IP Library › Granted Patent US 12,378,533
Granted Patent B2
US 12,378,533 · App. 18/471,524 · Granted Aug 5, 2025

Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

Inventors: Oscar Alvizo (Fremont, CA); Melissa Ann Mayo (Foster City, CA); Jeffrey C. Moore (Westfield, NJ)
Assignee: Codexis, Inc.
C12N9/0028C12P13/001C07K14/195
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,378,533
App. No.
18/471,524
Granted
Aug 5, 2025
Kind
B2
Abstract

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

Claims (22)

1. An engineered polypeptide comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8 and an amino acid residue other than lysine at the position corresponding to position X168 of SEQ ID NO: 8, wherein the engineered polypeptide has improved imine reductase activity as compared to SEQ ID NO: 8.

2. The engineered polypeptide of claim 1 , wherein the amino acid residue at the position corresponding to position X168 of SEQ ID NO:8 is methionine.

3. The engineered polypeptide of claim 2 , further comprising at least one additional substitution at a position corresponding to the position of SEQ ID NO:8 selected from X9, X12, X31, X33, X37, X39, X43, X44, X46, X48, X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153, X154, X155, X156, X159, X166, X167, X173, X177, X178, X184, X185, X195, X197, X198, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218, X220, X221, X223, X224, X226, X234, X239, X242, X245, X251, X253, X256, X257, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272, X273, X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290, X291, X292, X293, X294, X330, X341, and X349, and wherein the engineered polypeptide has improved imine reductase activity as compared to SEQ ID NO:8.

4. The engineered polypeptide of claim 2 , further comprising at least two additional substitutions at positions corresponding to SEQ ID NO:8 and selected from X9, X12, X31, X33, X37, X39, X43, X44, X46, X48, X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153, X154, X155, X156, X159, X166, X167, X173, X177, X178, X184, X185, X195, X197, X198, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218, X220, X221, X223, X224, X226, X234, X239, X242, X245, X251, X253, X256, X257, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272, X273, X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290, X291, X292, X293, X294, X330, X341, and X349, wherein the engineered polypeptide has improved imine reductase activity as compared to SEQ ID NO:8.

5. The engineered polypeptide of claim 1 , wherein the imine reductase activity comprises converting at least one of the following ketone and amine substrate compound pairs to the listed amine product compound under suitable reaction conditions:

(a) ketone substrate compound (1a) and amine substrate compound (2a) to produce compound (3a):

(b) ketone substrate compound (1b) and amine substrate compound (2b) to produce compound (3b);

(c) ketone substrate compound (1c) and amine substrate compound (2c) to produce compound (3c);

(d) ketone substrate compound (1d) and amine substrate compound (2d) to produce compound (3d);

(e) ketone substrate compound (1d) and amine substrate compound (2c) to produce compound (3e);

(f) ketone substrate compound (1d) and amine substrate compound (2c) to produce compound (3f); and

(g) ketone substrate compound (1d) and amine substrate compound (2c) to produce compound (3g)

6. The engineered polypeptide of claim 5 , wherein the imine reductase activity in converting the ketone and amine substrate compound pair to the listed amine product compound under suitable reaction conditions is increased at least 1.2-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS: 8, 360, 550, 872, 1020, 1188 and/or 1220.

7. The engineered polypeptide of claim 1 , in which the amino acid sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 522, 876, 888, 902, 904, 906, 924, 940, 942, 944, 946, 948, 960, 962, 966, 968, 974, 980, 982, 992, 998, 1000, 1010, 1014, 1062, 1082, 1102, 1106, 1168, 1172, 1174, 1178, 1182, 1184, 1188, 1190, 1192, 1194, 1196, 1198, 1200, 1202, 1204, 1206, 1208, 1210, 1212, 1214, 1216, 1218, 1220, 1222, 1224, 1226, 1228, 1230, 1232, 1234, 1236, 1238, 1240, 1242, 1244, 1246, 1248, 1250, 1252, 1254, 1256, 1258, 1260, 1262, 1264, 1266, 1268, 1270, 1272, 1274, 1276, 1278, 1280, 1282, 1284, 1286, 1288, 1290, 1292, 1294, 1296, 1298, and 1300.

8. The engineered polypeptide of claim 1 , further comprising at least one additional substitution at positions corresponding to SEQ ID NO:8 and selected from X9, X12, X31, X33, X37, X39, X43, X44, X46, X48, X55, X56, X65, X82, X93, X98, X108, X137, X138, X141, X142, X143, X153, X154, X155, X156, X159, X166, X167, X173, X177, X178, X184, X185, X195, X197, X198, X199, X200, X201, X203, X205, X206, X210, X213, X215, X216, X218, X220, X221, X223, X224, X226, X234, X239, X242, X245, X251, X253, X256, X257, X259, X260, X261, X262, X263, X265, X266, X267, X268, X269, X272, X273, X274, X276, X277, X278, X279, X280, X281, X282, X283, X284, X285, X289, X290, X291, X292, X293, X294, X330, X341, and X349, wherein the polypeptide has improved imine reductase activity as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS: 360, 550, 872, 1020, 1188, and/or 1220.

9. The engineered polypeptide of claim 5 , wherein the imine reductase activity in converting the ketone and amine substrate compound pair to the listed amine product compound under suitable reaction conditions is increased at least 2-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS: 8, 360, 550, 872, 1020, 1188 and/or 1220.

10. The engineered polypeptide of claim 5 , wherein the imine reductase activity in converting the ketone and amine substrate compound pair to the listed amine product compound under suitable reaction conditions is increased at least 2.5-fold as compared to the corresponding activity of the reference polypeptide of SEQ ID NOS: 8, 360, 550, 872, 1020, 1188 and/or 1220.

11. A polynucleotide encoding the engineered polypeptide of claim 1 .

12. The polynucleotide of claim 11 , in which the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 521, 875, 887, 901, 903, 905, 923, 939, 941, 943, 945, 947, 958, 961, 965, 967, 973, 979, 981, 991, 997, 999, 1009, 1013, 1061, 1081, 1101, 1105, 1167, 1171, 1173, 1177, 1181, 1183, 1187, 1189, 1191, 1193, 1195, 1197, 1199, 1201, 1203, 1205, 1207, 1209, 1211, 1213, 1215, 1217, 1219, 1221, 1223, 1225, 1227, 1229, 1231, 1233, 1235, 1237, 1239, 1241, 1243, 1245, 1247, 1249, 1251, 1253, 1255, 1257, 1259, 1261, 1263, 1265, 1267, 1269, 1271, 1273, 1275, 1277, 1279, 1281, 1283, 1285, 1287, 1289, 1291, 1293, 1295, 1297 and 1299.

13. An expression vector comprising the polynucleotide of claim 11 .

14. A host cell comprising the expression vector of claim 13 .

15. A method of preparing an engineered polypeptide having imine reductase activity, comprising culturing the host cell of claim 14 , under conditions suitable for expression of the engineered polypeptide, optionally further comprising isolating the engineered polypeptide.

Assignments (2)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 8, 2024
From: ALVIZO, OSCAR; MAYO, MELISSA; MOORE, JEFFREY C.; MERCK SHARPE & DOHME CORP.; MERCK SHARP & DOHME CORP.
To: CODEXIS, INC.
Reel/Frame 066420/0143 →
Continuity (7)
Continuation 17739028 · May 6, 2022
Continuation 17010709 · Sep 2, 2020
Continuation 16443250 · Jun 17, 2019
Division 15519949
Provisional Application 62084037 · Nov 25, 2014
Related Publication 20240132856A1 · Apr 25, 2024
Related Publication 20240228982A9 · Jul 11, 2024
References Cited (210)
US 5538867A · Durliat et al. · 1996 [cited by applicant]
US 5605793A · Stemmer · 1997 [cited by applicant]
US 5811238A · Stemmer et al. · 1998 [cited by applicant]
US 5830721A · Stemmer et al. · 1998 [cited by applicant]
US 5834252A · Stemmer et al. · 1998 [cited by applicant]
US 5837458A · Minshull et al. · 1998 [cited by applicant]
US 5928905A · Stemmer et al. · 1999 [cited by applicant]
US 6096548A · Stemmer · 2000 [cited by applicant]
US 6117679A · Stemmer · 2000 [cited by applicant]
US 6132970A · Stemmer · 2000 [cited by applicant]
US 6165793A · Stemmer · 2000 [cited by applicant]
US 6180406B1 · Stemmer · 2001 [cited by applicant]
US 6251674B1 · Tobin et al. · 2001 [cited by applicant]
US 6277638B1 · Stemmer · 2001 [cited by applicant]
US 6287861B1 · Stemmer et al. · 2001 [cited by applicant]
US 6287862B1 · delCardayre et al. · 2001 [cited by applicant]
US 6291242B1 · Stemmer · 2001 [cited by applicant]
US 6297053B1 · Stemmer · 2001 [cited by applicant]
US 6303344B1 · Patten et al. · 2001 [cited by applicant]
US 6309883B1 · Minshull et al. · 2001 [cited by applicant]
US 6319713B1 · Patten et al. · 2001 [cited by applicant]
US 6319714B1 · Crameri et al. · 2001 [cited by applicant]
US 6323030B1 · Stemmer · 2001 [cited by applicant]
US 6326204B1 · delCardayre et al. · 2001 [cited by applicant]
US 6335160B1 · Patten et al. · 2002 [cited by applicant]
US 6335198B1 · delCardayre et al. · 2002 [cited by applicant]
US 6344356B1 · Stemmer · 2002 [cited by applicant]
US 6352859B1 · delCardayre et al. · 2002 [cited by applicant]
US 6355484B1 · Patten et al. · 2002 [cited by applicant]
US 6358740B1 · Patten et al. · 2002 [cited by applicant]
US 6358742B1 · Stemmer · 2002 [cited by applicant]
US 6365377B1 · Patten et al. · 2002 [cited by applicant]
US 6365408B1 · Stemmer · 2002 [cited by applicant]
US 6368861B1 · Crameri et al. · 2002 [cited by applicant]
US 6372497B1 · Stemmer · 2002 [cited by applicant]
US 6376246B1 · Crameri et al. · 2002 [cited by applicant]
US 6379964B1 · delCardayre et al. · 2002 [cited by applicant]
US 6387702B1 · Stemmer · 2002 [cited by applicant]
US 6391552B2 · Stemmer · 2002 [cited by applicant]
US 6391640B1 · Minshull et al. · 2002 [cited by applicant]
US 6395547B1 · Stemmer · 2002 [cited by applicant]
US 6406855B1 · Patten et al. · 2002 [cited by applicant]
US 6406910B1 · Patten et al. · 2002 [cited by applicant]
US 6413745B1 · Patten et al. · 2002 [cited by applicant]
US 6413774B1 · Stemmer · 2002 [cited by applicant]
US 6420175B1 · Stemmer · 2002 [cited by applicant]
US 6423542B1 · Crameri et al. · 2002 [cited by applicant]
US 6426224B1 · Crameri et al. · 2002 [cited by applicant]
US 6436675B1 · Welch et al. · 2002 [cited by applicant]
US 6444468B1 · Stemmer et al. · 2002 [cited by applicant]
US 6455253B1 · Patten et al. · 2002 [cited by applicant]
US 6479652B1 · Crameri et al. · 2002 [cited by applicant]
US 6482647B1 · Stemmer · 2002 [cited by applicant]
US 6489146B2 · Stemmer et al. · 2002 [cited by applicant]
US 6495023B1 · Zeikus et al. · 2002 [cited by applicant]
US 6506602B1 · Stemmer · 2003 [cited by applicant]
US 6506603B1 · Stemmer · 2003 [cited by applicant]
US 6519065B1 · Colbourne et al. · 2003 [cited by applicant]
US 6521453B1 · Crameri et al. · 2003 [cited by applicant]
US 6528311B1 · delCardayre et al. · 2003 [cited by applicant]
US 6537746B2 · Arnold et al. · 2003 [cited by applicant]
US 6573098B1 · Stemmer · 2003 [cited by applicant]
US 6576467B1 · Stemmer · 2003 [cited by applicant]
US 6579678B1 · Patten et al. · 2003 [cited by applicant]
US 6586182B1 · Patten et al. · 2003 [cited by applicant]
US 6602986B1 · Stemmer et al. · 2003 [cited by applicant]
US 6613514B2 · Patten et al. · 2003 [cited by applicant]
US 6653072B1 · Patten et al. · 2003 [cited by applicant]
US 6716631B1 · delCardayre et al. · 2004 [cited by applicant]
US 6946296B2 · Patten et al. · 2005 [cited by applicant]
US 6961664B2 · Selifonov et al. · 2005 [cited by applicant]
US 6995017B1 · Stemmer · 2006 [cited by applicant]
US 7024312B1 · Selifonov et al. · 2006 [cited by applicant]
US 7058515B1 · Selifonov et al. · 2006 [cited by applicant]
US 7105297B2 · Minshull et al. · 2006 [cited by applicant]
US 7148054B2 · delCardayre et al. · 2006 [cited by applicant]
US 7288375B2 · Stemmer et al. · 2007 [cited by applicant]
US 7421347B2 · Selifonov et al. · 2008 [cited by applicant]
US 7430477B2 · Selifonov et al. · 2008 [cited by applicant]
US 7452704B2 · Esaki et al. · 2008 [cited by applicant]
US 7534564B2 · Patten et al. · 2009 [cited by applicant]
US 7620500B2 · Mundorff et al. · 2009 [cited by applicant]
US 7620502B2 · Selifonov et al. · 2009 [cited by applicant]
US 7629170B2 · delCardayre et al. · 2009 [cited by applicant]
US 7702464B1 · Emig et al. · 2010 [cited by applicant]
US 7747391B2 · Gustafsson et al. · 2010 [cited by applicant]
US 7747393B2 · Fox · 2010 [cited by applicant]
US 7751986B2 · Gustafsson et al. · 2010 [cited by applicant]
US 7776598B2 · Patten et al. · 2010 [cited by applicant]
US 7783428B2 · Gustafsson et al. · 2010 [cited by applicant]
US 7795030B2 · Minshull et al. · 2010 [cited by applicant]
US 7853410B2 · Selfinov et al. · 2010 [cited by applicant]
US 7868138B2 · Stemmer et al. · 2011 [cited by applicant]
US 7873499B2 · Selifonov et al. · 2011 [cited by applicant]
US 7904249B2 · Selifonov et al. · 2011 [cited by applicant]
US 7957912B2 · Selifonov et al. · 2011 [cited by applicant]
US 8383346B2 · Colbeck et al. · 2013 [cited by applicant]
US 8504498B2 · Fox · 2013 [cited by applicant]
US 8768871B2 · Fox · 2014 [cited by applicant]
US 8849575B2 · Gustafsson et al. · 2014 [cited by applicant]
US 9487760B2 · Chen et al. · 2016 [cited by applicant]
US 9695451B2 · Chen et al. · 2017 [cited by applicant]
US 10370648B2 · Alvizo et al. · 2019 [cited by applicant]
US 10793836B2 · Alvizo et al. · 2020 [cited by applicant]
US 11352609B2 · Alvizo et al. · 2022 [cited by applicant]
US 11802274B2 · Alvizo · 2023 [cited by examiner]
US 20050095619A1 · Davis et al. · 2005 [cited by applicant]
US 20050153417A1 · Davis et al. · 2005 [cited by applicant]
US 20080220990A1 · Fox · 2008 [cited by applicant]
US 20080318295A1 · Ching et al. · 2008 [cited by applicant]
US 20090093031A1 · Liang et al. · 2009 [cited by applicant]
US 20090155863A1 · Liang et al. · 2009 [cited by applicant]
US 20090162909A1 · Campopiano et al. · 2009 [cited by applicant]
US 20090191605A1 · Liang et al. · 2009 [cited by applicant]
US 20090312196A1 · Colbeck et al. · 2009 [cited by applicant]
US 20100055751A1 · Voladri et al. · 2010 [cited by applicant]
US 20100062499A1 · Mundorff et al. · 2010 [cited by applicant]
US 20110262977A1 · Nagasawa et al. · 2011 [cited by applicant]
US 20130302859A1 · Chen et al. · 2013 [cited by applicant]
US 20140005057A1 · Clark et al. · 2014 [cited by applicant]
US 20140214391A1 · Cope · 2014 [cited by applicant]
US 20140221216A1 · Cope et al. · 2014 [cited by applicant]
US 20150050658A1 · Cho · 2015 [cited by applicant]
US 20150132807A1 · Agard et al. · 2015 [cited by applicant]
US 20150133307A1 · Zhang et al. · 2015 [cited by applicant]
US 20150134315A1 · Sarmiento et al. · 2015 [cited by applicant]
EP 0641862B2 · 2001 [cited by applicant]
WO 9522625A1 · 1995 [cited by applicant]
WO 9533836A1 · 1995 [cited by applicant]
WO 9600787A1 · 1996 [cited by applicant]
WO 970078A1 · 1997 [cited by applicant]
WO 9735966A1 · 1997 [cited by applicant]
WO 9827230A1 · 1998 [cited by applicant]
WO 200042651A1 · 2000 [cited by applicant]
WO 2000053731A2 · 2000 [cited by applicant]
WO 200175767A2 · 2001 [cited by applicant]
WO 2005018579A2 · 2005 [cited by applicant]
WO 2009008908A2 · 2009 [cited by applicant]
WO 2009132921A1 · 2009 [cited by applicant]
WO 2009152336A1 · 2009 [cited by applicant]
WO 2010025238A2 · 2010 [cited by applicant]
WO 2010025287A2 · 2010 [cited by applicant]
WO 2015073555A1 · 2015 [cited by applicant]
Tiwari, S., et al., “Prediction of probable genes by Fourier analysis of genomic sequences,” Comput. Appl. Biosci. 13 (3):263-270 [1997]. [cited by applicant]
Truppo, M.D., et al., “Development of an Improved Immobilized CAL-B for the Enzymatic Resolution of a Key Intermediate to Odanacatib,” Organic Process Research & Development, 15:1033-1035 (2011). [cited by applicant]
Uberbacher, E.C., et al., “Discovering and Understanding Genes in Human DNA Sequence Using GRAIL,” Methods Enzymol., 266:259-281 [1996]. [cited by applicant]
Vaijayanthi, T., et al., “Asymmetric reduction of aryl imines using Candida parapsilosis ATCC 7330,” Tetrahedron: Asym., 19:93-96 [2008]. [cited by applicant]
Villa-Komaroff, L., et al., “A bacterial clone synthesizing proinsulin,” Proc. Natl Acad. Sci. USA, 75:3727-3731 (1978). [cited by applicant]
Wada, K., et al., “Codon usage tabulated from the GenBank genetic sequence data,” Nucl. Acids Res., 20:2111-2118 [1992]. [cited by applicant]
Wells, J.A., et al., “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites,” Gene, 34:315-323 (1985). [cited by applicant]
Wetmur, J. G., “Dna Probes: Applications of the Principles of Nucleic Acid Hybridization,” Crit Rev Biochem Mol Biol, 26(3/4):227-259 (1991). [cited by applicant]
Wright, F., “The ‘effective number of codons’ used in a gene,” Gene 87:23-29 [1990]. [cited by applicant]
Yi, S., et al., “Covalent immobilization of omega-transaminase from Vibrio fluvialis JS17 on chitosan beads,” Process Biochemistry 42(5): 895-898 (2007). [cited by applicant]
Zhang, J-H., et al., “Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening,” Proc. Nat. Acad. Sci., U.S.A., 94:4504-4509 (1997). [cited by applicant]
Zhao, H., et al., “Molecular evolution by staggered extension process (StEP) in vitro recombination,” Nat. Biotechnol., 16:258-261 (1998). [cited by applicant]
Kohls, H., et al., “Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis,” Current Opinion in Chemical Biology, 19:180-192 [2014]. [cited by applicant]
Scheller, P.N., et al., “Enzyme Toolbox: Novel Enantiocomplementary Imine Reductases,” Chembiochem—A European Journal of Chemical Biology, 15(15):2201-2204 [2014]. [cited by applicant]
UniProt Accession No. Q44297 dated Jul. 15, 1998. [cited by applicant]
Singh, R.K., et al., “Protein Engineering Approaches in the Post-Genomic Era,” Current Protein and Peptide Science, 18:1-11 [2017]. [cited by applicant]
Mitsukura, K., et al., “A NADPH-dependent (S)-imine reductase (SIR) from [cited by applicant]
Altschul, S., et al., “Basic local alignment search tool,” J. Mol. Biol., 215: 403-410 (1990). [cited by applicant]
Altschul, S.F., et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res., 25(17):3389-3402 (1997). [cited by applicant]
Baldino, Jr., F., et al., “High-Resolution in Situ Hybridization Histochemistry,” Methods Enzymology, 168:761-777 (1989). [cited by applicant]
Batzer, M.A., “Erratum: Structure and variability of recently inserted Alu family members”, Nucleic Acids Res 19:698-699 [1991]. [cited by applicant]
Beaucage, S.L., et al., “Deoxynucleoside phosphoamidites—A new class of key intermediates for deoxypolynucleotide synthesis,” Tetrahedron Letters, 22(20):1859-62 (1981). [cited by applicant]
Black, M.E., et al., “Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy,” Proc Natl Acad Sci USA, 93:3525-3529 (1996). [cited by applicant]
Bolton, E.T., et al., “A General Method for the lisolation of RNA Complementary to DNA,” Proc. Natl. Acad. Sci. USA 48:1390 ( 1962). [cited by applicant]
Botstein, D., et al., “Strategies and applications of in vitro mutagenesis,” Science, 229(4719):1193-1201, 1985. [cited by applicant]
Breslauer, K.J., et al., “Predicting DNA duplex stability from the base sequence,” Proc. Natl. Acad. Sci. USA, 83:3746-3750 (1986). [cited by applicant]
Britton, K.L., et al., “Crystal structure and active site location of N-(1-D-carboxyethyl)-L-norvaline dehydrogenase,” Nat. Struct. Biol., 5(7): 593-601 [1998]. [cited by applicant]
Caldwell, R.C., et al., “Mutagenic PCR,” PCR Methods Appl., 3:S136-S140 (1994). [cited by applicant]
Carter, P., “Site-directed mutagenesis,” Biochem. J., 237:1-7 (1986). [cited by applicant]
Christians, F.C., et al., “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling,” Nat. Biotechnol., 17:259-264 (1999). [cited by applicant]
Crameri, A., et al., “DNA shuffling of a family of genes from diverse species accelerates directed evolution”, Nature, 391:288-291 (1998). [cited by applicant]
Crameri, A., et al., “Improved green fluorescent protein by molecular evolution using DNA shuffling, ”Nat. Biotechnol., 14(3):315-319 (1996). [cited by applicant]
Crameri, A., et al., “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nat. Biotechnol., 15 (5):436-438 (1997). [cited by applicant]
Dairi, T., et al. “Cloning, nucleotide sequencing, and expression of an opine dehydrogenase gene from [cited by applicant]
Dale, S.J., et al., “Oligonucleotide-directed random mutagenesis using the phosphorothioate method,” Methods Mol. Biol., 57:369-74 (1996). [cited by applicant]
De Boer, H.A., et al., “The tac promoter: A functional hybrid derived from the trp and lac promoters,” Proc. Natl Acad. Sci. USA, 80: 21-25 (1983). [cited by applicant]
Fasman, G.D.,CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70 [1989]. [cited by applicant]
Freier, S.M., et al., “Improved free-energy parameters for predictions of RNA duplex stability,” Proc. Natl. Acad. Sci USA, 83:9373-9377 (1986). [cited by applicant]
Green, A. P., et al., “Chiral Amine Synthesis Using ω-Transaminases: An Amine Donor that Displaces Equilibria and Enables High-Throughput Screening,” Angew. Chem, Int. Ed., 53: 10714-10717 [2014]. [cited by applicant]
Guo, Z., et al., “3′-End-Forming Signals of Yeast mRNA,” Mol. Cell. Biol., 15(11):5983-5990 [1995]. [cited by applicant]
Henaut and Danchin in Neidhardt et al. [eds.], [cited by applicant]
Henikoff, S.,et al., “Amino acid substitution matrices from protein blocks,” Proc. Natl. Acad. Sci., 89:10915-10919 (1992). [cited by applicant]
Kierzek, R., et al., “Polymer-Supported RNA Synthesis and Its Application To Test the Nearest-Neighbor Model for Duplex Stability,” Biochemistry, 25:7840-7846 (1986). [cited by applicant]
Koszelewski, D., et al., “Immobilization of omega-transaminases by encapsulation in a sol-gel/celite matrix,” Journal of Molecular Catalysis B: Enzymatic, 63: 39-44 (2010). [cited by applicant]
Kramer, B., et al., “Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of [cited by applicant]
Li, H., et al., “A dynamic combinatorial screen for novel imine reductase activity,” Tetrahedron, 60(3):753-758 [2004]. [cited by applicant]
Ling, M., et al., “Approaches to DNA Mutagenesis:An Overview,” Anal. Biochem., 254:157-78 (1997). [cited by applicant]
Martin, A.R., et al., “Characterization of free and immobilized (S)-aminotransferase for acetophenone production,” Applied Microbiology and Biotechnology, 76(4): 843-851 (2007). [cited by applicant]
Mateo, C., et al., “Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment,” Biotechnology Progress 18(3):629-34 (2002). [cited by applicant]
Matthes, H.W.D., et al., “Simultaneous rapid chemical synthesis of over one hundred oligonucleotides on a microscale,” Embo J., 3(4):801-05 (1984). [cited by applicant]
McInerney, J.O., “GCUA: general codon usage analysis,” Bioinformatics, 14(4):372-73, 1998. [cited by applicant]
Mihara, H., et al., “N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily,” FEBS J., 272(5): 1117-1123 [2005]. [cited by applicant]
Minshull, J., et al., “Protein evolution by molecular breeding,” Curr. Op. Chem. Biol., 3(3):284-290 (1999). [cited by applicant]
Mitsukura, K., et al., “Asymmetric synthesis of chiral cyclic amine from cyclic imine by bacterial whole-cell catalyst of enantioselective imine reductase,” Org. Biomol.Chem., 8(20):4533-4535 [2010]. [cited by applicant]
Nakamura, Y., et al., “Codon usage tabulated from international DNA sequence databases: status for the year 2000,” Nucl. Acids Res., 28:292 [2000]. [cited by applicant]
Needleman, S., et al., “A general method applicable to the search for similarities in the amino acid sequence of two proteins,” J. Mol. Biol. 48:443-453 (1970). [cited by applicant]
Pearson, W.R., “Improved tools for biological sequence comparison,” Proc. Nat'l. Acad. Sci. USA, 85:2444-2448 (1988). [cited by applicant]
Romanos, M.A., et al., “Foreign gene expression in yeast: a review,” Yeast 8:423-488 [1992]. [cited by applicant]
Rychlik, W., et al., “Optimization of the annealing temperature for DNA amplification in vitro,” Nucleic Acids Res, 18 (21):6409-6412 (1990). [cited by applicant]
Simonen, M., et al., “Protein Secretion in Bacillus Species,” Microbiological Reviews, 57:109-137 (1993). [cited by applicant]
Smith, M., “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462 (1985). [cited by applicant]
Smith, T., et al., “Comparison of Biosequences,” Adv. Appl. Math, 2:482-489 (1981). [cited by applicant]
Stellwagon, E., “Dye Affinity Chromatography,” Current Protocols in Protein Science, Unit 9.2-9.2.16 [2001]. [cited by applicant]
Stemmer, W., “DNA Shuffling by Random Fragmentation and Reassembly: In vitro Recombination for Molecular Evolution,” Proc. Natl. Acad. Sci. USA, 91:10747-10751 (1994). [cited by applicant]
Stemmer, W.P.C., “Rapid evolution of a protein in vitro by DNA shuffling”, Nature, 370:389-391 (1994). [cited by applicant]
Stenico, M., et al., “Codon usage in Caenorhabditis elegans: delineation of translational selection and mutational biases,” Nucl. Acids Res. 22(13):2437-46 [1994]. [cited by applicant]
Suggs, S.V., et al., “Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences,” In Developmental Biology Using Purified Genes (Brown et al., eds.), pp. 683-693, Academic Press (1981… [cited by applicant]