TRANSAMINASES

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

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 and/or in position Y164.

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

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

4. 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.

5. 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.

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

7. 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 or which comprises a substitution selected from the group consisting of G51A, G51C, G51D, G51E, G51F, G51H, G51I, G51K, G51L, G51M, G51N, G51 P, G51Q, G51R, G51S, G51T, G51V, G51W, and G51Y.

8. 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.

9. (canceled)

10. (canceled)

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

12. The transaminase according to claim 1, 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, 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.

13. (canceled)

14. 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.

15. (canceled)

16. 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 engineered 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.

17. (canceled)

18. The transaminase according to claim 1, 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 as N161.

19. The transaminase according to claim 1, 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-r.

20. The transaminase according to claim 1, 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 claim 1, 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 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.

22. The transaminase according to claim 1, which is engineered 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.

23. The transaminase according to claim 1, 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, 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.

24. The transaminase according to claim 1, 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 (i) a ketone substrate according to general formula (I) ##STR00019## to an amine product according to general formula (II) ##STR00020## and/or the preferably concomittant conversion of (ii) a cosubstrate according to general formula (III) ##STR00021## to a ketone coproduct according to general formula (IV) ##STR00022## 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 C.sub.1-12-hydrocarbon residues; unsubstituted or mono- or polysubstituted C.sub.6-10-aromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted heteroaromatic hydrocarbon residues, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-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, OC.sub.1-12-alkyl, OC.sub.6-10-aryl, O-heteroaryl, OCOC.sub.1-12-alkyl, OCOC.sub.6-10-aryl, OCO-heteroaryl, SH, SC.sub.1-12-alkyl, SC.sub.6-10-aryl, S-heteroaryl, S(O).sub.1-2OH, NO, NO.sub.2, N.sub.3, NH.sub.2, NH(C.sub.1-12-alkyl), N(C.sub.1-12-alkyl).sub.2, NH(C.sub.6-10-aryl), N(C.sub.6-10-aryl).sub.2, NH(heteroaryl), N(heteroaryl).sub.2, CN, CHO, CO.sub.2H, COC.sub.1-2-alkyl, COC.sub.6-10-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 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 and/or in position Y164.

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

Description

EXAMPLE 1: IDENTIFICATION OF A NEW ATA GENE CORRESPONDING TO SEQ ID NO:1

[0363] The gene of the new aminotransferase was detected during a screening for new aminotransferases by selective enrichment of wild type strains expressing aminotransferases. Soil samples from different habitats were randomly collected and five grams of each sample were suspended in 20 ml of 0.9% NaCl. A non-selective rich medium TSB (15 g/L peptone from casein, 5 g/L peptone from soymeal, 5 g/L NaCl) was inoculated by these suspensions and incubated at 30 C. for several hours for obtaining a first pre-culture. This first pre-culture has been used to inoculate a second pre-culture in defined minimal medium MA (50 mM potassium phosphate pH 7, 100 mM glycerol, 0.2 mM CaCl.sub.2, 4 mM MgSO.sub.4, 1 g/L NH.sub.4Cl, 10 ml/L 100 trace element stock solution [10 mg/L ZnCl.sub.2, 10 mg/ml MnSO.sub.44 H.sub.2O, 2 mg/L H.sub.3BO.sub.3, 10 mg/L CuSO.sub.45 H.sub.2O, 5 mg/L CoCl.sub.2, 10 mg/L NiSO.sub.46 H.sub.2O, 200 mg/L Na.sub.2MoO.sub.4, 400 mg/L FeSO.sub.47 H.sub.2O]). After overnight cultivation this second pre-culture was washed with 0.9% NaCl for remove of residual sources of nitrogen. The washed cells were used to inoculate a selective enrichment medium consisting of the defined minimal medium MA where the original nitrogen-source has been replaced by a collection of different amines (e.g. R/S--Methylbenzylamine or R/S-1-Methyl-3-phenylpropylamine). The amines have been added at concentrations of 50 mM. Enrichment has been ensured by repetitive dilution of grown culture into new enrichment medium. After third dilution of enriched cultures, the enriched cells were plated on agar plates containing enrichment medium MA with the respective nitrogen source in order to select single clones. Single clones have been further verified by liquid cultivation in enrichment medium as well as by direct detection of transaminase activity.

[0364] Transaminase activity was detected by culturing selected strains in 400 l half concentrated LB medium for two days at 30 C. Cells were harvested by centrifugation and resuspended in 200 l substrate solution consisting of 5 mM 4-phenyl-2-butanone, 100 mM 1-phenylethan-1-amine (MBA), 50 mM potassium phosphate pH 7.4, and 1 mM pyridoxalphosphate. The reaction went overnight at 30 C., was stopped by adding 200 l methanol, and analyzed by HPLC as described in Example 3.

[0365] Identification of the corresponding transaminase genes, genomic libraries have been built from active strains. The DNA of selected microorganisms grown in a 96-well format was isolated, mechanically fragmented to the desired size range, and cloned into pF2F4 (WO2010/075956 A1). The resulting plasmids were transformed to E. coli BL21(DE3)placI(+) cells. Screening of the library was done with cluster screening (WO2005/040376 A2) with cluster sizes of about 300, 000 to 350, 000 clones per plate also by selective enrichment.

[0366] For expression of the genomic library cells were cultivated in defined minimal medium supplemented with kanamycin (50 mg/1) and chloramphenicol (34 mg/1)). Expression of the genes of the genomic library was induced at logarithmic phase either by IPTG (0.5 mM) or arabinose (0.1 (v/v)). Cultivations were carried out at 30 C. for 16 hours after which cells were washed with 0.9% NaCl remove the residual sources of nitrogen. Washed cells were cultivated in defined minimal medium with the original nitrogen-source being replaced by the respective amine for enrichment. Enrichment was accomplished in three repetitive dilutions. After third dilution plasmids have been purified from grown enriched cultures and the transaminase gene sequences have been determined.

EXAMPLE 2: EXPRESSION OF A NEW ATA GENE CORRESPONDING TO SEQ ID NO:1 AND 2

[0367] The newly found ATA gene sequences corresponding to SEQ ID NO:1 was codon optimized for E. coli expression while simultaneously decreasing the GC-content, corresponding to SEQ ID NO:2. Both genes according to SEQ ID NO:1 and SEQ ID NO:2 were cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmids were used for transformation of E. coli BL21(DE3) cells.

[0368] For expression of the new ATA corresponding to SEQ ID NO:1 and SEQ. ID NO:2 cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37 C. Expression of the gene was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30 C. for 16-18 hours.

[0369] Cells were harvested by centrifugation (3220g, 20 min, 4 C.) and disrupted by resuspending corresponding to an optical density, measured at 600 nm (OD.sub.600) of 100 with cell lysis buffer (50 mM Tris-HCl pH 7.0; 2 mM MgCl2, 1 CelLytic B (Sigma); DNA nuclease 0.02 U, lysozyme 0.5 mg/ml). The crude extracts were separated from cell debris by centrifugation (3220g 30 min, 4 C.), resulting in enzymatic active preparations of the ATA of SEQ ID NO:3. For detection of the enzymatic activity of an ATA of the invention, a lyophilisate of the active preparation of the ATA may be obtained. The crude extract lyophilisate was investigated regarding the ATA activity using the Transaminase Standard Assay.

[0370] The Transaminase Standard Assay monitors the conversion of racemic 1-phenylethan-1-amine (MBA) and pyruvate to 1-phenylethanone (Acetophenone) and L-alanine. The reaction is performed at 30 C. in 50 mM phosphate buffer (pH 7, 4) and 0.1 mM pyridoxalphosphate (PLP) using 10 mM racemic 1-phenylethan-1-amine and 10 mM sodium-pyruvate as substrates. The production of 1-phenylehanone is followed photometrically at 300 nm. One unit (U) liberates 1 mol 1-phenylethanone (Acetophenone) per minute. Specific activity refers to units per milligram crude extract lyophilisate (U/mg)

EXAMPLE 3: CHARACTERIZATION OF ENZYMATIC PROPERTIES OF NEW ATA OF SEQ ID NO:3

[0371] An enzymatic active preparation of ATA of SEQ ID NO:3 was prepared as described in Example 2. For comparison of its enzymatic properties, enzymatic active preparations of several other omega-transaminases were prepared in a similar way. These include omega-transaminase well known in the literature (J. S. Shin et al., Appl. Microbiol. Biotechnol. 2003, 61 463-471. WO 2010/081053. J. H. Seo, et al. Biotechnol. J 3 (5):676-686, 2008. S. Schatzle, et al. Anal. Chem. 81 (19):8244-8248, 2009.).

[0372] The enzymatic activity of these enzymes was analyzed with the Transaminase Standard Assay as described in Example 2. Furthermore, the thermostability of each enzyme was analyzed as described in Example 5.

TABLE-US-00002 Activity (Transaminase No. of Tm (80%) standard assay Example 2) Entry transaminase origin mutations [ C.] [U/mg] seq reference 1 Example 1 none, 60 1.1 SEQ ID NO: 3 wildtype 2 Vibrio fluvialis none, 61 0.1 WP_040602310 wildtype 3 Agrobacterium none, 57 0.04 WP_010972924 tumefaciens wildtype 4 Rhodobacter none, 53 0.05 WP_002720543 spheroides wildtype 5 Bradyrhizobium none, 57 0.1 WP_011086907 wildtype 6 Arthrobacter citreus One 57 0.2 SEQ ID NO: 148 7 SEQ ID NO: 147 10 59 0.1 SEQ ID NO: 147

[0373] Additionally the conversion properties of these enzymes were investigated in Transaminase Conversion Assays.

[0374] Transaminase Conversion Assays monitors enzyme properties at different reaction conditions that are relevant for a preparative synthesis application at high concentration of different amine donors, isopropylamine (IPA), racemic 1-phenylethan-1-amine (MBA) or (S)-1-phenylethan-1-amine (S-MBA) or racemic alanine, respectively. The ketone acceptor was 4-phenyl-2-butanone (BA). The reaction was performed at 30 C. in 50 mM phosphate buffer (pH 7, 4) and 0.1 mM pyridoxalphosphate (PLP).

[0375] Conversion from BA to 1-methyl-3-phenylpropylamine was analyzed by HPLC after a given time of reaction, typically 6 h or 20 h. Analytical conditions are: [0376] Column: Gemini 5 C18, 1504.6 mm (Phenomenex); [0377] Eluents: A) dH2O, 0.1% trifluoroacetic acid (TFA); B) Acetonitrile, 0.1% TFA; [0378] Flow: 1 ml/min; gradient: 20% B to 80% B in 6 min, hold for 1 min, to 20% B in 1 min, hold for 3 min; [0379] Oven temperature: 35 C.; [0380] Detection: 210 nm. [0381] The retention times of the analytes are 6.52 min for BA and 3.65 min for 1-methyl-3-phenylpropylamine.

[0382] Overall, Transaminase Conversion Assays may be done at conditions differing in the type of amine donor (IPA, MBA, and alanine) and the respective concentration of the reactants.

Condition A: 50 mM BA, 100 mM IPA; after 6 h
Condition B: 50 mM BA, 200 mM rac. MBA; after 6 h
Condition C: 50 mM BA, 500 mM IPA; after 6 h
Condition D: 50 mM BA, 1000 mM rac. MBA; after 6 h
Condition E: 50 mM BA, 200 mM rac. alanine; after 6 h
Condition F: 10 mM BA, 50 mM IPA; after 6 h
Condition G: 150 mM BA, 300 mM IPA; after 20 h
Condition H: 150 mM BA, 300 mM (S)-MBA; after 20 h
Condition I: 150 mM BA, 1000 mM IPA; after 20 h
Condition J: 50 mM BA, 1000 mM IPA; after 20 h

[0383] The transaminases under this example were analyzed at the Conditions A, B, C, D, and E.

TABLE-US-00003 % conversion % conversion % conversion % conversion % conversion Entry transaminase origin cond. A cond. B cond. C cond. D cond. E 1 see Example 1 11.9 71.0 25.8 3.7 3.2 2 Vibrio fluvialis 5.2 1.7 3.3 0.0 1.6 3 Agrobacterium 0.3 0.0 0.0 0.0 0.7 tumefaciens 4 Rhodobacter 2.8 0.6 2.0 0.0 2.0 spheroides 5 Bradyrhizobium 5.0 2.1 1.0 0.0 1.8 6 Arthrobacter 15.9 8.2 27.0 0.0 1.6 citreus 7 SEQ ID NO: 147 33.7 88.8 70.7 54.8 1.6

[0384] Among the wild type enzymes analyzed, the new transaminase enzyme SEQ ID NO:3 shows the highest transaminase standard activity and the best properties with respect to substrate conversion at the different process relevant conditions.

EXAMPLE 4: ANALYSIS OF STEREOSELECTIVITY OF ENGINEERED ATAS

[0385] Several ATA variants that had been engineered in comparison to SEQ ID NO:3 were analyzed regarding their stereoselectivity in the formation of (S)-1-methyl-3-phenylpropylamine. For analysis of stereoselectivity, Transaminase Conversion Assays with each ATA were performed as described in Example 3 under Condition F.

[0386] The products were analyzed by chiral HPLC analysis. Analytical conditions are: [0387] Column: chiralpak IB (Daicel); [0388] Eluent: 98% n-hexane, 2% isopropanol, 0.1% ethylenediamine; [0389] Flow 1 ml/min; [0390] Oven temperature: 35 C.; [0391] Detection: 267 nm. [0392] The retention times of the analytes are 5.6 min for (R)-1-methyl-3-phenylpropylamine and 5.9 min for (S)-1-methyl-3-phenylpropylamine.

[0393] Calculation of the enantiomeric excess (% ee) of the (S) enantiomer is done by integrating the peak areas of the enantiomers using the formula:

% ee=[(S)enantiomer(R)enantiomer]/[(S)enantiomer+(R)enantiomer]

[0394] It was found, that the engineered ATAs showed an improved stereoselectivity in comparison to the wild type ATA SEQ ID No:3: The stereoselectivities of mutants are listed in the table below.

TABLE-US-00004 SEQ ID NO % ee Mutation 1 Mutation 2 Mutation 3 Mutation 4 3 59 96 99.1 I417V I422S 97 97.8 I417V I422C 98 95.3 I417A I422S 99 88.8 I417F I422V 100 78.0 I417A I422C 101 97.1 N161M Y164L 102 96.6 N161I Y164L 103 94.2 N161Q Y164L 104 92.6 N161I Y164M 105 90.3 N161M Y164M 106 89.4 N161Q Y164M 107 87.7 N161I Y164F 108 67.2 N146Y N161Q 109 93.1 G51S N161I Y164L 110 96.2 N161I Y164L A288G 111 93.5 A67N N161I Y164L 112 93.5 Q44R N161I Y164L 113 95.6 A68P N161I Y164L 114 94.4 N161I Y164L A349G 115 93.6 N161I Y164L T430N 116 93.3 N161I Y164L K420H 117 93.2 N161I Y164L Q300E 118 92.6 L73M N161I Y164L 119 92.4 G33Y N161I Y164L 120 91.8 N161I Y164L K420S 121 91.2 Q44N N161I Y164L 122 90.5 N161I Y164L K420N 144 75.3 M16W N161I Y164L V230A 145 70.3 M16F N161M Y164I V230A 146 95.8 M16W W54A N161Q Y164L

EXAMPLE 5: DETECTION OF THERMOSTABILITY OF ENGINEERED ATAS

[0395] Several ATA variants that had been engineered in comparison to SEQ ID NO:3 were analyzed regarding their thermostability. Crude extracts of a respective ATA were obtained as described in Example 2. Melting profiles of these active enzymatic active preparations of a respective ATA were recorded by incubation the crude extract for 15 minutes at different temperatures in a PCR cycler. Afterwards the crude extracts were incubated on ice for 30 minutes. Insoluble proteins were separated by centrifugation and the supernatants were analyzed regarding their remaining ATA activity in a Transaminase Standard Assay as described in Example 2.

[0396] Thermostability was expressed as the temperature at which 80% of the initial activity of the ATA variant remains after 15 min of incubation [Tm(80%)]. The initial activity is the activity of the respective ATA variant without any high temperature treatment, i.e. with 15 min incubation on ice instead of incubation at different temperatures in a PCR cycler.

[0397] It was found, that the engineered ATAs showed an improved thermostability in comparison to the wild type ATA SEQ ID No:3: The melting temperatures of mutants are listed in the table below.

TABLE-US-00005 Thermostability SEQ ID T.sub.m (80%) Mutation Mutation Mutation Mutation NO [ C.] 1 2 3 4 3 61 4 62 M16F 16 67 W54A 47 65 Y164L 48 62 Y164M 87 62 I417F 101 77 N161M Y164L 102 78 N161I Y164L 103 67 N161Q Y164L 104 73 N161I Y164M 105 71 N161M Y164M 106 62 N161Q Y164M 107 73 N161I Y164F 108 67 N146Y N161Q 109 81 G51S N161I Y164L 110 76 N161I Y164L A288G 123 79 R45K N161I Y164L 124 79 N161I Y164L Q391K 125 79 N161I Y164L Y366H 126 79 N161I Y164L Q354F 127 75 N161I Y164L E433D 146 72 M16W W54A N161Q Y164L

EXAMPLE 6: EVALUATION FOR IMPROVED CONVERSION UNDER RELEVANT PROCESS CONDITIONS

[0398] Several ATA variants that had been engineered in comparison to SEQ ID NO:3 were analyzed in Transaminase Conversion Assays for properties relevant for a preparative synthesis application at high concentration of amine donors, isopropylamine (IPA) and racemic 1-phenylethan-1-amine (MBA), respectively. Reactions were performed and analysed for conversion as described in Example 3, with analysis of conversion after a reaction time of 20 h. For the reaction 20% of the total reaction volume was crude extract of the respective ATA, obtained as described in Example 2. Different Conditions were used varying the concentrations of reactants and type of amino donor as indicated for each table.

TABLE-US-00006 SEQ Conversion ID Number Condition G NO Mutations Mutation 1 Mutation 2 Mutation 3 Mutation 4 [%] Conversion Condition H [%] 3 5 34 101 2 N161M Y164L 8 52 102 2 N161I Y164L 8 57 103 2 N161Q Y164L 9 53 104 2 N161I Y164M 6 44 105 2 N161M Y164M 8 51 106 2 N161Q Y164M 7 47 107 2 N161I Y164F 5 41 146 4 M16W W54A N161Q Y164L 6 37 SEQ ID NO Mutation 1 Mutation 2 Mutation 3 Conversion Condition I [%] Conversion Condition J [%] 3 0 0 102 N161I Y164L 49 89 109 G51S N161I Y164L 66 88 110 N161I Y164L A288G 68 78 111 A67N N161I Y164L 65 91 112 Q44R N161I Y164L 63 90 113 A68P N161I Y164L 47 86 114 N161I Y164L A349G 51 87 115 N161I Y164L T430N 51 88 116 N161I Y164L K420H 52 86 117 N161I Y164L Q300E 56 88 118 L73M N161I Y164L 58 90 119 G33Y N161I Y164L 56 89 120 N161I Y164L K420S 46 86 121 Q44N N161I Y164L 54 90 122 N161I Y164L K420N 45 86 123 R45K N161I Y164L 55 88 124 N161I Y164L Q391K 50 88 125 N161I Y164L Y366H 49 84 126 N161I Y164L Q354F 44 85 127 N161I Y164L E433D 57 92 128 N161I Y164L A353R 61 87 129 N161I Y164L Y366F 61 88 130 Q44H N161I Y164L 61 89 131 V29L N161I Y164L 59 87 132 H87T N161I Y164L 58 90 133 E9R N161I Y164L 57 88 134 G147S N161I Y164L 56 87 135 N7L N161I Y164L 55 88 136 R140K N161I Y164L 55 87 137 N161I Y164L H165R 54 88 138 H87N N161I Y164L 53 89 139 V29I N161I Y164L 53 92 140 A71G N161I Y164L 52 92 141 G114S N161I Y164L 51 88 142 V109I N161I Y164L 51 86 143 N161I Y164L Q391E 47 83

EXAMPLE 7 CHARACTERIZATION OF ENZYMATIC PROPERTIES OF ENGINEERED ATA OF SEQ ID NO:148

[0399] An enzymatic active preparation of ATA of SEQ ID NO:148 and SEQ ID NO147 were prepared as described in Example 2. Analysis of their properties in the Transaminase Standard Assay has been shown in Example 3

[0400] Active enzyme preparations of SEQ ID NO:147 and SEQ ID NO:148 have been further subjected to Transaminase Conversion Assays as described in Example 3, but at conditions:

Condition K (100 mM BA; 500 mM rac. MBA), or
Condition L (200 mM BA; 500 mM rac. MBA), or

Condition M (10 g/L BA, 900 mM IPA)

[0401]

TABLE-US-00007 transaminase % conversion % conversion % conversion T.sub.m (80%) Entry origin cond. K cond. L cond. M [ C.] ee % 1 SEQ ID NO: 148 0.0 0.0 0.0 51 2 SEQ ID NO: 147 74 53 80 58 >99