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Laboratory Evolution of Laccase for Substrate Specificity
Author(s) -
Gupta Nirupama,
Lee Frederick S,
Farinas Edgardo T
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.645.3
Subject(s) - abts , laccase , chemistry , bacillus subtilis , substrate (aquarium) , biochemistry , directed evolution , thermostability , mutant , enzyme , biology , antioxidant , bacteria , ecology , dpph , gene , genetics
A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild‐type CotA oxidizes ABTS (ABTS = diammonium 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonate) and SGZ (SGZ = 4‐hydroxy‐3,5‐ dimethoxy‐benzaldehyde azine), and it was engineered for increased specificity for ABTS. Laccases have several applications in biotechnology that include pulp bleaching, biosensors, bioremediation, and biofuel cells. Based on the ABTS‐bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half‐life for the heat inactivation ( t 1/2 ) at 80 o C was increased by 62 min for the mutant, and this was surprising since this property was not included in the substrate specificity screen. In short, the substrate‐binding pocket of CotA is moldable and it can be tailored to oxidize a variety of target molecules for specific purposes.