公开(公告)号：US20030228673A1

公开(公告)日：2003-12-11

申请号：US10375909

申请日：2003-02-27

Applicant: CALIFORNIA INSTITUTE OF TECHNOLOGY

Inventor： Frances H. Arnold ,  Lian-Hong Sun ,  Ioanna P. Petrounia

IPC: G06F019/00 ,  G01N033/48 ,  G01N033/50 ,  C12N009/02 ,  C12N001/16 ,  C12P021/02 ,  C07H021/04

CPC classification number: C12N9/0006 ,  C12Y101/03004 ,  C12Y101/03009

Abstract: Glucose oxidase enzymes are provided, including novel variants of galactose oxidase enzymes. The polynucleotides that encode these novel variants can be expressed in recombinant host cell expression systems. The novel variant oxidase enzymes are capable of oxidizing compounds towards which wild-type galactose oxidase (e.g. D-galactose: oxygen 6-oxidoreductase, GAO; EC 1.1.3.9) has little or no activity. Preferred galactose oxidase variants are those which that have improved capability to oxidize secondary alcohols and/or D-glucose relative to the wild-type enzyme.

Abstract translation: 提供了葡萄糖氧化酶，包括半乳糖氧化酶的新型变体。 编码这些新变体的多核苷酸可以在重组宿主细胞表达系统中表达。 新型变体氧化酶能够氧化化合物，野生型半乳糖氧化酶（例如D-半乳糖：氧-6-氧化还原酶，GAO; EC1.1.3.9）具有很少或没有活性。 优选的半乳糖氧化酶变体是那些相对于野生型酶具有改善氧化仲醇和/或D-葡萄糖的能力的变体。

公开(公告)号：US20030207345A1

公开(公告)日：2003-11-06

申请号：US10453104

申请日：2003-06-02

Applicant: California Institute of Technology

Inventor： Frances H. Arnold ,  Hyun Joo

IPC: C12Q001/26

CPC classification number: C12N9/0071 ,  C07K2319/02 ,  C12N9/0065 ,  C12N9/0069 ,  C12N9/0077 ,  C12N15/102 ,  C12N15/1034 ,  C12N15/67 ,  C12N15/815 ,  C12Q1/26 ,  C12Q1/28 ,  G01N2333/795 ,  G01N2333/80 ,  G01N2333/90245

Abstract: A method for detecting the presence of an oxygenated compound which is produced when a substrate is reacted with an oxygenase for the substrate. The method involves reacting a coupling enzyme with the oxygenated compound to form a polymeric oxygenated compound which is fluorescent or luminescent. Measurement of the fluorescence or luminescence of the polymeric oxygenated compound provides indirect detection of the oxygenated compound produced by reaction of the oxygenase with the substrate. The method is carried out in a whole cell environment wherein the cell is transformed to express both the oxygenase being screened and the coupling enzyme. The method can be used to measure the activity of monooxygenases and dioxygenases on aromatic substrates. The method is amenable to large scale screening of enzyme mutants to isolate those with maximum oxygenase activity.

Abstract translation: 用于检测当底物与用于底物的加氧酶反应时产生的含氧化合物的存在的方法。 该方法包括使偶联酶与含氧化合物反应以形成荧光或发光的聚合的氧化化合物。 聚合物氧化化合物的荧光或发光的测量提供了通过加氧酶与底物的反应产生的氧合化合物的间接检测。 该方法在整个细胞环境中进行，其中转化细胞以表达被筛选的加氧酶和偶联酶。 该方法可用于测量单加氧酶和双加氧酶在芳香族底物上的活性。 该方法适用于大规模筛选酶突变体以分离具有最大加氧酶活性的那些。

公开(公告)号：US20010051855A1

公开(公告)日：2001-12-13

申请号：US09795500

申请日：2001-02-16

Applicant: California Institute of Technology

Inventor： Zhen-Gang Wang ,  Christopher A. Voigt ,  Stephen L. Mayo ,  Frances H. Arnold

IPC: G06F019/00 ,  G01N033/48 ,  G01N033/50

CPC classification number: G16B15/00 ,  G16B20/00

Abstract: The invention relates to improved methods for directed evolution of polymers, including directed evolution of nucleic acids and proteins. Specifically, the methods of the invention include analytical methods for identifying nullstructurally tolerantnull residues of a polymer. Mutations of these, structurally tolerant residues are less likely to adversely affect desirable properties of a polymer sequence. The invention further provides improved methods for directed evolution wherein the structurally tolerant residues of a polymer are selectively mutated. Computer systems for implementing analytical methods of the invention are also provided.

Abstract translation: 本发明涉及聚合物定向进化的改进方法，包括核酸和蛋白质的定向进化。 具体地，本发明的方法包括用于鉴定聚合物的“结构上耐受性”残留物的分析方法。 这些结构上耐受性残基的突变不太可能不利地影响聚合物序列的期望性质。 本发明还提供了用于定向进化的改进方法，其中聚合物的结构耐受性残基被选择性突变。 还提供了用于实现本发明的分析方法的计算机系统。

公开(公告)号：US20030153042A1

公开(公告)日：2003-08-14

申请号：US10274793

申请日：2002-10-21

Applicant: California Institute of Technology

Inventor： Frances H. Arnold ,  Zhanglin Lin

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

CPC classification number: C12N9/0071 ,  C07K2319/02 ,  C12N9/0065 ,  C12N15/102 ,  C12N15/1034 ,  C12N15/67 ,  C12N15/815 ,  C12Q1/26 ,  C12Q1/28 ,  G01N2333/795 ,  G01N2333/80 ,  G01N2333/90245

Abstract: This invention relates to the improved expression of evolved polynucleotide and polypeptide sequences encoding for eukaryotic enzymes, particularly peroxidase enzymes, in conventional or facile expression systems. Various methods for directed evolution of polynucleotide sequences can be used to obtain the improved sequences. The improved characteristics of the polypeptides or proteins generated in this maruer include improved folding, without formation of inclusion bodies, and retained functional activity. In a particular embodiment, the invention relates to improved expression of the horseradish peroxidase (HRP) gene and HRP enzymes. HRP mutants that are highly expressed, highly active, and/or thermostable, are disclosed.

Abstract translation: 本发明涉及在常规或简单表达系统中改良表达的编码真核生物酶，特别是过氧化物酶的进化多核苷酸和多肽序列。 可以使用用于多核苷酸序列的定向进化的各种方法来获得改进的序列。 在该嫁妆者中产生的多肽或蛋白质的改进特征包括改善的折叠，而不形成包涵体，并保留功能活性。 在一个具体实施方案中，本发明涉及辣根过氧化物酶（HRP）基因和HRP酶的改良表达。 公开了高表达，高活性和/或热稳定的HRP突变体。

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：US20020051998A1

公开(公告)日：2002-05-02

申请号：US09733759

申请日：2000-12-08

Applicant: CALIFORNIA INSTITUTE OF TECHNOLOGY

Inventor： Claudia Schmidt-Dannert ,  Frances H. Arnold

IPC: G01N033/53 ,  C12P023/00 ,  C12N005/04 ,  C12N005/06

CPC classification number: C12N9/0004 ,  C12N15/1027 ,  C12N15/52 ,  C12P23/00

Abstract: The present invention relates to engineering new biosynthetic pathways into microorganisms, in particular biosynthetic carotenoid pathways. New and improved catalytic functions of metabolic pathways are created by, for example, site-specific mutation or gene shuffling techniques, to provide for efficient biosynthesis of carotenoids. By applying the described directed evolution techniques, almost any carotenoid could be produced, in a host cell, from one or a few sets of genes. In addition, the described techniques are useful for creating gene or protein libraries for new and uncharacterized carotenoids.

Abstract translation: 本发明涉及将新的生物合成途径工程化成微生物，特别是生物合成类胡萝卜素途径。 通过例如位点特异性突变或基因改组技术产生新的和改进的代谢途径的催化功能，以提供类胡萝卜素的有效生物合成。 通过应用所描述的定向进化技术，可以在宿主细胞中从一组或几组基因产生几乎任何类胡萝卜素。 此外，所描述的技术可用于产生新的和未表征的类胡萝卜素的基因或蛋白质文库。

公开(公告)号：US20020005354A1

公开(公告)日：2002-01-17

申请号：US09928590

申请日：2001-08-13

Applicant: California Institute of Technology

Inventor： Charles F. Spence ,  Anne Y. Fu ,  Stephen R. Quake ,  Frances H. Arnold

IPC: C12M001/36 ,  C12M001/00 ,  C12M001/34 ,  C12M001/42 ,  G01N027/26 ,  G01N027/447

CPC classification number: G01N27/26 ,  B01L3/502761 ,  B01L2200/0647 ,  B01L2300/0864 ,  B01L2300/123 ,  B01L2400/0406 ,  B01L2400/0415 ,  B01L2400/0418 ,  B01L2400/0421 ,  B01L2400/0424 ,  B01L2400/0454 ,  B01L2400/0481 ,  B01L2400/0487 ,  B01L2400/0655 ,  G01N15/14 ,  G01N2015/149

Abstract: The invention provides a microfabricated device for sorting cells based on a desired characteristic, for example, reporter-labeled cells can be sorted by the presence or level of reporter on the cells. The device includes a chip having a substrate into which is microfabricated at least one analysis unit. Each analysis unit includes a main channel, having a sample inlet channel, typically at one end, and a detection region along a portion of its length. Adjacent and downstream from the detection region, the main channel has a discrimination region or branch point leading to at least two branch channels. The analysis unit may further include additional inlet channels, detection points, branch points, and branch channels as desired. A stream containing cells is passed through the detection region, such that on average one cell occupies the detection region at a given time. The cells can be sorted into an appropriate branch channel based on the presence or amount of a detectable signal such as an optical signal, with or without stimulation, such as exposure to light in order to promote fluorescence.

Abstract translation: 本发明提供了一种用于基于所需特征分选细胞的微制造装置，例如，报道分子标记的细胞可以通过细胞上报告物的存在或水平进行分选。 该装置包括具有基板的芯片，微基板将至少一个分析单元微加工。 每个分析单元包括主通道，具有通常在一端的样品入口通道和沿其长度的一部分的检测区域。 相邻和下游的检测区域，主通道具有通向至少两个分支通道的识别区域或分支点。 分析单元还可以根据需要进一步包括额外的入口通道，检测点，分支点和分支通道。 包含细胞的流通过检测区域，使得平均一个细胞在给定时间占据检测区域。 基于可检测信号（例如光信号）的存在或量，可以将细胞分类到适当的分支通道中，具有或不具有刺激，例如暴露于光以促进荧光。

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。