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.
Stephens
Dawn Elizabeth
DE
Department of Biotechnology and Food Technology, ML Sultan Campus, Durban University of Technology, Durban, South Africa.
Singh
Suren
S
Permaul
Kugen
K
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007221:Industry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods*
D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases