Altered thermostability of enzymes

Abstract

Provided is a method of screening gene libraries derived from a mixed population of organisms for a bioactivity or biomolecule of interest. The mixed population of organisms can be a cultured population or an uncultured population from, for example, the environment. Also provided are methods of screening isolates or enriched populations of organisms, which isolates include a population that is spatially, temporally, or hierarchical, for example, of a particular species, genus, family, or class of organisms. Identified clones containing a biomolecule or bioactivity of interest can be further variegated or the DNA contained in the clone can be variegated to create novel biomolecules or bioactivities of interest.

What is claimed is:  
     
         1 . A method for obtaining a bioactive protein having a thermostability that is altered as compared to that of the corresponding wild-type protein, comprising: 
 a) variegating a nucleic acid sequence encoding the wild-type protein; and    b) comparing the bioactivity after variegation with the bioactivity of the wild-type protein, wherein a difference in the bioactivity is indicative of an effect of sequence variegation, thereby providing the a bioactive protein having a thermostability that is altered as compared to that of the corresponding wild-type protein.    
     
     
         2 . The method of  claim 1 , further comprising comparing the variegated nucleic acid sequence of interest to the non-variegated nucleic acid sequence of (c), thereby identifying the nucleotide sequence variegation.  
     
     
         3 . The method of  claim 2 , wherein the comparison is performed using a sequence comparison algorithm.  
     
     
         4 . The method of  claim 1 , wherein the bioactivity is an enzymatic activity.  
     
     
         5 . The method of  claim 4 , wherein the enzymatic activity is provided by an enzyme selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, dehydrogenases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, epozide hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.  
     
     
         6 . The method of  claim 1 , wherein the bioactivity is identified from an expression library.  
     
     
         7 . The method of  claim 6 , wherein the library contains DNA obtained from an environmental sample.  
     
     
         8 . The method of  claim 6 , wherein the library contains nucleic acid obtained from extremophiles.  
     
     
         9 . The method of  claim 8 , wherein the extremophiles are thermophiles.  
     
     
         10 . The method of  claim 8 , wherein the extremeophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.  
     
     
         11 . The method of  claim 6 , wherein the bioactivity is identified by screening comprising contacting a clone with a substrate labeled with a detectable molecule wherein interaction of the substrate with the bioactivity contained in the clone produces a detectable signal.  
     
     
         12 . The method of  claim 11 , wherein the substrate is a bioactive substrate.  
     
     
         13 . The method of  claim 11 , wherein the bioactive substrate comprises C12FDG.  
     
     
         14 . The method of  claim 11 , wherein the substrate comprises a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, wherein modulation of the interaction of the first test protein linked to the DNA binding moiety with the second test protein linked to the transcription activation moiety results in a change in the expression of a detectable protein.  
     
     
         15 . The method of  claim 1 , further comprising, prior to (b), obtaining nucleic acids from the clone containing the specified bioactivity or biomolecule.  
     
     
         16 . The method of  claim 15 , wherein obtaining the nucleic acids contained in the clone comprises contacting the clone with a complementary nucleic acid, or fragment thereof, thereby allowing hybridization of the clone nucleic acids with the complementary nucleic acid and isolation thereof.  
     
     
         17 . The method of  claim 16 , wherein the complementary nucleic acid or fragment thereof comprises a solid phase bound hybridization probe.  
     
     
         18 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by a method selected from the group consisting of error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, ligation reassembly, GSSM and any combination thereof.  
     
     
         19 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by error-prone PCR.  
     
     
         20 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by shuffling.  
     
     
         21 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by oligonucleotide-directed mutagenesis.  
     
     
         22 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by assembly PCR.  
     
     
         23 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by sexual PCR mutagenesis.  
     
     
         24 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by in vivo mutagenesis.  
     
     
         25 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by cassette mutagenesis.  
     
     
         26 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by recursive ensemble mutagenesis.  
     
     
         27 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by exponential ensemble mutagenesis.  
     
     
         28 . The method of  claim 1 , wherein the nucleic acid sequence is variegated by site-specific mutagenesis.  
     
     
         29 . The method of  claim 6 , comprising screening a clone of the library for a further specified protein or enzymatic activity, prior to variegating the nucleic acids.  
     
     
         30 . The method of  claim 6 , wherein the library is generated in a prokaryotic cell.  
     
     
         31 . The method of  claim 6 , wherein the library is generated in a Streptomyces sp.  
     
     
         32 . The method of  claim 31 , wherein the Streptomyces is  Streptomyces venezuelae.    
     
     
         33 . The method of  claim 30 , wherein the prokaryotic cell is gram negative.  
     
     
         34 . The method of  claim 30 , wherein the prokaryotic cell is a Bacillus sp.  
     
     
         35 . The method of  claim 30 , wherein the prokaryotic cell is a Pseudomonas sp.  
     
     
         36 . The method of  claim 6 , wherein the library is screened by contacting or encapsulating a clone of the library with bioactive substrate, wherein a bioactivity or biomolecule produced by the clone is detectable by a difference in the substrate prior to contacting with the clone as compared to after contacting.  
     
     
         37 . The method of  claim 6 , wherein the library is normalized before screening the library.  
     
     
         38 . The method of  claim 1 , wherein the bioactivity is a gene cluster or fragment thereof.  
     
     
         39 . The method of  claim 1 , wherein the bioactivity is a polypeptide in a metabolic pathway.  
     
     
         40 . The method of  claim 1 , wherein the thermostability is lower in the variegated protein as compared to the wild-type protein.  
     
     
         41 . The method of  claim 1 , wherein the thermostability is higher in the variegated protein as compared to the wild-type protein.