Glucose dehydrogenase and production thereof

Abstract

The invention relates to novel PQQ-dependent soluble glucose dehydrogenases (sPQQGDH) from Acinetobacter and to a process for their preparation by overexpression in suitable microbial expression systems.

1 .- 8 . (canceled) 
     
     
         9 . A soluble pyrroloquinoline quinone glucose dehydrogenase (sPQQGDH) having the amino acid sequence of wild-type  Acinetobacter  LMD 79:41 (numbering as shown in SEQ ID NO: 2), wherein compared to the amino acid sequence of wild-type  Acinetobacter  LMD 79:41 (numbering as shown in SEQ ID NO: 2):
 a) the following amino acids are exchanged:
 i) position 21 has a N; 
 ii) position 41 has an S; 
 iii) position 47 has an L; 
 iv) position 121 has a V or an A; 
 v) position 149 has an A; 
 vi) position 213 has an S; 
 vii) position 244 has an I; 
 viii) position 320 has a G; 
 ix) position 391 has an S; 
 x) position 452 has an S; 
 xi) position 474 has an R; and 
 xii) position 480 has a Q; and 
   b) at least one of the following amino acids are also exchanged:
 i) position 16 has an H; 
 ii) position 18 has an L; 
 iii) position 20 has an F; 
 iv) position 40 has a G; 
 v) position 48 has an I; 
 vi) position 61 has an A; 
 vii) position 111 has a T; 
 viii) position 154 has a D; 
 ix) position 190 has an E; 
 x) position 293 has an A; 
 xi) position 311 has an S; 
 xii) position 314 has an A; 
 xiii) position 324 has an L; 
 xiv) position 333 has an M; 
 xv) position 339 has an S; 
 xvi) position 355 has a G; 
 xvii) position 366 has a D; 
 xviii) position 417 has an N; or 
 xix) position 418 has an A. 
   
     
     
         10 . An isolated and purified DNA sequence encoding a sPQQGDH enzyme according to  claim 9 . 
     
     
         11 . A vector comprising a DNA sequence according to  claim 11 . 
     
     
         12 . A transformed microorganism expressing a DNA sequence according to  claim 11 . 
     
     
         13 . Process for preparing a sPQQGDH enzyme, which comprises cultivating a microorganism according to  claim 12 , and then isolating the sPQQGDH enzyme. 
     
     
         14 . Reagent for detecting glucose comprising one or more sPQQGDH enzymes according to  claim 9 . 
     
     
         15 . Sensor for detecting glucose comprising one or more sPQQGDH enzymes according to  claim 9 . 
     
     
         16 . A method for detecting glucose in a sample comprising contacting the sample with one or more sPQQGDH enzymes according to  claim 9 .