Alteration of the pH optimum of a family 11 xylanase, XynB6 of Dictyoglomus thermophilum

We reported previously that the activities of several glycosyl hydrolase family 11 xylanases claimed to be active under alkaline conditions, were found to have optima in the pH 5-6 range when assayed under optimal conditions One enzyme, BadX, had enhanced activity at pHs greater than 7 compared to other family 11 xylanases Gene shuffling between badX and Dictyoglomus thermophilum xvnB6 was performed in an attempt to elucidate regions conferring alkaline activity to BadX, and potentially, to increase the alkaline activity of the highly thermophilic XynB6 Segment substitution using degenerate oligonucleotide gene shuffling (DOGS) experiments with combinations of input parental gene fragments from xynB6 and badX was not able to improve the activity of XynB6 at alkaline pH With one exception, the replacement of a single segment of BadX with the equivalent segment from XynB6 reduced the alkaline activity BadX The results indicate that it might not be possible to alter significantly the alkaline pH characteristics of family 11 xylanases by one or a few mutations and that family 1 l xylanases showing enhanced activity at alkaline pH's require multiple sequence adaptations across the protein