Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase

A thermostability screening assay was developed using an Escherichia coli e xpression system to express Streptomyces lividans xylanase A (XInA). The sc reening system was tested using mutants randomized at position 49 of the S. lividans XInA gene, a position previously shown to confer thermostability with a 149P point mutation. The library was cloned into an E. coli expressi on vector and transformed into XL1- Blue bacteria. The resulting crones wer e screened for increased thermostability with respect to wild-type XInA. Us ing this assay, we isolated the 149P mutant previously shown to be thermost able, as well as novel 149A and 149C mutants. The 149A and 149C mutants wer e shown to have 2.8- to 8-fold increase in thermostability over that of wil d-type XInA. The results show that the screening assay can selectively enri ch for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.