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 expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.
Ebanks
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R
Centre de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, Laval, Québec, Canada H7N 4Z3.
Dupont
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M
Shareck
F
F
Morosoli
R
R
Kluepfel
D
D
Dupont
C
C