Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

Characterization of the thermostability of xylanase produced by new isolates of Thermomyces lanuginosus

“…An actively growing 3-day old colony of each T. lanuginosus strain (1 or 2 agar blocks) grown on yeast glucose medium was inoculated into synthetic medium (15 ml) consisting of (per litre of water) 2 g of KH 2 PO 4 , 0.3 g of CaCl 2 , 0.3 g of MgSO 4 , 5 g of peptone, 3 g of yeast extract, 3 g of malt extract and 10 g of oat spelt in 50 ml Erlenmeyer flasks. The flasks were shaken at 120 rpm and incubated at 45 °C for 5 days.…”

“…3). Stephens et al 4 improved the thermostability of the xylanase from T. lanuginosus DSM 5826 by directed evolution using error-prone PCR. The amino acid sequence of xylanase from the mutants with enhanced thermostability differed in 3 amino acids for mutant 2B7-6 and had single mutation for mutants 2B11-16 and 2B7-10.…”

“…Only one amino acid substitution of D72G and Y58F of xylanases from mutant 2B11-16 and mutant 2B7-10 increased the half-lives at 70 °C by 2 and 2.5 times, respectively. Most amino acid substitution for the mutants, except mutant 2B7-10, occurred within the β-sheet of the enzyme which forms the hydrophobic region of the enzyme 4 . The single amino acid substitution of xylanase (V96G) of T. lanuginosus THKU-49 and substitution (Y58F) of xylanase in mutant 2B7-10 occurred on the outer surface of the β-sheet, which resulted in an increase of the hydrophilicity of the enzyme increasing the thermostability.…”

“…The wild type had a half-life at 70 °C (pH 8.0) for 89 min. By using this technique, single amino acid substitutions (Y58F and D72G) of xylanases of mutant 2B7-10 and mutant 2B11-16 increased the half-life to 215 and 168 min, respectively 4 . Evolution in nature may give rise to strains producing enzymes with different properties including their thermostability.…”

Wood production, wood technology, and biotechnological impacts

“…However, it is also possible to enhance enzyme yields in homologous and heterologous filamentous fungi by replacing the natural xylan-inducible promoter by more efficiently working promoters underlying different regulation schemes (de Faria et al 2002, Rose & van Zyl 2002, Levasseur et al 2005. Recombinant protein production has another positive consequence for applications: site-directed mutagenesis and directed evolution are used in molecular gene engineering to optimise the enzyme properties (see for examples on xylanases see Xion et al 2004, Fenel et al 2006, Sriprang et al 2006, Stephens et al 2007). …”