US 12,258,605 B2
Alkane oxidation by modified hydroxylases
Frances Arnold, Pasadena, CA (US); Peter Meinhold, Pasadena, CA (US); Matthew W. Peters, Pasadena, CA (US); Rudi Fasan, Brea, CA (US); and Mike M. Y. Chen, Pasadena, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by The California Institute of Technology, Pasadena, CA (US)
Filed on Dec. 23, 2021, as Appl. No. 17/560,753.
Application 14/270,268 is a division of application No. 11/697,404, filed on Apr. 6, 2007, granted, now 8,715,988, issued on May 6, 2014.
Application 17/560,753 is a continuation of application No. 16/872,275, filed on May 11, 2020, granted, now 11,214,817.
Application 16/872,275 is a continuation of application No. 15/942,001, filed on Mar. 30, 2018, granted, now 10,648,006, issued on May 12, 2020.
Application 15/942,001 is a continuation of application No. 15/224,900, filed on Aug. 1, 2016, granted, now 9,963,720, issued on May 8, 2018.
Application 15/224,900 is a continuation of application No. 14/788,365, filed on Jun. 30, 2015, granted, now 9,404,096, issued on Aug. 2, 2016.
Application 14/788,365 is a continuation of application No. 14/270,268, filed on May 5, 2014, granted, now 9,074,178, issued on Jul. 7, 2015.
Application 11/697,404 is a continuation in part of application No. PCT/US2006/011273, filed on Mar. 28, 2006.
Claims priority of provisional application 60/900,243, filed on Feb. 8, 2007.
Claims priority of provisional application 60/700,781, filed on Jul. 20, 2005.
Claims priority of provisional application 60/698,872, filed on Jul. 13, 2005.
Claims priority of provisional application 60/665,903, filed on Mar. 28, 2005.
Prior Publication US 2022/0112524 A1, Apr. 14, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/00 (2006.01); C07K 14/32 (2006.01); C12N 9/02 (2006.01); C12P 7/04 (2006.01); C12P 7/06 (2006.01)
CPC C12P 7/04 (2013.01) [C07K 14/32 (2013.01); C12N 9/0042 (2013.01); C12N 9/0071 (2013.01); C12N 9/0077 (2013.01); C12P 7/06 (2013.01); C12Y 106/02004 (2013.01); C12Y 114/14001 (2013.01); C12Y 114/15 (2013.01); C12Y 114/15003 (2013.01); Y02E 50/10 (2013.01); Y02P 20/52 (2015.11)] 8 Claims
 
1. An isolated or recombinant polypeptide comprising residues 456-1048 of the amino acid sequence set forth in SEQ ID NO: 1 with up to 65 conservative amino acid substitutions excluding residues 464, 631, 645, 710 and 968, wherein the amino acid sequence comprises residues selected from the group consisting of:
(a) at position 464, a Z1, Z3, Z4, or Z5 amino acid residue;
(b) at positions 631 and 710, a Z1 amino acid residue;
(c) at position 645, a Z3 amino acid residue; and
(d) at position 968, a Z2 amino acid residue,
wherein: Z1 is an amino acid residue selected from the group consisting of glycine (G), asparagine (N), glutamine (Q), serine(S), threonine (T), tyrosine (Y), and cysteine (C); Z2 is an amino acid residue selected from the group consisting of alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), and methionine (M); Z3 is an amino acid residue selected from the group consisting of lysine (K), and arginine (R); Z4 is an amino acid residue selected from the group consisting of tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H); and is an amino acid residue selected from the group consisting of threonine (T), valine (V), and isoleucine (I), wherein when the isolated or recombinant polypeptide is operably linked to the heme domain of a cytochrome P450 enzyme to form a chimeric polypeptide the chimeric polypeptide has cytochrome P450 activity.