Compositions and methods comprising a lipolytic enzyme variant

Patent metadata

Field	Value
Publication number	US-10870839-B2
Application number	US-201916381295-A
Country	US
Kind code	B2
Filing date	Apr 11, 2019
Priority date	Jul 19, 2013
Publication date	Dec 22, 2020
Grant date	Dec 22, 2020

Abstract

The present invention provides lipolytic enzyme variants. Specifically, the present invention provides lipolytic enzyme variants having two, three, or more modifications as compared to a parent lipolytic enzyme and having at least one improved property. In addition, the present invention provides compositions comprising a lipolytic enzyme variant of the invention. The present invention also provides methods of cleaning using compositions comprising a lipolytic enzyme variant of the invention.

First claim

We claim:

1. A lipolytic enzyme variant or an active fragment thereof comprising an amino acid substitution at a position 187, wherein the lipolytic enzyme variant has at least 85% sequence identity to SEQ ID NO: 1 and the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of Thermomyces lanuginosa lipase set forth in SEQ ID NO: 1, and wherein the amino acid substitution is selected from the group consisting of V187N, V187H, V187Q and V187T.

2. The lipolytic enzyme variant or active fragment thereof of claim 1 , wherein the lipolytic enzyme variant further comprises amino acid substitutions selected from the group consisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73, 75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 189, 227, 233, 252, 256, and 264.

3. The lipolytic enzyme variant or active fragment thereof of claim 2 , wherein the variant or active fragment thereof comprises amino acid modifications: G023/D027/F051/E056/S058/L075/G091/D130N187/1252/L264; G023/E056/L075/D130/V187/L264; A018/G023/K024/L075/V077/N094/D130/V154/G156/V187; A018/G023/K024/L075/N094/D111/D130N154/G156N187/T189/L264; or N011/G023/D027/E056/L075/N094/D111/D130N154/G156N187/T189/L264; or A018/G023/E045/A049L075/V077/G156/V187/T189, and wherein the V187 amino acid modification is an amino acid substitution selected the group consisting of V187N, V187H, V187Q and V187T.

4. The lipolytic enzyme variant or active fragment thereof of claim 1 , wherein the variant or active fragment has lipolytic activity.

5. The lipolytic enzyme variant or active fragment thereof of claim 1 , wherein the variant or active fragment has a performance index (pI) relative to the parent lipolytic enzyme for hydrolysis of p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenyl palmitate that is greater than 1.0.

6. The lipolytic enzyme variant or active fragment thereof of claim 5 , wherein the performance index is measured using the p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenyl palmitate assay of Example 1, and, optionally, at a pH of 8.

7. The lipolytic enzyme variant or active fragment thereof of any of claim 1 or 2 , wherein the variant or active fragment thereof comprises
(i) the amino acid modifications selected from: D130A/V187N, V187T/L264R, A049V/V187T/T189D, D130A/V187T/L264R, G023K/E056K/V187T, G023Q/L075Q/V187T, L075G/D130A/V187H, L075Q/G156W/V187N, L075Q/V187N/L264R, L075R/D130A/V187T, L075R/V187T/L264R, V187N/T189Q/L264R, A018K/G023Q/E045F/V187T, A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/V187T, E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T, G023K/L075Q/G156W/V187N, G023Q/V154I/G156W/V187N, K024A/L075R/G156W/V187N, L075G/G091Q/V187N/L264R, L075Q/D130A/G156W/V187N, L075Q/V077I/G156W/V187N, A018K/A049V/L075Q/V187T/T189D, A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/L075Q/G156W/V187T, A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/V187T/T189D, A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N, A018K/L075Q/N094R/G156W/V187N, A018K/V077I/G156W/V187T/T189D, D027S/E056K/D111A/V187N/L264R, D027S/L075Q/D111A/D130A/V187N, E045F/A049V/L075Q/V187T/T189D, G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R, G023K/L075Q/D111A/D130A/V187T, G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T, G023Q/K024A/L075Q/G156W/V187Q, K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q, K024A/L075R/V154I/G156W/V187Q, L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T, L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V187T/T189Q/L264R, A018K/G023K/L075Q/D130A/G156W/V187N, A018K/G023K/L075Q/V077I/G156W/V187Q, A018K/G023K/L075R/N094R/D130A/V187N, A018K/G023Q/A049V/V077I/V187T/T189D, A018K/G023Q/E045F/A049V/G156W/V187T, A018K/G023Q/E045F/G156W/V187T/T189D, A018K/G023Q/E045F/L075Q/G156C/V187T, A018K/G023Q/L075Q/G156W/V187T/T189D, A018K/G023Q/N094R/D130A/G156W/V187Q, A018K/K024A/L075Q/N094R/V154I/V187N, A018K/K024A/L075Q/V077I/D130A/V187N, A018K/K024A/L075R/D130A/V187N/T189Q, A018K/L075Q/D130A/V187N/T189Q/L264R, A018K/N094R/D111A/D130A/V154I/V187N, D027Q/F051T/L075Q/D130A/V187H/L264R, D027Q/S058M/L075R/D130A/V187T/I252Q, D027S/E056K/S058M/V187T/I252Q/L264R, G023K/D027S/L075R/D130A/V187T/I252Q, G023K/E056K/L075R/D130A/V187N/L264R, G023K/F051T/D130A/V187N/I252Q/L264R, G023K/F051T/G091Q/D130A/V187H/L264R, G023K/K024A/L075Q/V154I/G156W/V187Q, G023K/K024A/L075R/D130A/V154I/V187N, G023K/N094R/D111A/G156W/V187T/L264R, G023Q/E056K/D111A/D130A/V187N/T189Q, G023Q/F051T/D130A/V187T/I252Q/L264R, G023Q/L075R/V154I/G156W/V187N/L264R, K024A/L075Q/D111A/V154I/V187N/T189Q, K024A/L075Q/V077I/N094R/D130A/V187N, K024A/L075Q/V154I/V187T/T189Q/L264R, L075Q/D130A/V154I/G156W/V187N/L264R, L075R/D130A/V154I/G156W/V187N/L264R, A018K/E045F/L075Q/V077I/G156W/V187T/T189D, A018K/G023K/K024A/L075Q/D130A/G156W/V187N, A018K/G023K/K024A/L075Q/N094R/G156W/V187Q, A018K/G023K/K024A/L075Q/N094R/V154I/V187Q, A018K/G023K/K024A/L075R/V154I/G156W/V187Q, A018K/G023K/K024A/N094R/V154I/V187T/L264R, A018K/G023Q/E045F/A049V/L075Q/G156W/V187T, A018K/G023Q/E045F/L075Q/G156W/V187T/T189D, A018K/G023Q/K024A/L075R/N094R/G156W/V187N, A018K/G023Q/L075R/V077I/N094R/G156W/V187N, A018K/K024A/L075Q/D130A/V154I/G156W/V187T, A018K/L075Q/N094R/D111A/V154I/V187T/L264R, D027Q/F051T/L075G/D130A/V187H/I252Q/L264R, D027S/E056K/L075R/D111A/G156W/V187N/L264R, D027S/F051T/L075G/G091Q/V187T/I252Q/L264R, E056K/L075Q/D111A/D130A/G156W/V187N/T189Q, G023K/D027Q/F051T/L075G/D130A/V187H/L264R, G023K/D027S/F051T/S058M/L075Q/D130A/V187N, G023K/K024A/N094R/V154I/G156W/V187N/T189Q, G023Q/E056K/L075Q/D111A/G156W/V187N/L264R, G023Q/E056K/L075R/D111A/D130A/V187N/T189Q, G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R, G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R, G023Q/L075Q/V077I/N094R/D130A/G156W/V187N, N011K/D027S/D111A/D130A/V154I/V187N/T189Q, A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q, A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q, G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q, G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R, G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R, G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R, G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q, G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q, G023Q/K024A/L075Q/V077I/N094R/D130A/V15411187N, K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R, A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N, A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q, A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H, A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D, G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q, G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R, G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R, G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R, N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R, N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q, A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T, A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H, A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q, N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R, A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R, A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D, A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T, A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D, optionally wherein the variant or active fragment has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for hydrolysis of p-nitrophenyl butyrate that is greater than 1.0;
(ii) the amino acid modifications selected from: A018K/L075Q/V187T, A049V/V187T/T189D, G023K/V187N/L264R, G023

Assignees

• Danisco Us Inc

Inventors

• Graycar Thomas P
• Poulose Ayrookaran J
• Estell David A

Classifications

• C12Y301/01003

  Triacylglycerol lipase (3.1.1.3) · CPC title

• C12N9/20 Primary

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

• C11D3/38681

  Chemically modified or immobilised enzymes · CPC title

• C12P7/20

  Glycerol · CPC title

• C11D3/38627

  containing lipase · CPC title