# US 10,370,693 B2 · Transaminases

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- Number: US 10,370,693 B2
- Document ID: US10370693B2
- Status: Granted
- Kind: B2
- Application number: 15/735,165
- Filed: 2016-06-10
- Published: 2018-10-18
- Granted: 2019-08-06
- Earliest claimed: 2015-06-12
- Assignees: C-LEcta GmbH
- Applicants: C-LEcta GmbH
- Inventors: Andreas Vogel; Daniel Schwarze; Rico Czaja; Sally Bayer; Sebastian Bartsch
- Web page: https://patentgrep.com/patent/US10370693B2

## CPC

- C12P 41/006: {by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures}
- C12N 9/1096: {transferring nitrogenous groups (2.6)}
- C12P 7/26: Ketones
- C12P 13/001: {Amines; Imines}
- C12Y 206/01: Transaminases (2.6.1)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The invention relates to transaminases that are particularly useful for catalyzing the conversion of amine substrates to ketone products and/or vice versa.

## Claims

### Claim 1

A transaminase comprising an amino acid sequence with at least 80% identity to SEQ ID NO:3, wherein the transaminase is substituted compared to SEQ ID NO:3 at least in position N161 and/or in position Y164, and has a transaminase activity.

### Claim 2 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in position Y164.

### Claim 3 (depends on 1)

The transaminase according to claim 1, which is additionally substituted compared to SEQ ID NO:3 in position G51.

### Claim 4 (depends on 1)

The transaminase according to claim 1, which comprises at least two substitutions selected from the group consisting of N161A, N161F, N161M, N161Y, N161Q, N161I, Y164L, Y164M, Y164A, Y164F, Y164I, and G51S.

### Claim 5 (depends on 1)

The transaminase according to claim 1, which comprises a substitution selected from the group consisting of N161A, N161C, N161 D, N161E, N161G, N161I, N161K, N161L, N161M, N161P, N161Q, N161R, N161S, N161T, N161V, N161W, and N161Y.

### Claim 6 (depends on 5)

The transaminase according claim 5, which comprises a substitution selected from the group consisting of N161A, N161F, N161M, N161Y, N161Q, and N161I.

### Claim 7 (depends on 1)

The transaminase according to claim 1, which comprises a substitution selected from the group consisting of Y164A, Y164C, Y164D, Y164E, Y164F, Y164G, Y164H, Y164I, Y164K, Y164L, Y164M, Y164N, Y164P, Y164Q, Y164R, Y164S, Y164T, Y164V, and Y164W and/or
which comprises a substitution selected from the group consisting of G51A, G51C, G51 D, G51E, G51F, G51H, G51I, G51K, G51L, G51M, G51N, G51P, G51Q, G51R, G51S, G51T, G51V, G51W, and G51Y.

### Claim 8 (depends on 7)

The transaminase according to claim 7, which comprises a substitution selected from the group consisting of Y164L, Y164M, Y164A, Y164F, and Y164I or which comprises the substitution G51S.

### Claim 9 (depends on 1)

The transaminase according to claim 1, wherein the identity to SEQ ID NO:3 is at least 90%.

### Claim 10 (depends on 1)

The transaminase according to claim 1, which is additionally substituted compared to SEQ ID NO:3 in at least one or more positions selected from the group consisting of the positions N7, E9, M16, V29, G33, Q44, R45, L53, W54, A67, A68, A71, L73, F82, H87, V109, G114, R140, N146, G147, Y148, Y151, H165, E222, A227, G228, V230, V257, V258, A288, Q300, A349, A353, Q354, Y366, M378, Q391, R415, I417, K420, I422, T430, and E433.

### Claim 11 (depends on 1)

The transaminase according claim 1, which has
(A) a temperature stability of at least 50° C., and/or
(B) a specific activity of at least 0.5 U/mg in Transaminase Standard Assays; and/or
(C) a high conversion activity under different reaction conditions involving high amine concentrations.

### Claim 12 (depends on 1)

The transaminase according to claim 1, comprising at least one or more additional substitutions selected from the group consisting of N7L, E9R, M16F, M16C, M16V, M16L, M16A, M16W, V29L, V29I, G33Y, Q44R, Q44N, Q44H, R45K, L53F, L53W, L53V, L53A, L53S, L53G, W54A, W54I, W54L, W54Y, W54S, W54C, W54F, W54V, A67N, A68P, A71G, L73M, F82V, F82A, F82G, F82L, F82Y, H87T, H87N, V109I, G114S, R140K, N146Y, N146D, N146S, G147S, Y148F, Y148S, Y148G, V151A, V151W, V151I, V151F, V151Y, V151S, H165R, E222S, E222A, E222D, A227Y, A227V, A227I, A227G, A227F, A227M, G228A, G228I, V230A, V230G, V230I, V230L, V257A, V258A, V258I, A288G, Q300E, A349G, A353R, Q354F, Y366H, Y366F, M378L, M378V, M378I, M378F, M378Y, M378T, M378A, M378C, Q391K, Q391E, R415A, R415V, R415L, R415G, R415Y, R415T, R415C, I417T, I417C, I417F, I417V, I417Y, I417A, K420H, K420S, K420N, I422V, I422S, I422A, I422L, I422C, T430N, and E433D or
being substituted in at least one other position selected from the group consisting of N7, E9, M16, V29, G33, Q44, R45, L53, W54, A67, A68, A71, L73, F82, H87, V109, G114, R140, N146, G147, Y148, Y151, H165, E222, A227, G228, V230, V257, V258, A288, Q300, A349, A353, Q354, Y366, M378, Q391, R415, I417, K420, I422, T430, and E433.

### Claim 13 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in at least two positions selected from the group consisting of N161 as well as Y164; and N146 as well as N161.

### Claim 14 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in at least two positions such that it comprises
(i) at position N161 a substitution selected from the group consisting of N161A, N161F, N161M, N161Y, N161I, or N161Q; as well as
at position Y164 a substitution selected from the group consisting of Y164L, Y164M, Y164A, Y164F, or Y164I; and/or
(ii) at position N146 a substitution selected from the group consisting of N146Y, N146D, or N146S; as well as
at position N161 a substitution selected from the group consisting of N161A, N161F, N161M, N161Y, N161I, or N161Q.

### Claim 15 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in at least two positions such that it comprises at least two substitutions selected from the group consisting of the substitutions N161M as well as Y164L; N161I as well as Y164L; N161Q as well as Y164L; N161I as well as Y164M; N161M as well as Y164M; N161Q as well as Y164M; N161I as well as Y164F; N161M as well as Y164I; and N146Y as well as N161Q.

### Claim 16 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in
(i) at least two positions selected from the group consisting of N161 as well as Y164; or N146 as well as N161; and
(ii) in addition in at least one other position selected from the group consisting of N7, E9, M16, V29, G33, Q44, R45, G51, L53, W54, A67, A68, A71, L73, F82, H87, V109, G114, R140, N146, G147, Y148, Y151, Y164, H165, E222, A227, G228, V230, V257, V258, A288, Q300, A349, A353, Q354, Y366, M378, Q391, R415, I417, K420, I422, T430, and E433.

### Claim 17 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in
(i) at least two positions wherein the transaminase comprises at least two substitutions selected from the group consisting of N161M as well as Y164L; N161I as well as Y164L; N161Q as well as Y164L; N161I as well as Y164M; N161M as well as Y164M; N161Q as well as Y164M; N161I as well as Y164F; N161M as well as Y164I and N146Y as well as N161Q; and
(ii) in addition in at least one other position the transaminase comprises at least one other substitution selected from the group consisting of N7L, E9R, M16F, M16C, M16V, M16L, M16A, M16W, V29L, V29I, G33Y, Q44R, Q44N, Q44H, R45K, G51S, L53F, L53W, L53V, L53A, L53S, L53G, W54A, W54I, W54L, W54Y, W54S, W54C, W54F, W54V, A67N, A68P, A71G, L73M, F82V, F82A, F82G, F82L, F82Y, H87T, H87N, V109I, G114S, R140K, N146Y, N146D, N146S, G147S, Y148F, Y148S, Y148G, V151A, V151W, V151I, V151F, V151Y, V151S, H165R, E222S, E222A, E222D, A227V, A227I, A227G, A227F, A227Y, A227M, G228A, G228I, V230G, V230A, V230I, V230L, V257A, V258A, V258I, A288G, Q300E, A349G, A353R, Q354F, Y366H, Y366F, M378L, M378V, M378I, M378F, M378Y, M378T, M378A, M378C, Q391K, Q391E, R415A, R415V, R415L, R415G, R415Y, R415T, R415C, I417T, I417C, I417F, I417V, I417Y, I417A, K420H, K420S, K420N, I422V, I422S, I422A, I422L, I422C, T430N, and E433D.

### Claim 18 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in at least in three positions such that it comprises at least three substitutions, wherein
(i) the first substitution of said at least three substitutions is selected from the group consisting of N161I, N161M, and N161Q; and
(ii) the second substitution of said at least three substitutions is selected from the group consisting of Y164L, and Y164I; and
(iii) the third substitution of said at least three substitutions is selected from the group consisting of N7L, E9R, M16F, M16W, V29L, V29I, G33Y, Q44R, Q44N, Q44H, R45K, G51S, W54A, A67N, A68P, A71G, L73M, H87T, H87N, V109I, G114S, R140K, G147S, H165R, V230A, A288G, Q300E, A349G, A353R, Q354F, Y366H, Y366F, Q391K, Q391E, K420H, K420S, K420N, T430N, and E433D.

### Claim 19 (depends on 1)

The transaminase according to claim 1, which comprises an amino acid sequence of at least 85% identity to the SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57.58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150.

### Claim 20 (depends on 1)

A method for the conversion of
(i) a ketone substrate according to general formula (I)
to an amine product according to general formula (II)
and/or the preferably concomittant conversion of
(ii) a cosubstrate according to general formula (III)
to a ketone coproduct according to general formula (IV)
or vice versa;
wherein X and Y as well as A and B, in either case, independently of one another, are selected from saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic or alicyclic C1-12-hydrocarbon residues; unsubstituted or mono- or polysubstituted C6-10-aromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted heteroaromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C1-12-hydrocarbon residue; and sugar residues or desoxysugar residues in each case comprising mono-, di- or oligosaccharides;
wherein one of residues X or Y, as well as one of residues A or B, in either case, independently of one another, may be hydrogen;
wherein mono- or polysubstituted means independently substituted with one or more functional groups selected from -halo, —OH, ═O, —OC1-12-alkyl, —OC6-10-aryl, —O-heteroaryl, —OCOC1-12-alkyl, —OCOC6-10-aryl, —OCO-heteroaryl, —SH, —SC1-12-alkyl, —SC6-10-aryl, —S-heteroaryl, —S(═O)1-2OH, —NO, —NO2, —N3, —NH2, —NH(C1-12-alkyl), —N(C1-12-alkyl)2, —NH(C6-10-aryl), —N(C6-10-aryl)2, —NH(heteroaryl), —N(heteroaryl)2, —CN, —CHO, —CO2H, CO—C1-2-alkyl, —CO—C6-10-aryl and —CO-heteroaryl;
wherein the method comprises the step of reacting a ketone substrate according to general formula (I) and/or an amine product according to general formula (II) and/or a amine cosubstrate according to general formula (III) and/or a ketone coproduct according to general formula (IV) in the presence of the transaminase of claim 1.

### Claim 21 (depends on 1)

The transaminase according to claim 1, which is substituted compared to SEQ ID NO:3 in position N161.