Polyvinyl alcohol-degrading enzyme and process for producing the same

Abstract

The objects of the present invention are to provide a novel PVA-degrading enzyme that the entity of which is revealed at the amino acid sequence level, a process for producing the same, a DNA encoding the enzyme, a recombinant DNA comprising the DNA, and a transformant having the recombinant DNA. The present invention solves the above objects by providing a polyvinyl alcohol-degrading enzyme having the following characteristics (1) to (3), a process for producing the same, a DNA encoding the enzyme, a recombinant DNA comprising the DNA, and a transformant having the recombinant DNA:(1) having an activity of oxidizing polyvinyl alcohol and forming hydrogen peroxide;(2) having an activity of hydrolyzing β-diketone, and(3) exhibiting a molecular weight of 100,000±20,000 in SDS-polyacrylamide gel electrophoresis.

1 . A polyvinyl alcohol-degrading enzyme having the following characteristics (1) to (3):
 (1) having an activity of oxidizing polyvinyl alcohol and forming hydrogen peroxide;   (2) having an activity of hydrolyzing β-diketone; and   (3) exhibiting a molecular weight of 100,000±20,000 in SDS-polyacrylamide gel electrophoresis.   
     
     
         2 . The polyvinyl alcohol-degrading enzyme of  claim 1 , wherein said activity of oxidizing polyvinyl alcohol has the following characteristics (4) to (7):
 (4) Optimum temperature: 35 to 40° C. under the conditions of 60 min-reaction at pH 7.0;   (5) Optimum pH: pH6.5 to 8.0 under the conditions of 60 min-reaction at 27° C.;   (6) Thermal stability: Stable up to 45° C. under the conditions of holding for 60 min at pH 7.0; and   (7) pH stability: Stable in a range of pH 4.5 to 10.5 under the condition of holding for 24 hours at 4° C.   
     
     
         3 . The polyvinyl alcohol-degrading enzyme of  claim 1 , further having the following characteristic (8):
 (8) having an amino acid sequence of SEQ ID NO: 1 as N-terminal amino acid sequence.   
     
     
         4 . The polyvinyl alcohol-degrading enzyme of  claim 1 , which has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or an amino acid sequence having deletion, replacement, or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 with holding the polyvinyl alcohol-degrading activity and having 84% or higher homology (sequence identity) to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         5 . The polyvinyl alcohol-degrading enzyme of  claim 1 , which is derived from a microorganism of the genus  Pseudomonas.    
     
     
         6 . A DNA encoding the polyvinyl alcohol-degrading enzyme of  claim 4 . 
     
     
         7 . The DNA of  claim 6 , which has a nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:5, or a nucleotide sequence having deletion, replacement, or addition of one or more nucleotides in the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 with holding the encoded polyvinyl alcohol-degrading activity and having 82% or higher homology (sequence identity) to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or complementary nucleotide sequences thereof. 
     
     
         8 . The DNA of  claim 6 , which is obtainable by replacing one or more nucleotides of SEQ ID NO:4 or SEQ ID NO:5 with other nucleotide(s) without altering the amino acid sequence encoded thereby based on the degeneracy of genetic code. 
     
     
         9 . A replicable recombinant DNA, comprising the DNA of  claim 6  and an autonomously replicable vector. 
     
     
         10 . A transformant, prepared by introducing the recombinant DNA of  claim 9  into an appropriate host. 
     
     
         11 . A process for producing a polyvinyl alcohol-degrading enzyme, comprising the steps of:
 culturing a microorganism capable of producing the polyvinyl alcohol-degrading enzyme of  claim 1  in a nutrient medium; and   collecting the polyvinyl alcohol-degrading enzyme of  claim 1  from the resulting culture.   
     
     
         12 . The process of  claim 11 , wherein said microorganism belongs to the genus  Pseudomonas.    
     
     
         13 . A process for producing a recombinant polyvinyl alcohol-degrading enzyme, comprising the steps of:
 culturing the transformant of  claim 10  in a nutrient medium; and   collecting the recombinant polyvinyl alcohol-degrading enzyme from the resulting culture.