Polyvinyl alcohol-degrading enzyme and process for producing the same

1. A process for producing a polyvinyl alcohol-degrading enzyme, comprising the steps of:

culturing a microorganism capable of producing the polyvinyl alcohol-degrading enzyme in a nutrient medium;

removing cells from the resulting culture to obtain a supernatant; and

collecting the polyvinyl alcohol-degrading enzyme from the supernatant or collecting the supernatant as a crude enzyme preparation containing the polyvinyl alcohol-degrading enzyme;

wherein said polyvinyl alcohol-degrading enzyme comprises the following characteristics (1) to (3):

(1) comprising an activity of oxidizing polyvinyl alcohol and forming hydrogen peroxide;

(2) comprising an activity of hydrolyzing β-diketone; and

(3) exhibiting a molecular weight of 100,000±20,000 in SDS-polyacrylamide gel electrophoresis,

and wherein said polyvinyl alcohol-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or an amino acid sequence comprising a deletion, a replacement, or an addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 while retaining the polyvinyl alcohol-degrading activity and comprising 84% or higher sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

2. The process of claim 1 , wherein said activity of oxidizing polyvinyl alcohol comprises 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:

pH 6.5 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 process of claim 1 , wherein said polyvinyl alcohol-degrading enzyme further comprises the following characteristic (8):

(8) comprises the amino acid sequence of SEQ ID NO: 1 as the N-terminal amino acid sequence.

4. The process of claim 1 , wherein said polyvinyl alcohol-degrading enzyme is encoded by the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a nucleotide sequence comprising a deletion, a replacement, or an addition of one or more nucleotides in the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 while retaining the encoded polyvinyl alcohol-degrading activity and having 82% or higher sequence identity to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or complementary nucleotide sequences thereof.

5. The process of claim 1 , wherein said microorganism belongs to the genus Pseudomonas.

6. A process for producing a recombinant polyvinyl alcohol-degrading enzyme, comprising the steps of:

transforming a microorganism by introducing a replicable recombinant DNA, which comprises a DNA encoding a polyvinyl alcohol-degrading enzyme and an autonomously replicable vector, into the microorganism to obtain a transformant capable of expressing the polyvinyl alcohol-degrading enzyme encoded by the DNA;

culturing the transformant in a nutrient medium; and

collecting the recombinant polyvinyl alcohol-degrading enzyme from the resulting culture,

wherein said recombinant polyvinyl alcohol-degrading enzyme comprises the following characteristics (1) to (3):

(1) comprising an activity of oxidizing polyvinyl alcohol and forming hydrogen peroxide;

(2) comprising an activity of hydrolyzing β-diketone; and

(3) exhibiting a molecular weight of 100,000±20,000 in SDS-polyacrylamide gel electrophoresis,

and wherein said polyvinyl alcohol-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or an amino acid sequence comprising a deletion, a replacement, or an addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 while retaining the polyvinyl alcohol-degrading activity and comprising 84% or higher (sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

7. The process of claim 6 , wherein said activity of oxidizing polyvinyl alcohol comprises 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:

pH 6.5 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.

8. The process of claim 6 , wherein said DNA encodes a polyvinyl alcohol-degrading enzyme comprising the amino acid sequence of SEQ ID NO: 1 as the N-terminal amino acid sequence.

9. The process of claim 6 , wherein said DNA comprises the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or a nucleotide sequence comprising a deletion, a replacement, or an addition of one or more nucleotides in the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 while retaining the encoded polyvinyl alcohol-degrading activity and having 82% or higher sequence identity to the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or complementary nucleotide sequences thereof.

10. The process of claim 6 , wherein said DNA is obtainable by replacing one of more nucleotides of SEQ ID NO: 4 or SEQ ID NO: 5 with other nucleotides without altering the amino acid sequence encoded thereby based on the degeneracy of genetic code.