Modified lipase and use thereof

Patent metadata

Field	Value
Publication number	US-11299722-B2
Application number	US-201816639871-A
Country	US
Kind code	B2
Filing date	Aug 17, 2018
Priority date	Sep 1, 2017
Publication date	Apr 12, 2022
Grant date	Apr 12, 2022

Abstract

The present invention has an object of providing a modified lipase having excellent stability and reactivity at a high temperature. Provided is a modified lipase with improved reactivity and/or stability at a high temperature, the modified lipase having an amino acid sequence including one or more amino acid substitutions which are selected from the group consisting of T130C-S153C, A249P, F259Y, S282P, S283Y and S300P in the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 90% or more sequence identity therewith.

First claim

The invention claimed is:

1. A modified lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, the modified lipase having the amino acid sequence of SEQ ID NO: 1 except as having one or more amino acid substitutions selected from the group consisting of T130C-S153C, A249P, F259Y, 5282P, 5283Y, and S300P,
or alternatively the modified lipase having an amino acid sequence having 92% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions selected from the group consisting of T130C-S153C, A249P, F259Y, S282P, S283Y, and S300P, with the proviso that the amino acid sequence of the modified lipase is different from the amino acid sequence of SEQ ID NO: 1 at a position or positions other than the position(s) at which the amino acid substitution(s) is(are) made;
wherein the high temperature is a temperature above 40 Celsius degree.

2. The modified lipase according to claim 1 , wherein the amino acid substitution contained in the amino acid sequence of the modified lipase is T130C-S153C or S283Y, and the modified lipase has improved reactivity at a high temperature.

3. The modified lipase according to claim 1 , wherein the amino acid substitution contained in the amino acid sequence of the modified lipase is A249P, S283Y, or S300P, and the modified lipase has improved stability at a high temperature.

4. A lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, comprising the amino acid sequence of any of SEQ ID NOs: 2 to 7, or an amino acid sequence having 92% or more sequence identity with the amino acid sequence of any of SEQ ID Nos: 2 to 7, with the proviso that the amino acid sequence is different at a position or positions other than those corresponding to cysteine at positions 130 and 153 in the amino acid sequence of SEQ ID NO: 2, proline at position 249 in the amino acid sequence of SEQ ID NO: 3, tyrosine at position 259 in the amino acid sequence of SEQ ID NO: 4, proline at position 282 in the amino acid sequence of SEQ ID NO: 5, tyrosine at position 283 in the amino acid sequence of SEQ ID NO: 6, and proline at position 300 in the amino acid sequence of SEQ ID NO: 7, when the respective amino acid sequences are used as a reference sequence to determine the sequence identity;
wherein the high temperature is a temperature above 40 Celsius degree.

5. A gene encoding the modified lipase according to claim 1 .

6. The gene according to claim 5 , comprising a base sequence of any of SEQ ID NOs: 8 to 19.

7. A recombinant DNA containing the gene according to claim 5 .

8. A microorganism carrying the recombinant DNA according to claim 7 .

9. The microorganism according to claim 8 , wherein the host is Escherichia coli, Candida cylindracea, Aspergillus oryzae, Bacillus subtilis , or Pichia pastoris.

10. An enzyme preparation comprising the modified lipase according to claim 1 .

11. A method for degradation of fats and oils comprising allowing the modified lipase according to claim 1 or an enzyme preparation comprising the modified lipase according to claim 1 to act on the fats and oils to carry out an enzyme reaction.

12. The method for degradation according to claim 11 , wherein the enzyme reaction is carried out at 30° C. to 70° C.

13. A modified lipase with improved reactivity and/or stability at a high temperature, as compared to a lipase consisting of the amino acid sequence of SEQ ID NO: 1, the modified lipase having the amino acid sequence of SEQ ID NO: 1 except as having the amino acid substitutions is T130C-S153C,
or alternatively the modified lipase having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions of T130C-S153C, with the proviso that the amino acid sequence of the modified lipase is different from the amino acid sequence of SEQ ID NO: 1 at a position or positions other than the position(s) at which the amino acid substitution is made;
wherein the high temperature is a temperature above 40 Celsius degree.

Assignees

• Amano Enzyme Inc

Inventors

• Yoshida Kazunori
• Takahashi Tetsuya
• Ishikawa Kazuhiko

Classifications

• C12Y301/01003 Primary

  Triacylglycerol lipase (3.1.1.3) · CPC title

• C12P7/64

  Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats · CPC title

• C12N9/20 Primary

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C11B3/003

  by enzymes or microorganisms, living or dead · CPC title

• C12R2001/72

  Candida · CPC title