Maltose-1-phosphate-producing enzyme

Abstract

The present invention provides an M-1-P-producing enzyme which employs, as starting materials, an oligosaccharide or polysaccharide having a glucose polymerization degree of 5 or more and containing an α-1,4-glycosidic bond such as maltooligosaccharide, dextrin, or starch, and which enables production of a large amount of M-1-P. The M-1-P-producing enzyme can produce M-1-P from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide having a glucose polymerization degree of 5 or more and containing an α-1,4-glycosidic bond.

1 . A method for producing a maltose-1-phosphate-producing enzyme, comprising culturing a microorganism capable of producing a maltose-1-phosphate-producing enzyme which is capable of producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more in a medium containing a phosphoric acid or a salt thereof in an amount of 50 mM or more, and collecting the maltose-1-phosphate-producing enzyme produced in the medium.  
     
     
         2 . The method for producing a maltose-1-phosphate-producing enzyme according to  claim 1 , wherein the microorganism is a transformant containing a recombinant vector expressing a gene encoding said enzyme or protein.  
     
     
         3 . The method according to  claim 1 , wherein the microorganism is a bacterium belonging to the family  Corynebacterium.    
     
     
         4 . The method according to  claim 1 , wherein the maltose-1-phosphate-producing enzyme has the following enzymatic characteristics; 
 1) Action: producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more,    2) Substrate specificity: in the presence of a phosphoric acid or a salt thereof, effectively acting on oligosaccharide or polysaccharide containing an α-1,4-glucosidic bond and having a glucose polymerization degree of 6 or more, or a degraded product thereof, thereby producing maltose-1-phosphate; weakly acting on an oligosaccharide having a glucose polymerization degree of 5; and exhibiting substantially no action on an oligosaccharide having a polymerization degree of 2 to 4,    3) Molecular weight: about 75 kDa (SDS-PAGE),    4) Optimum pH: 6.5 to 8.0, and    5) Optimum temperature: 35 to 50° C.    
     
     
         5 . The method according to  claim 1 , wherein the maltose-1-phosphate-producing enzyme is a protein selected from the group consisting of: 
 (a) A protein having the amino acid sequence of SEQ ID NO: 2,    (b) A protein having the amino acid sequence of SEQ ID NO: 2 in which one to several amino acid residues are deleted, substituted, or added, and which is capable of producing maltose-1-phosphate,    (c) A protein which has an amino acid sequence exhibiting 60% or more homology with that of SEQ ID NO: 2 and which is capable of producing a maltose-1-phosphate, and    (d) A protein which has an amino acid sequence exhibiting 38% or more homology with that of SEQ ID NO: 2, which is capable of producing a maltose-1-phosphate, and which contains an amino acid sequence represented by Ala-Glu-Asn-Pro-Pro-Lys-Lys(or Arg)-Tyr-Gln(or Glu)-Asp-Ile (SEQ ID NO: 11) or Phe-Arg-Val(or Ile)-Asp-Asn-Pro-His-Thr-Lys-Pro (SEQ ID NO: 12).    
     
     
         6 . A medium for producing a maltose-1-phosphate-producing enzyme which is capable of producing a maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more, comprising a phosphoric acid or a salt thereof in an amount of 50 mM or more.  
     
     
         7 . A method for producing maltose-1-phosphate, comprising reacting a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide having a glucose polymerization degree of 5 or more and containing an α-1,4-glycosidic bond with a maltose-1-phosphate-producing enzyme which is capable of producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more.  
     
     
         8 . The method according to  claim 7 , wherein the maltose-1-phosphate-producing enzyme has the following enzymatic characteristics; 
 1) Action: producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more,    2) Substrate specificity: in the presence of a phosphoric acid or a salt thereof, effectively acting on oligosaccharide or polysaccharide containing an α-1,4-glucosidic bond and having a glucose polymerization degree of 6 or more, or a degraded product thereof, thereby producing maltose-1-phosphate; weakly acting on an oligosaccharide having a glucose polymerization degree of 5; and exhibiting substantially no action on an oligosaccharide having a polymerization degree of 2 to 4,    3) Molecular weight: about 75 kDa (SDS-PAGE),    4) Optimum pH: 6.5 to 8.0, and    5) Optimum temperature: 35 to 50° C.    
     
     
         9 . The method according to  claim 7 , wherein the maltose-1-phosphate-producing enzyme is a protein selected from the group consisting of: 
 (a) A protein having the amino acid sequence of SEQ ID NO: 2,    (b) A protein having the amino acid sequence of SEQ ID NO: 2 in which one to several amino acid residues are deleted, substituted, or added, and which is capable of producing maltose-1-phosphate,    (c) A protein which has an amino acid sequence exhibiting 60% or more homology with that of SEQ ID NO: 2 and which is capable of producing a maltose-1-phosphate, and    (d) A protein which has an amino acid sequence exhibiting 38% or more homology with that of SEQ ID NO: 2, which is capable of producing a maltose-1-phosphate, and which contains an amino acid sequence represented by Ala-Glu-Asn-Pro-Pro-Lys-Lys(or Arg)-Tyr-Gln(or Glu)-Asp-Ile (SEQ ID NO: 11) or Phe-Arg-Val(or Ile)-Asp-Asn-Pro-His-Thr-Lys-Pro (SEQ ID NO: 12).    
     
     
         10 . A method for producing maltose-1-phosphate, comprising culturing a microorganism capable of producing a maltose-1-phosphate-producing enzyme which is capable of producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more in a medium containing 1 mM or more of a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide having a glucose polymerization degree of 5 or more and containing an α-1,4-glycosidic bond, and collecting maltose-1-phosphate produced and accumulated in the medium.  
     
     
         11 . The method according to  claim 10 , wherein the maltose-1-phosphate-producing enzyme has the following enzymatic characteristics; 
 1) Action: producing maltose-1-phosphate from a phosphoric acid or a salt thereof and an oligosaccharide or polysaccharide containing an α-1,4-glycosidic bond and having a glucose polymerization degree of 5 or more,    2) Substrate specificity: in the presence of a phosphoric acid or a salt thereof, effectively acting on oligosaccharide or polysaccharide containing an α-1,4-glucosidic bond and having a glucose polymerization degree of 6 or more, or a degraded product thereof, thereby producing maltose-1-phosphate; weakly acting on an oligosaccharide having a glucose polymerization degree of 5; and exhibiting substantially no action on an oligosaccharide having a polymerization degree of 2 to 4,    3) Molecular weight: about 75 kDa (SDS-PAGE),    4) Optimum pH: 6.5 to 8.0, and    5) Optimum temperature: 35 to 50° C.    
     
     
         12 . The method according to  claim 10 , wherein the maltose-1-phosphate-producing enzyme is a protein selected from the group consisting of: 
 (a) A protein having the amino acid sequence of SEQ ID NO: 2,    (b) A protein having the amino acid sequence of SEQ ID NO: 2 in which one to several amino acid residues are deleted, substituted, or added, and which is capable of producing maltose-1-phosphate,    (c) A protein which has an amino acid sequence exhibiting 60% or more homology with that of SEQ ID NO: 2 and which is capable of producing a maltose-1-phosphate, and    (d) A protein which has an amino acid sequence exhibiting 38% or more homology with that of SEQ ID NO: 2, which is capable of producing a maltose-1-phosphate, and which contains an amino acid sequence represented by Ala-Glu-Asn-Pro-Pro-Lys-Lys(or Arg)-Tyr-Gln(or Glu)-Asp-Ile (SEQ ID NO: 11) or Phe-Arg-Val(or Ile)-Asp-Asn-Pro-His-Thr-Lys-Pro (SEQ ID NO: 12).    
     
     
         13 . The method according to  claim 10 , wherein the microorganism is a transformant comprising a recombinant vector encoding said enzyme or protein.  
     
     
         14 . The method according to  claim 10 , wherein the microorganism is a bacterium belonging to the family  Corynebacterium.    
     
     
         15 . The method according to  claim 10 , wherein the concentration of a phosphoric acid or a salt thereof is 50 mM to 2,000 mM.  
     
     
         16 . A gene encoding the protein selected from the group consisting of: 
 (a) A protein having the amino acid sequence of SEQ ID NO: 2,    (b) A protein having the amino acid sequence of SEQ ID NO: 2 in which one to several amino acid residues are deleted, substituted, or added, and which is capable of producing maltose-1-phosphate,    (c) A protein which has an amino acid sequence exhibiting 60% or more homology with that of SEQ ID NO: 2 and which is capable of producing a maltose-1-phosphate, and    (d) A protein which has an amino acid sequence exhibiting 38% or more homology with that of SEQ ID NO: 2, which is capable of producing a maltose-1-phosphate, and which contains an amino acid sequence represented by Ala-Glu-Asn-Pro-Pro-Lys-Lys(or Arg)-Tyr-Gln(or Glu)-Asp-Ile (SEQ ID NO: 11) or Phe-Arg-Val(or Ile)-Asp-Asn-Pro-His-Thr-Lys-Pro (SEQ ID NO: 12).    
     
     
         17 . A recombinant vector containing a gene as recited in  claim 16 .  
     
     
         18 . A transformant containing a recombinant vector as recited in  claim 17 .  
     
     
         19 . The transformant according to  claim 18 , wherein the host is a microorganism.  
     
     
         20 . A maltose-1-phosphate-producing enzyme gene selected from the group consisting of: 
 (a) A DNA which has a nucleotide sequence represented by SEQ ID NO: 1    (b) A DNA which is capable of hybridizing a DNA fragment having the nucleotide sequence of (a) under stringent conditions and which encodes a protein capable of producing maltose-1-phosphate; and    (c) A DNA which exhibits 60% or more homology with the nucleotide sequence of (a) and which encodes a protein capable of producing maltose-1-phosphate.    
     
     
         21 . A recombinant vector containing a gene as recited in  claim 20 .  
     
     
         22 . A transformant containing a recombinant vector as recited in  claim 21 .  
     
     
         23 . The transformant according to  claim 22 , wherein the host is a microorganism.