IP Library › Granted Patent US 12,312,609
Granted Patent B2
US 12,312,609 · App. 17/343,058 · Granted May 27, 2025

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

Inventors: Nicholas J. Agard (San Francisco, CA); Oscar Alvizo (Fremont, CA); Melissa Ann Mayo (Foster City, CA); Stefanie Ng Minor (Redwood City, CA); James Nicholas Riggins (San Francisco, CA); Jeffrey C. Moore (Westfield, NJ)
Assignee: Codexis, Inc.
C12N9/0028C12P13/001C12P17/10C12Y105/01028
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Quick Facts
Patent No.
US 12,312,609
App. No.
17/343,058
Granted
May 27, 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 (49)

1. An engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO:6 and two or more residue differences as compared to SEQ ID NO:6 selected from X153Y, X283V, X82P, X141W, X154F, X259I/L/M, X274L/M, and X296N/V;

wherein the polypeptide has imine reductase activity.

2. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one residue difference as compared to SEQ ID NO:6 selected from X12M, X37P, X111A, X156N/M, X223S, X256E, X260D, X261H, X262P, X263C/E/Q, X267G, X277L, X281A, X284P/S, and X292E.

3. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X256E, X93G/Y, X94N, X96C, X111A/H, X142A, X159L, X163V, X259R, X273C, and X284P/S.

4. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises at least two residue differences as compared to the reference sequence of SEQ ID NO:6 selected from X82C/P/T, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T, X260G, X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N, X326V.

5. The engineered polypeptide of claim 1 , wherein the amino acid sequence comprises at least a combination of residue differences as compared to the reference sequence of SEQ ID NO:6 selected from:

(a) X153Y, and X283V;

(b) X141W, X153Y, and X283V;

(c) X141W, X153Y, X274L/M, and X283V;

(d) X141W, X153Y, X154F, X274L/M, and X283V;

(e) X141W, X153Y, X154F, and X283V;

(f) X141W, X153Y, X283V, and X296N/V;

(g) X141W, X153Y, X274L/M, X283V, and X296N/V:

(h) X111A, X153Y, X256E, X274M, and X283V;

(i) X111A, X141W, X153Y, X273C, X274M, X283V, and X284S;

(j) X111A, X141W, X153Y, X273C, and X283V;

(k) X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N;

(l) X111A, X141W, X153Y, X256E, X273W, X274L, X283V, X284S, and X296N;

(m) X111H, X141W, X153Y, X273W, X274M, X284S, and X296N;

(n) X111H, X141W, X153Y, X154F, X273W, X274L, X283V, X284S, and X296N;

(o) X82P, X141W, X153Y, X256E, X274M, and X283V;

(p) X82P, X111A, X141W, X153Y, X256E, X274M, X283V, M284S, and E296V;

(q) X94N, X143W, X159L, X163V, X259M, and X279L;

(r) X141W, X153Y, X154F, and X256E; and

(s) X153Y, X256E, and X274M.

6. The engineered polypeptide of claim 5 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V, X651/V, X74W, X82C/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S, X138L, X140M, X141M/N, X142A, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N, X254R, X256A/E/I/L/S/T, X257Q, X259C/R, X260A/D/N/Q/V/Y, X261E/F/H/L/P/Q/Y, X262P, X262F/G/V, X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V, X267E/G/H/I/N/S, X270L, X272D, X273C, X274L/S, X276L, X277H/L, X278E/H/K/N/R/S/W, X279T, X281A, X282A/R, X284C/F/H/P/Q/S, X291E, X292E/P, X295F, and X352Q.

7. The engineered polypeptide of claim 5 , wherein the amino acid sequence further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:6 selected from X12M, X37P, X82T, X111A, X141W, X153Y, X154F/S, X156N/M, X223S, X256E, X259I, X260D, X261H, X262P, X263C/E/Q, X267G, X274M, X277L, X281A, X283V, X284P/S, X292E, and X296N.

8. The engineered polypeptide of claim 1 , wherein the amino acid sequence comprises the residue differences X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N and at least residue difference or a combination of residue differences as compared to the reference sequence of SEQ ID NO:6 selected from:

(a) X156N;

(b) X37P, X82T, and X156N;

(c) X37P, X82T, X156N, and X259I;

(d) X259L/M;

(e) X82T, X156N, X223S, X259L, X267G, and X281A;

(f) X263C;

(g) X12M, X261H, X263C, X277L, and X292E;

(h) X154S; and

(i) X154S, X156M, X260D, X261H, X262P, X263E, and X284P.

9. The engineered polypeptide of claim 1 , wherein the amino acid sequence does not include a residue difference as compared to the reference sequence of SEQ ID NO:6 at a residue position selected from X29, X137, X157, X184, X197, X198, X201, X220, X232, X261, X266, X279, X280, X287, X288, X293, X295, X311, X324, X328, X332, and X353.

10. The engineered polypeptide of claim 1 , wherein the amino acid sequence further comprises a residue difference as compared to the reference sequence of SEQ ID NO:6 selected from: X4H/L/R, X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111M/Q/R/S, X124L/N, X136G, X137N, X141W, X143W, X149L, X153E/V/Y, X154F/M/Q/Y, X156G/I/Q/S/T/V, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R, X185V, X186K/R, X1971/P, X198A/E/H/P/S, X201L, X220D/H, X223T, X226L, X232G/A/R, X243G, X246W, X256V, X258D, X259E/H/I/L/M/S/T/V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T, X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X280L, X283M/V, X284K/L/M/Y, X287S/T, X288G/S, X292C/G/I/P/S/T/V/Y, X293H/I/K/L/N/Q/T/V, X294A/I/V, X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V, X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V, X353E, and X356R.

11. The engineered polypeptide of claim 1 , wherein the amino acid sequence does not include a residue difference as compared to the reference sequence of SEQ ID NO:6 at the following residue positions X29, X137, X157, X184, X197, X198, X201, X220, X232, X261, X266, X279, X280, X287, X288, X293, X295, X311, X324, X328, X332, and X353.

12. 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:

13. The engineered polypeptide of claim 12 , 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 NO:6.

14. The engineered polypeptide of claim 1 , in which the amino acid sequence comprises a sequence selected from the group consisting of the even-numbered sequence identifiers of SEQ ID NOS:8-924.

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

16. The polynucleotide of claim 15 , in which the polynucleotide comprises a nucleic acid sequence selected from the group consisting of the odd-numbered sequence identifiers of SEQ ID NOS:7-923.

17. An expression vector comprising the polynucleotide of claim 15 .

18. A host cell comprising the polynucleotide of claim 15 .

19. A host cell comprising the expression vector of claim 17 .

20. A method of preparing the engineered polypeptide having imine reductase activity, comprising culturing the host cell of claim 18 , under conditions suitable for expression of the polypeptide, optionally further comprising isolating the engineered polypeptide.

Assignments (1)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
Continuity (7)
Continuation 16525834 · Jul 30, 2019
Division 15783657 · Oct 13, 2017
Division 14539690 · Nov 12, 2014
Provisional Application 62022315 · Jul 9, 2014
Provisional Application 62022323 · Jul 9, 2014
Provisional Application 61903772 · Nov 13, 2013
Related Publication 20210301266A1 · Sep 30, 2021
References Cited (202)
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 · Selifonov 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 9822346B2 · Agard et al. · 2017 [cited by applicant]
US 10407668B2 · Agard et al. · 2019 [cited by applicant]
US 20050095619A1 · Davis et al. · 2005 [cited by applicant]
US 20050153417A1 · Davis et al. · 2005 [cited by applicant]
US 20060195947A1 · Davis et al. · 2006 [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]
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 2013170050A1 · 2013 [cited by applicant]
Muller et al (FEBS J. Dec. 2007;274(24):6329-39) (Year: 2007). [cited by examiner]
Umeno et al (Nucleic Acids Res. Aug. 15, 2003;31(16):e91) (Year: 2003). [cited by examiner]
Singh et al (Curr. Protein Pept. Sci, 18:1-11; 2017) (Year: 2017). [cited by examiner]
Nisthal et al (Proc Natl Acad Sci U S A. Aug. 13, 2019;116(33):16367-16377) (Year: 2019). [cited by examiner]
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]
Zhong, S., et al., “5-Chloro-7-iodo-8-quinolinolatomanganese(III) with the Feature of pH-Regulated Molecular Switches as a Highly Efficient Catalyst for Epoxidation of Olefins with Hydrogen Peroxide,” Adv. Synth. Catal.… [cited by applicant]
Zhu, D., et al., “Biocatalytic asymmetric amination of carbonyl functional groups—a synthetic biology approach to organic chemistry,” Biotechnol J., 4(10):1420-31 (2009). [cited by applicant]
UniProtKB Accession No. P00386, entry name: DHNO_AGRT7 dated Jul. 21, 1986. [cited by applicant]
UniProtKB Accession No. P38997, entry name: LYS1_YARLI dated Feb. 1, 1995. [cited by applicant]
Asano, Y., et al., “A new NAD+-dependent opine dehydrogenase from [cited by applicant]
Dairi, “Cloning, nucleotide sequencing, and expression of an opine dehydrogenase gene from [cited by applicant]
Kato, Y., et al., “Stereoselective synthesis of opine-type secondary amine carboxylic acids by a new enzyme opine dehydrogenase use of recombinant enzymes,” Journal of Molecular Catalysis B: Enzymatic, 1(3-6):151-160 [1… [cited by applicant]
Schrittwieser, J., et al., “Biocatalytic Imine Reduction and Reductive Amination of Ketones,” Adv. Synth. Catal., 357(8):1655-1685 [2015]. [cited by applicant]
Grogan, G., et al., “InspIRED by Nature: NADPH-Dependent Imine Recutases (IREDS) as Catalysts for the Preparation of Chiral Amines,” Chem. Eur. J., 22(6):1900-1907 [2015]. [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]
Cho, B.K., et al., “Redesigning the substrate specificity of omega-aminotransferase for the kinetic resolution of aliphatic chiral amines,” Biotechnol Bioeng. 99(2):275-84 [2008]. [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]
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]
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]
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 [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 sequenes,” In Developmental Biology Using Purified Genes (Brown et al., eds.), pp. 683-693, Academic Press (1981). [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]