High throughput fluorescence-based screening for novel enzymes

Abstract

Disclosed is a process for identifying clones having a specified activity of interest, which process comprises (i) generating one or more expression libraries derived from nuclei acid directly isolated from the environment; and (ii) screening said libraries utilizing a fluorescence activated cell sorter to identify said clones. More particularly, this is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; (ii) exposing said libraries to a particular substrate or substrates of interest; and (iii) screening said exposed libraries utilizing a fluorescence activated cell sorter to identify clones which react with the substrate or substrates. Also provided is a process for identifying clones having a specified activity of interest by (i) generating one or more expression libraries derived from nucleic acid directly or indirectly isolated from the environment; and (ii) screening said exposed libraries utilizing an assay requiring a binding event or the covalent modification of a target, and a fluorescence activated cell sorter to identify positive clones.

What is claimed is:  
     
         1 . A method for high throughput screening of prokaryotic genomic DNA samples to identify one or more enzymes encoded by the prokaryotic DNA of said sample, comprising the steps of : 
 a) generating a normalized, multispecific, prokaryotic expression library;    b) inserting bioactive substrates into samples of the library;    c) screening the samples with a fluorescent analyzer that detects bioactive fluorescence;    d) separating samples detected as positive for bioactive fluorescence; and    e) determining the DNA sequence of positive samples;    wherein the DNA sequence identifies and encodes an enzyme that catalyzes the bioactive substrate detected in step d).    
     
     
         2 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.  
     
     
         3 . The method of  claim 1 , wherein the prokaryotic expression library contains at least of about 2×10 6  clones.  
     
     
         4 . The method of  claim 1 , wherein the sample is a prokaryotic cell.  
     
     
         5 . The method of  claim 4 , wherein the prokaryotic cell is gram negative.  
     
     
         6 . The method of  claim 1 , wherein the sample is encapsulated in a gel microdrop.  
     
     
         7 . The method of  claim 1 , wherein the high-throughput screening step c) screens up to about 35 million samples per hour.  
     
     
         8 . The method of  claim 1 , wherein the prokaryotic expression library contains extremophiles.  
     
     
         9 . The method of  claim 3 , wherein the extremophiles are thermophiles.  
     
     
         10 . The method of  claim 3 , wherein the extremeophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.  
     
     
         11 . The method of  claim 1 , wherein the bioactive substrate comprises C12FDG.  
     
     
         12 . The method of  claim 10 , wherein the bioactive substrate further comprises a lipophilic tail.  
     
     
         13 . The method of  claim 1 , wherein the the samples are heated before step b).  
     
     
         14 . The method of  claim 13 , wherein the heating is in the range of about 70° C.  
     
     
         15 . The method of  claim 14 , wherein the heating occurs in the range of about 30 minutes.  
     
     
         16 . The method of  claim 1 , wherein the fluorescent analyzer comprises a FACS apparatus.  
     
     
         17 . The method of  claim 1 , wherein the prokaryotic expression library is biopanned before step b).  
     
     
         18 . The method of  claim 1 , including the additional steps of: 
 subjecting an enzyme encoded by the DNA identified in step d) to directed evolution comprising the steps of: 
 a) subjecting the enzyme to non-directed mutagenesis; and  
 b) screening mutant enzymes produced in step a) for a mutant enzyme that is stable at a temperature of at least in the range of about 60° C. and that has functioning enzymatic activity at a temperature at least 10° C. below its optimal temperature range and that catalyzes a greater amount of a catalytic substrate per a defined unit of time than the enzyme of step a).