Mutant kanamycin nucleotidyltransferase and a method of screening thermophilic bacteria using the same

Abstract

To obtain a selective marker suitable for screening of thermophilic bacteria such as Thermus thermophilus. T. thermophilus are good research materials for investigating the interrelation between enzyme structures and functions since they are stable at extreme pH, crystallize easily and are easy-to-handle. To provide a novel kanamycin nucleotidyltransferase with markedly improved thermostability, a selective marker using the same, and, a screening method for thermophilic bacteria such as Thermus thermophilus using said selective marker.

1 . A mutant kanamycin nucleotidyltransferase having one or more point mutations selected from a group consisting of Met57Leu, Ala62Val, Ser94Pro, Ser203Pro, Asp206Val, His207Gln, Ser220Pro, Ile234Val and Thr238Ala as against the protein comprising the amino acid sequence indicated by SEQ ID NO: 1, and having improved thermostability.  
     
     
         2 . A mutant kanamycin nucleotidyltransferase with improved thermostability, wherein it comprises the amino acid sequence indicated by SEQ ID NO: 2.  
     
     
         3 . The kanamycin nucleotidyltransferase according to  claim 1 , wherein it comprises the amino acid sequence indicated in SEQ ID NO: 3.  
     
     
         4 . A kanamycin nucleotidyltransferase gene encoding the kanamycin nucleotidyltransferase according to any one of  claims 1  to  3 .  
     
     
         5 . A plasmid comprising the gene according to  claim 4 .  
     
     
         6 . A transformant comprising the plasmid according to  claim 5 .  
     
     
         7 . A selective marker for thermophilic bacteria wherein it is the gene according to  claim 4 .  
     
     
         8 . A method for screening thermophilic bacteria, wherein it the selective marker according to  claim 7  is used.  
     
     
         9 . The screening method according to  claim 8 , wherein said thermophilic bacteria is  Thermus thermophilus.