Method for producing scyllo-inositol

Abstract

It is intended to provide a novel NAD + -independent myo-inositol 2-dehydrogenase which converts myo-inositol into scyllo-inosose in the absence of NAD + ; a novel enzyme scyllo-inositol dehydrogenase which stereospecifically reduces scyllo-inosose into scyllo-inositol in the presence of NADH or NADPH; and a novel microorganism which belongs to the genus Acetobacter or Burkholderia and can convert myo-inositol into scyllo-inositol. By using these enzymes or the microorganism, scyllo-inositol is produced. Furthermore, scyllo-inositol is purified by adding boric acid and a metal salt to a liquid mixture containing scyllo-inositol and a neutral saccharide other than scyllo-inositol to form a scyllo-inositol/boric acid complex, separating the complex from the liquid mixture, dissolving the thus separated complex in an acid to give an acidic solution or an acidic suspension and then purifying scyllo-inositol from the acidic solution or the acidic suspension.

What is claimed is: 
     
         1 . A method of producing scyllo-inositol comprising: contacting a microorganism capable of converting myo-inositol into scyllo-inositol and belonging to the genus  Acetobacter  or  Burkholderia  with myo-inositol in a solution comprising myo-inositol to produce and accumulate scyllo-inositol in the solution; and collecting the scyllo-inositol from the solution. 
     
     
         2 . The method according to  claim 1 , wherein the solution comprising myo-inositol is a liquid medium containing myo-inositol, and the microorganism is contacted with myo-inositol by culturing the microorganism in the liquid medium. 
     
     
         3 . The method according to  claim 1 , wherein cells obtained by culturing the microorganism are contacted with myo-inositol in the solution. 
     
     
         4 . A method for producing scyllo-inositol according to  claim 1 , comprising: culturing  Acetobacter  sp. AB10281 strain (FERM BP-10119) or a mutant thereof in a medium comprising myo-inositol to produce scyllo-inosose; reacting the scyllo-inosose with a reducing agent to generate scyllo-inositol; and separating and isolating the generated scyllo-inositol from the medium. 
     
     
         5 . A scyllo-inositol dehydrogenase having the following physiological properties:
 Reaction: as shown in the following formula, catalyzing an oxidation-reduction reaction between scyllo-inositol and scyllo-inosose and stereospecifically reducing scyllo-inosose to scyllo-inositol in the presence of NADH or NADPH   
       
         
           
           
               
               
           
         
       
     
     
         6 . The scyllo-inositol dehydrogenase according to  claim 5 , further having the following physiological properties:
 (1) Molecular weight and association property: 38 to 46 k Dalton, forming a dimer or a trimer;   (2) Coenzyme: requiring NAD +  or NADP + , or NADH or NADPH as a coenzyme;   (3) Activating heavy metals: activated in the presence of Co 2+  ion;   (4) Inhibiting heavy metals: inhibited in the presence of Sn 2+  ion; and   (5) Optimum pH: having an activity at pH of 5 to 9.   
     
     
         7 . The scyllo-inositol dehydrogenase according to  claim 5 , which is a protein represented by the following (A) or (B):
 (A) A protein comprising an amino acid sequence of SEQ ID NO: 28, or   (B) A protein comprising an amino acid sequence of SEQ ID NO: 28, whereby one or plural of amino acids are substituted, deleted, inserted, and/or added, and catalyzing the oxidation-reduction reaction between scyllo-inositol and scyllo-inosose and stereospecifically reducing scyllo-inosose into scyllo-inositol in the presence of NADH or NADPH.   
     
     
         8 . A DNA encoding a scyllo-inositol dehydrogenase according to  claim 7 . 
     
     
         9 . The DNA according to  claim 8  which is represented by the following (a) or (b):
 (a) A DNA comprising a coding region of the nucleotide sequence of SEQ ID NO: 27, or 
 (b) A DNA which hybridizes under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence complementary thereto, and encodes a protein that catalyzes the oxidation-reduction reaction between scyllo-inositol and scyllo-inosose and stereospecifically reduces scyllo-inosose into scyllo-inositol. 
 
     
     
         10 . A vector comprising the DNA according to  claim 8 . 
     
     
         11 . A transformant microorganism comprising the DNA according to  claim 8 . 
     
     
         12 . A transformant microorganism comprising the vector according to  claim 10 . 
     
     
         13 . The transformant microorganism according to  claim 11  which is  Escherichia coli.    
     
     
         14 . A method for producing scyllo-inositol dehydrogenase, comprising:
 culturing the transformant microorganism according to  claim 11 ; and   collecting scyllo-inositol dehydrogenase from the culture product thereof.   
     
     
         15 . A method for producing scyllo-inositol comprising: oxidizing myo-inositol at pH 6.0 to 8.5 in the presence of NAD +  or NADP + , in a solution which comprises the scyllo-inositol dehydrogenase according to  claim 5  and myo-inositol dehydrogenase (EC 1.1.1.18), wherein the myo-inositol dehydrogenase catalyzes an oxidation reaction with myo-inositol to generate scyllo-inosose in the presence of NAD +  and NADP + . 
     
     
         16 . A method of producing scyllo-inositol, comprising:
 1) obtaining a liquid mixture containing myo-inositol and scyllo-inositol by reducing scyllo-inosose with a metal salt of boron hydride in a solution comprising scyllo-inosose;   2) dissolving a scyllo-inositol/boric acid complex in the liquid mixture by adding an acid to the liquid mixture and adjusting the solution to be an acidic solution of 0.01 N or more; and   3) precipitating only scyllo-inositol by adding an aqueous organic solvent to the acidic solution in an amount such that the myo-inositol is not precipitated.