公开(公告)号：US20030100744A1

公开(公告)日：2003-05-29

申请号：US10201213

申请日：2002-07-22

Applicant: California Institute of Technology

Inventor： Edgardo T. Farinas ,  Frances H. Arnold ,  Ulrich Schwaneberg ,  Anton Glieder

IPC: C07H021/04 ,  C12N009/02 ,  C12P021/02 ,  C12N005/06

CPC classification number: C12N9/0071 ,  C12P7/02 ,  C12P7/04 ,  C12P7/16 ,  C12P17/02 ,  Y02E50/10 ,  Y02P20/52

Abstract: Nucleic acids encoding cytochrome P450 variants are provided. The cytochrome P450 variants of have a higher alkane-oxidation capability, alkene-oxidation capability, and/or a higher organic-solvent resistance than the corresponding wild-type or parent cytochrome P450 enzyme. A preferred wild-type cytochrome P450 is cytochrome P450 BM-3. Preferred cytochrome P450 variants include those having an improved capability to hydroxylate alkanes and epoxidate alkenes comprising less than 8 carbons, and have amino acid substitutions corresponding to V78A, H236Q, and E252G of cytochrome P450 BM-3. Preferred cytochrome P450 variants also include those having an improved hydroxylation activity in solutions comprising co-solvents such as DMSO and THF, and have amino acid substitutions corresponding to T235A, R471A, E494K, and S1024E of cytochrome P450 BM-3.

Abstract translation: 提供了编码细胞色素P450变体的核酸。 细胞色素P450变体具有比相应的野生型或亲本细胞色素P450酶更高的烷烃氧化能力，烯烃氧化能力和/或较高的有机溶剂耐受性。 优选的野生型细胞色素P450是细胞色素P450 BM-3。 优选的细胞色素P450变体包括具有改善含有少于8个碳原子的烷烃和环氧化烯烃羟基化能力以及对应于细胞色素P450 BM-3的V78A，H236Q和E252G的氨基酸取代的那些变体。 优选的细胞色素P450变体还包括在包含共溶剂如DMSO和THF的溶液中具有改善的羟基化活性的那些，并且具有对应于细胞色素P450 BM-3的T235A，R471A，E494K和S1024E的氨基酸取代。

公开(公告)号：US20010055786A1

公开(公告)日：2001-12-27

申请号：US09828599

申请日：2001-04-05

Applicant: CALIFORNIA INSTITUTE OF TECHNOLOGY

Inventor： Frances H. Arnold ,  John Joern ,  Takeshi Sakamoto ,  Ulrich Schwaneberg

IPC: C12Q001/26

CPC classification number: C12Q1/26 ,  C12Q1/32

Abstract: The present invention relates to screening methods for oxidation enzymes, particularly mono- and dioxygenases. According to the methods of the invention, a product of an oxidation reaction is converted into a phenol or a catechol, which is easily detected by a Gibbs assay. This conversion allows for a sensitive and efficient assay. Both high-throughput liquid-phase and solid-phase methods using these principles are provided. Also described are method for detecting phenolic ether-products and sulfhydryl products from oxidation reactions, also using a Gibbs assay.

Abstract translation: 本发明涉及氧化酶，特别是单加氧酶和双加氧酶的筛选方法。 根据本发明的方法，将氧化反应的产物转化成苯酚或邻苯二酚，其可以通过吉布斯测定容易地检测。 该转化允许敏感和有效的测定。 提供了使用这些原理的高通量液相和固相方法。 还描述了也使用吉布斯测定法从氧化反应中检测酚醚产物和巯基产物的方法。