Protein having nadh and/or nadph oxidase activity

Patent metadata

Field	Value
Publication number	US-2016145585-A1
Application number	US-201615011989-A
Country	US
Kind code	A1
Filing date	Feb 1, 2016
Priority date	Jan 20, 2010
Publication date	May 26, 2016
Grant date	—

Abstract

Water-forming NADH oxidase derived from Streptococcus mutans should be further improved in terms of stability for practical use in industrial production. An object of the present invention is to provide an enzyme that is obtained through modification of a water-forming NADH oxidase, which is useful as an NAD+ regeneration system for stereoselective oxidation catalyzed by an oxidoreductase, by protein engineering techniques so that the enzyme can withstand long-term use without exhibiting a reduction of its activity for the regeneration of NAD+, that is, an enzyme having improved stability, and to provide a method for efficiently producing a useful substance such as an optically active alcohol or amino acid. The present invention relates to an enzyme modification method that can improve the stability of water-forming NADH oxidase derived from Streptococcus mutans by appropriately introducing mutation.

First claim

1 . A protein having an amino acid sequence that has at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and further contains at least one amino acid substitution selected from (a) to (g):
(a) a substitution of an amino acid residue at a position corresponding to position 42 of SEQ ID NO:1 with an amino acid having a side-chain surface area of 100 to 200 (Å 2 ); (b) a substitution of an amino acid residue at a position corresponding to position 46 of SEQ ID NO: 1 with a neutral amino acid having a side-chain surface area of 100 to 150 (Å 2 ) or an acidic amino acid having a side-chain surface area of 100 to 150 (Å 2 ); (c) a substitution of an amino acid residue at a position corresponding to position 96 of SEQ ID NO:1 with a basic amino acid; (d) a substitution of an amino acid residue at a position corresponding to position 172 of SEQ ID NO:1 with an amino acid having a smaller side-chain surface area than Tyr; (e) a substitution of an amino acid residue at a position corresponding to position 196 of SEQ ID NO: 1 with a basic amino acid; (f) a substitution of an amino acid residue at a position corresponding to position 312 of SEQ ID NO:1 with an amino acid having a larger side-chain surface area than Ala; and (g) a substitution of an amino acid residue at a position corresponding to position 371 of SEQ ID NO:1 with an aliphatic amino acid, an acidic amino acid, or an amino acid having a hydroxyl group-bearing side chain; and wherein the protein has NADH activity or NADPH oxidase activity or both, and has improved stability as compared to the stability of the protein having the amino acid sequence of SEQ ID NO:1.

2 . The protein according to claim 1 ,
wherein the amino acid sequence contains at least one amino acid substitution selected from (a) to (g): (a) a substitution of an amino acid residue at a position corresponding to position 42 of SEQ ID NO:1 with Met; (b) a substitution of an amino acid residue at a position corresponding to position 46 of SEQ ID NO:1 with Ile; (c) a substitution of an amino acid residue at a position corresponding to position 96 of SEQ ID NO:1 with Arg or His; (d) a substitution of an amino acid residue at a position corresponding to position 172 of SEQ ID NO:1 with Ala or Ser; (e) a substitution of an amino acid residue at a position corresponding to position 197 of SEQ ID NO:1 with His; (f) a substitution of an amino acid residue at a position corresponding to position 312 of SEQ ID NO:1 with Ile; and (g) a substitution of an amino acid residue at a position corresponding to position 371 of SEQ ID NO: 1 with Ala, Val, Ile, Glu, Ser, Thr, or Tyr.

3 . A protein comprising an amino acid sequence of SEQ ID NO:1 which further contains at least one amino acid substitution selected from (a) to (g):
(a) a substitution of Leu-42 with an amino acid having a side-chain surface area of 100 to 200 (Å 2 ); (b) a substitution of Met-46 with a neutral amino acid having a side-chain surface area of not more than 150 (Å 2 ) or an acidic amino acid having a side-chain surface area of not more than 150 (Å 2 ); (c) a substitution of Asn-96 with a basic amino acid; (d) a substitution of Tyr-172 with an amino acid having a smaller side-chain surface area than Tyr; (e) a substitution of Thr-196 with a basic amino acid; (f) a substitution of Ala-312 with an amino acid having a larger side-chain surface area than Ala; and (g) a substitution of Phe-371 with an aliphatic amino acid, an acidic amino acid, or an amino acid having a hydroxyl group-bearing side chain, and contains the amino acid sequence of SEQ ID NO:1 except for the amino acid substitution.

4 . The protein according to claim 3 ,
wherein the amino acid sequence contains at least one amino acid substitution selected from (a) to (g): (a) a substitution of Leu-42 with Met; (b) a substitution of Met-46 with Ile; (c) a substitution of Asn-96 with Arg or His; (d) a substitution of Tyr-172 with Ala or Ser; (e) a substitution of Thr-196 with His; (f) a substitution of Ala-312 with Ile; and (g) a substitution of Phe-371 with Ala, Val, Ile, Glu, Ser, Thr, or Tyr, and contains the amino acid sequence of SEQ ID NO:1 except for the amino acid substitution.

5 . The protein according to claim 4 ,
wherein the protein has an amino acid sequence selected from the amino acid sequences of SEQ ID NO:2 and 4 to 19.

6 . An enzyme variant-containing product obtained by processing a culture of a transformant obtained by transformation of a host cell with a vector comprising an isolated DNA encoding a protein
wherein said protein has NADH oxidase activity or NADPH oxidase activity or both and has improved stability compared to the protein having the amino acid sequence of SEQ ID NO:1, and which has an amino acid sequence that has at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1 and further contains at least one amino acid substitution selected from (a) to (g): (a) a substitution of an amino acid residue at a position corresponding to position 42 of SEQ ID NO: 1 with an amino acid having a side-chain surface area of 100 to 200 Å 2 ; (b) a substitution of an amino acid residue at a position corresponding to position 46 of SEQ ID NO: 1 with a neutral amino acid having a side-chain surface area of 100 to 150 Å 2 or an acidic amino acid having a side-chain surface area of 100 to 150 Å 2 : (c) a substitution of an amino acid residue at a position corresponding to position 96 of SEQ ID NO:1 with a basic amino acid; (d) a substitution of an amino acid residue at a position corresponding to position 172 of SEQ ID NO: 1 with an amino acid having a smaller side-chain surface area than Tyr; (e) a substitution of an amino acid residue at a position corresponding to position 196 of SEQ ID NO: 1 with a basic amino acid; (f) a substitution of an amino acid residue at a position corresponding to position 312 of SEQ ID NO: 1 with an amino acid having a larger side-chain surface area than Ala; and (g) a substitution of an amino acid residue at a position corresponding to position 371 of SEQ ID NO:1 with an aliphatic amino acid, an acidic amino acid, or an amino acid having a hydroxyl group-bearing side chain.

Assignees

• Kaneka Corp

Inventors

• Yoshida Shinichi
• Iwasaki Akira
• Washida Motohisa
• Nishiyama Tozo
• Moriyama Daisuke
• Taoka Naoaki

Classifications

• C12N9/0065 Primary

  acting on hydrogen peroxide as acceptor (1.11) · CPC title

• C12N9/0036 Primary

  acting on NADH or NADPH (1.6) · CPC title

• C12Y106/03

  with oxygen as acceptor (1.6.3) · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title