Error-prone PCR of a fungal xylanase for improvement of its alkaline and thermal stability

Random mutagenesis was used to improve the alkaline and thermal stability of the xylanase (XynA) from Thermomyces lanuginosus. Error-prone PCR reactions were carried out; the PCR products were cloned into Escherichia coli and a library of 960 clones was selected on xylan-containing agar plates. The crude filtrates of positive xylanase producers were screened at 80 degrees C and tested separately at pH 10 for alkaline tolerance. The native XynA lost 80% activity after 90 min at 80 degrees C and lost 70% activity at pH 10. Conversely, the most thermostable variant, G41, retained 75% activity after 90 min at 80 degrees C and the best alkali-stable variant, G53, retained 93% activity at pH 10. Sequence analysis revealed four amino acid substitutions in G41 and a single substitution in G53. These variants, therefore, have improved thermal and alkaline stability and are ideal candidates for DNA shuffling experiments to produce a robust xylanase for industrial application.