Method of enhancing enzyme activity
AssigneesNovozymes A/S, The Scripps Research Institute
InventorsRobert G. Blazej, Brian M. Paegel
Life of this patent
Estimating the term…
Abstract
Described are methods and means for enhancing enzyme activity toward insoluble substrates. This is achieved by means of in vitro compartmentalization in which an insoluble microparticle functions both as the enzyme substrate and as a structure for negative selection. Enhanced enzymes expressed from a microparticle-linked polynucleotide library preferentially degrade the microparticle releasing specific gene variants into solution. Gene variants encoding less active enzyme variants remain linked to the microparticle and may be removed through centrifugation, thus enriching the polynucleotide library for more active enzyme variants. These methods may be used to enhance cellulase and ligninase activity toward insoluble cellulosic biomass.
Claims17
A selection method for enhanced enzyme activity on an insoluble substrate, the method comprising:
- (a) providing a plurality of polynucleotides encoding variants of one or more enzyme(s) that act(s) on an insoluble substrate, wherein the plurality of polynucleotides is linked to a plurality of solid phases and the linker or the solid phases comprise a substrate for the one or more enzyme(s);
- (b) suspending the polynucleotide-linked solid phases in an aqueous phase comprising components for in vitro transcription/translation;
- (c) forming a water-in-oil emulsion, wherein the polynucleotide-linked solid phases are compartmentalized in aqueous droplets in an oil continuous phase;
- (d) carrying out in vitro transcription/translation to express enzyme variants within aqueous droplets of the emulsion, wherein an active enzyme variant in an aqueous droplet releases the polynucleotide(s) from the solid phase-in that droplet;
- and
- (e) separating the aqueous phase from the solid and oil phases to recover
- polynucleotides in the aqueous phase that have been released from the solid phases.
Dependencies are inferred from the claim text.