Method of Enhancing Enzyme Activity

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.

What is claimed is: 
     
         1 . 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; and   (e) separating the aqueous phase from the solid and oil phases to recover polynucleotides that have been released from the solid phases.   
     
     
         2 . The method of  claim 1 , wherein the plurality of polynucleotides comprises at least 10 6  different polynucleotides. 
     
     
         3 . The method of  claim 1 , wherein each polynucleotide-linked solid phase comprises no more than 6 different polynucleotides, each of which is present in one or more copies. 
     
     
         4 . The method of  claim 3 , wherein said no more than 6 different polynucleotides encode 2 to 6 different types of enzymes. 
     
     
         5 . The method of  claim 1 , wherein each polynucleotide-linked solid phase comprises only one polynucleotide, which is present in one or more copies. 
     
     
         6 . The method of  claim 1 , wherein the solid phases comprise microbeads or particles. 
     
     
         7 . The method of  claim 1 , wherein the linker comprises a substrate for the enzyme. 
     
     
         8 . The method of  claim 1 , wherein the solid phases comprise an insoluble substrate for the enzyme. 
     
     
         9 . The method of  claim 1 , wherein the water-in-oil emulsion is formed under conditions wherein at least about 20% of aqueous droplets comprise 1 or less than 1 polynucleotide-linked solid phase. 
     
     
         10 . The method of  claim 9 , wherein each polynucleotide-linked solid phase comprises 2 to 6 polynucleotides, each of which is present in one or more copies, whereby, 2 to 6, respectively, enzyme variants are expressed per aqueous droplet containing a polynucleotide-linked solid phase. 
     
     
         11 . The method of  claim 9 , wherein each polynucleotide-linked solid phase comprises only one polynucleotide, which is present in one or more copies. 
     
     
         12 . The method of  claim 1 , wherein the emulsion comprises at least about 10 9  aqueous droplets/mL of emulsion. 
     
     
         13 . The method of  claim 1 , wherein the aqueous droplets in the emulsion have an average diameter between about 1 μm and about 100 μm, inclusive. 
     
     
         14 . The method of  claim 1 , wherein the aqueous droplets in the emulsion have an average volume of between about 1 femtoliter and about 1 nanoliter, inclusive. 
     
     
         15 . The method of  claim 1 , wherein the aqueous phase from the solid and oil phases by sedimentation using a centrifuge. 
     
     
         16 . The method of  claim 1 , wherein recovered polynucleotides are amplified and linked to a plurality of solid phases, wherein the linker or the solid phases comprise a substrate for the one or more enzyme(s), and steps (b)-(e) are repeated. 
     
     
         17 . The method of  claim 1 , wherein recovered polynucleotides are mutagenized and then linked to a plurality of solid phases, wherein the linker or the solid phases comprise a substrate for the one or more enzyme(s), and steps (b)-(e) are repeated. 
     
     
         18 . The method of  claim 1 , wherein the one or more of the recovered polynucleotides are amplified. 
     
     
         19 . The method of  claim 1 , wherein the one or more of the recovered polynucleotides are translated in vitro to produce one or more enzyme variants. 
     
     
         20 . The method of  claim 1 , wherein one or more of the recovered polynucleotides are cloned into a vector. 
     
     
         21 . The method of  claim 20 , wherein the vector comprises an expression vector, and the method additionally comprises expressing one or more of the recovered polynucleotides. 
     
     
         22 . The method of  claim 21 , wherein said expression results in the production of one or more enzyme variants, which is/are recovered from the culture. 
     
     
         23 . The method of  claim 22 , wherein the one or more recovered enzyme variants is/are contacted with an insoluble substrate. 
     
     
         24 . The method of  claim 23 , wherein the insoluble substrate comprises biomass. 
     
     
         25 . The method of  claim 24 , wherein the biomass comprises particulate matter, wherein the average particle diameter is in the range of 1 to100 microns. 
     
     
         26 . The method of  claim 24 , wherein said contacting is carried out in a process for producing a biofuel. 
     
     
         27 . The method of  claim 1 , wherein said one or more enzyme(s) comprise one or more cellulases. 
     
     
         28 . The method of  claim 27 , wherein said one or more cellulases comprise one or more of the cellulases selected from the group consisting of an endoglucanase, an exoglucanase, and a β-glucosidase. 
     
     
         29 . The method of  claim 28 , wherein said one or more cellulases comprise an endoglucanase, an exoglucanase, and a β-glucosidase, wherein each polynucleotide-linked solid phase comprises a polynucleotide encoding a variant of each type of enzyme. 
     
     
         30 . The method of  claim 27 , wherein the solid phases comprise cellulosic microbeads.