Polyvinyl alcohol-degrading enzyme and process for producing the same

Patent metadata

Field	Value
Publication number	US-11859229-B2
Application number	US-202117449888-A
Country	US
Kind code	B2
Filing date	Oct 4, 2021
Priority date	Mar 2, 2017
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

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.

First claim

What is claimed is:

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.

Assignees

• Hayashibara Co

Inventors

• Yamanaka Akihiro
• Matsuo Naoki
• Mori Tetsuya
• Nishimoto Tomoyuki

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12Y307/01007

  Beta-diketone hydrolase (3.7.1.7) · CPC title