-1,4-beta-xylanase (afxynG1)from Aspergillus fumigatus RT-1 using error-prone PCR. Since the wild type enzyme has an optimum temperature stability at 50 degrees 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 degrees C for 30 min. Four mutants (T16NT391/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/L176Qshowed highest stability at 70 degrees 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 Q4713/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/T391/L176Qincreased up to 70 degrees C, followed by A60D increased up to 60 degrees C while the rest remained the same, similar to the wild type enzyme. The mutant T16A/T391/L176Q had the highest half-life time (tip) of 42 min at 70 degrees C, which is a 3.5-fold increase compared to the wild type enzyme which only showed a tip of 12 min at 70 degrees C. This is followed by mutant A60D, tip of 31 min (2.7-fold), S68R, tip of 29 min (2.4-fold) and Q4713/S159R, tip 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. (C) 2016 Elsevier B.V. All rights reserved.