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1. (WO2016198660) TRANSAMINASES
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

Patent claims:

1. A transaminase comprising an amino acid sequence with at least 80% homology to SEQ ID NO: 3, wherein the transaminase is engineered compared to SEQ ID NO:3 at least in position N161.

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

3. The transaminase according to claim 1 or 2, which is additionally engineered compared to SEQ ID NO:3 in position G51.

4. The transaminase according to any of the preceding claims, 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.

5. The transaminase according to any of the preceding claims, which comprises a substitution selected from the group consisting of N161A, N161C, N161D, N161E, N161G, N161I, N161K, N161L, N161M, N161P, N161Q, N161R, N161S, N161T, N161V, N161W, and N161Y.

6. The transaminase according claim 5, which comprises a substitution selected from the group consisting of N161A, N161F, N161M, N161Y, N161Q, andN161I.

7. The transaminase according to any of the preceding claims, 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.

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

9. The transaminase according to any of the preceding claims, which comprises a substitution selected from the group consisting of G51A, G51C, G51D, G51E, G51F, G51H, G51I, G51K, G51L, G51M, G51N, G51P, G51Q, G51R, G51S, G51T, G51V, G51W, and G51Y.

10. The transaminase according to claim 9, which comprises the substitution G51 S.

11. The transaminase according to any of the preceding claims, wherein the homology to SEQ ID NO:3 is at least 90%.

12. The transaminase according to any of the preceding claims, which is additionally engineered 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, Gl 14, R140, N146, G147,

Y148, Y151, H165, E222, A227, G228, V230, V257, V258, A288, Q300, A349, A353, Q354, Y366, M378, Q391, R415, 1417, K420, 1422, T430, and E433.

The transaminase according to any of the preceding claims, which is capable of catalyzing the conversion of

(i) a ketone substrate according to general formula (I)


to an amine product according to general formula (II)


(ii);

and/or the preferably concomittant conversion of

(ii) an amine cosubstrate according to general formula (III)


(HI)

to a ketone coproduct according to general formula (IV)


(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 Ci-12- hydrocarbon residues; unsubstituted or mono- or polysubstituted C6-io-aromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic Ci-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 Ci-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, =0, -OCi-12-alkyl, -OC6-io-aryl, -O-heteroaryl, -OCOCi-12-alkyl, -OCOCe-io-aryl, -OCO-heteroaryl, -SH, -SCi-12-alkyl, -SCe-io-aryl, -S-heteroaryl, -S(=0)i-2OH, -NO, -NO2, -N3, -NH2, -NH(Ci-i2-alkyl), -N(Ci-i2-alkyl)2, -NH(C6-io-aryl), -N(C6-io-aryl)2, -NH(heteroaryl), -N(heteroaryl)2, -CN, -CHO, -C02H, CO-Ci-2-alkyl, -CO-C6-io-aryl and -CO- heteroaryl.

14. The transaminase according to any of the preceding claims, which is characterized by either

(A) a temperaturestability of at least 50 °C, preferably 55°C, and most preferably of 60°C, and/or

(B) a specific activity of at least 0.5 U/mg, preferably, 075 U/mg, more preferably of 1 U/mg, and most preferably at 1, 1 U/mg in Transaminase Standard Assays; and/or

(C) a high conversion activity under different reaction conditions involving high amine concentrations.

15. The transaminase according to any of the preceding claims, which is engineered compared to SEQ ID NO: 3 in at least one or more positions such that

(A) the stereoselectivity of the engineered transaminase is higher than that of the wildtype transaminase of SEQ ID NO: 3; and/or

(B) the thermostability of the engineered transaminase is higher than that of the wildtype transaminase of SEQ ID NO: 3; and/or

(C) the conversion of the engineered transaminase is higher than that of the wildtype transaminase of SEQ ID NO: 3.

16. The transaminase according to any of the preceding claims, which is engineered compared to SEQ ID NO: 3 in at least one or more positions such that it comprises 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.

The transaminase according to any of the preceding claims, which is engineered compared to SEQ ID NO: 3 in

(I) position N 161 ; and

(II) in addition in at least one other position selected from the group consisting of N7, E9, Ml 6, 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, 1417, K420, 1422, T430, and E433.

18. The transaminase according to any of the preceding claims, which is engineered 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 asN161.

19. The transaminase according to any of the preceding claims, which is engineered 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.

20. The transaminase according to any of the preceding claims, which is engineered 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.

21. The transaminase according to any of the preceding claims, which is engineered compared to SEQ ID NO: 3 in

(i) at least two positions selected from the group consisting of N161 as well as Y164; or N 146 as well as N161; and

(ii) in addition in at least one other position selected from the group consisting of N7, E9, Ml 6, 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, 1417, K420, 1422, T430, and E433.

22. The transaminase according to any of the preceding claims, which is engineered compared to SEQ ID NO: 3 in

(i) at least two positions such that is comprises at least two substitutions selected from the group consiting 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 andN146Y as well as N161Q; and

(ii) in addition in at least one other position that is 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.

23. The transaminase according to any of the preceding claims, which is engineered 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, andN161Q; and

(ii) the second substitution of said at least three substitutions is selected from the group consisting of Y164L, and Yl 641; 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.

24. The transaminase according to any of the preceding claims, which comprises an amino acid sequence of at least 85% homology 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.

25. A method for the conversion of

a ketone substrate according to general formula (I)


to an amine product according to general formula (II)


(ii);

and/or the preferably concomittant conversion of

(ii) a cosubstrate according to general formula (III)


(HI)

to a ketone coproduct according to general formula (IV)


(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 Ci-12- hydrocarbon residues; unsubstituted or mono- or polysubstituted C6-io-aromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic Ci-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 Ci-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, =0, -OCi-12-alkyl, -OC6-io-aryl, -O-heteroaryl, -OCOCi-12-alkyl, - OCOCe-io-aryl, -OCO-heteroaryl, -SH, -SCi-12-alkyl, -SC6-io-aryl, -S-heteroaryl, -NO,

-NO2, -N3, -NH2, -NH(Ci-i2-alkyl), -N(Ci-i2-alkyl)2, -NH(C6-io-aryl), -N(C6-io-aryl)2, - NH(heteroaryl), -N(heteroaryl)2, -CN, -CHO, -C02H, CO-Ci-2-alkyl, -CO-Ce-io-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 a transaminase according to any of claims 1 to 24.