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1. 发明授权

US08603949B2 Libraries of optimized cytochrome P450 enzymes and the optimized P450 enzymes 有权
标题翻译：优化的细胞色素P450酶和优化的P450酶的文库
公开(公告)号：US08603949B2
公开(公告)日：2013-12-10
申请号：US10869813
申请日：2004-06-15
申请人： Frances H. Arnold , Christopher R. Otey
发明人： Frances H. Arnold , Christopher R. Otey
IPC分类号： C40B50/02
CPC分类号： C12N9/0071
摘要： The present disclosure teaches that the recombination of homologous sequences of P450 enzymes, with the aid of SCHEMA to predict a resulting protein structure, is able to generate libraries of chimeras with significant functional diversity. Additionally, the members of these libraries demonstrate superior or unexpected new properties, which correlate with other factors that are observable in the library. Thus, the making of libraries of optimized P450 enzymes, the analysis of libraries to identify an optimized subset, and the optimized chimeras with improved or altered functionalities are all taught in the present disclosure.
摘要翻译：本公开内容指出，借助于SCHEMA来预测所得蛋白质结构的P450酶的同源序列的重组能够产生具有显着功能多样性的嵌合体库。此外，这些图书馆的成员展示了优秀或意想不到的新属性，这与库中可观察到的其他因素相关。因此，在本公开内容中教导了制备优化的P450酶的文库，分析库以鉴定优化的子集，以及具有改进或改变的功能的优化的嵌合体。

2. 发明申请

US20110229872A1 Microfabricated Cell Sorter 审中-公开
标题翻译：微型细胞分选机
公开(公告)号：US20110229872A1
公开(公告)日：2011-09-22
申请号：US12954409
申请日：2010-11-24
申请人： CHARLES F. SPENCE , ANNE Y. FU , STEPHEN R. QUAKE , FRANCES H. ARNOLD
发明人： CHARLES F. SPENCE , ANNE Y. FU , STEPHEN R. QUAKE , FRANCES H. ARNOLD
IPC分类号： C12Q3/00 , G01N27/447 , C12M1/36
CPC分类号： 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
摘要： 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.
摘要翻译：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。

3. 发明申请

US20100304464A1 STABLE, FUNCTIONAL CHIMERIC CELLOBIOHYDROLASES 审中-公开
标题翻译：稳定的，功能性的二氧化碳氢化合物
公开(公告)号：US20100304464A1
公开(公告)日：2010-12-02
申请号：US12723597
申请日：2010-03-12
申请人： Frances H. Arnold , Pete Heinzelman , Jeremy Minshull , Sridhar Govindarajan , Alan Villalobos
发明人： Frances H. Arnold , Pete Heinzelman , Jeremy Minshull , Sridhar Govindarajan , Alan Villalobos
IPC分类号： C12N9/42 , C07H21/00 , C12N15/63 , C12N1/00
CPC分类号： C12N9/2437 , C12Y302/01091 , C12Y302/01099
摘要： The present disclosure relates to CBH II chimera fusion polypeptides, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.
摘要翻译：本公开涉及CBH II嵌合融合多肽，编码多肽的核酸和用于产生多肽的宿主细胞。

4. 发明申请

US20090142821A1 Peroxide-driven cytochrome P450 oxygenase variants 有权
标题翻译：过氧化物驱动的细胞色素P450加氧酶变体
公开(公告)号：US20090142821A1
公开(公告)日：2009-06-04
申请号：US12287681
申请日：2008-10-11
申请人： Patrick C. Cirino , Frances H. Arnold
发明人： Patrick C. Cirino , Frances H. Arnold
IPC分类号： C12N9/02
CPC分类号： C12N9/0071 , C12Y114/14001
摘要： The invention relates to novel variants of cytochrome P450 oxygenases. These variants have an improved ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. These variants also have an improved thermostability as compared to the cytochrome P450 BM-3 F87A mutant. Preferred variants include cytochrome P450 BM-3 heme domain mutants having 158V, F87A, H100R, F107L, A135S, M145A/V, N239H, S274T, L3241, I366V, K434E, E442K, and/or V446I amino acid substitutions.
摘要翻译：本发明涉及细胞色素P450加氧酶的新变体。与相应的野生型酶相比，这些变体具有改善使用过氧化物作为供氧体的能力。与细胞色素P450 BM-3 F87A突变体相比，这些变体也具有改善的热稳定性。优选的变体包括具有158V，F87A，H100R，F107L，A135S，M145A / V，N239H，S274T，L3241，I366V，K434E，E442K和/或V446I氨基酸取代的细胞色素P450 BM-3血红素结构域突变体。

5. 发明授权

US07465567B2 Peroxide-driven cytochrome P450 oxygenase variants 有权
标题翻译：过氧化物驱动的细胞色素P450加氧酶变体
公开(公告)号：US07465567B2
公开(公告)日：2008-12-16
申请号：US10125640
申请日：2002-04-16
申请人： Patrick C. Cirino , Frances H. Arnold
发明人： Patrick C. Cirino , Frances H. Arnold
IPC分类号： C12N9/02 , C12N15/00 , C12N1/20 , C12Q1/26 , C12Q1/68 , C12P21/04 , C07H21/04
CPC分类号： C12N9/0071 , C12Y114/14001
摘要： The invention relates to novel variants of cytochrome P450 oxygenases. These variants have an improved ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. These variants also have an improved thermostability as compared to the cytochrome P450 BM-3 F87A mutant. Preferred variants include cytochrome P450 BM-3 heme domain mutants having I58V, F87A, H100R, F107L, A135S, M145A/V, N239H, S274T, L324I, I366V, K434E, E442K, and/or V446I amino acid substitutions.
摘要翻译：本发明涉及细胞色素P450加氧酶的新变体。与相应的野生型酶相比，这些变体具有改善使用过氧化物作为供氧体的能力。与细胞色素P450 BM-3 F87A突变体相比，这些变体也具有改善的热稳定性。优选的变体包括具有I58V，F87A，H100R，F107L，A135S，M145A / V，N239H，S274T，L324I，I366V，K434E，E442K和/或V446I氨基酸取代的细胞色素P450 BM-3血红素结构域突变体。

6. 发明授权

US07435570B2 Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use 有权
标题翻译：耐热过氧化物驱动的细胞色素P450加氧酶变体和使用方法
公开(公告)号：US07435570B2
公开(公告)日：2008-10-14
申请号：US10916369
申请日：2004-08-11
申请人： Frances H. Arnold , Patrick C. Cirino
发明人： Frances H. Arnold , Patrick C. Cirino
IPC分类号： C12N9/02 , C12N15/00 , C12Q1/26 , C12P21/04 , C07H21/04
CPC分类号： C12N9/0077
摘要： The invention relates to novel variants of cytochrome P450 oxygenases. These variants have at least one mutation improving their ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. The variants also have at least one mutation improving thermostability as compared to the parent enzyme or corresponding wild-type enzyme. Preferred variants include cytochrome P450 BM-3 heme domain variants having L52I, I58V, F87A, H100R, S106R, F107L, A135S, M145A/V, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and/or V446I amino acid substitutions.
摘要翻译：本发明涉及细胞色素P450加氧酶的新变体。与相应的野生型酶相比，这些变体具有提高其使用过氧化物作为供氧体的能力的至少一个突变。与亲本酶或相应的野生型酶相比，变体还具有提高热稳定性的至少一个突变。优选的变体包括具有L52I，I58V，F87A，H100R，S106R，F107L，A135S，M145A / V，A184V，N239H，S274T，L324I，V340M，I366V，K434E，E442K和/或V446I的细胞色素P450 BM-3血红素结构域变体氨基酸取代。

7. 发明授权

US07115403B1 Directed evolution of galactose oxidase enzymes 失效
标题翻译：半乳糖氧化酶的定向进化
公开(公告)号：US07115403B1
公开(公告)日：2006-10-03
申请号：US09571553
申请日：2000-05-16
申请人： Frances H. Arnold , Ioanna P. Petrounia , Lianhong Sun
发明人： Frances H. Arnold , Ioanna P. Petrounia , Lianhong Sun
IPC分类号： C12N9/02 , C12N1/20 , C12N15/00 , C12Q1/68 , C12Q1/00 , C07H21/04 , C12P21/04
CPC分类号： C12N9/0006 , C12N15/102
摘要： This invention relates to the expression of improved polynucleotide and polypeptide sequences encoding for eukaryotic enzymes, particularly oxidase enzymes. The enzymes are advantageously produced 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 manner include improved expression, enhanced activity toward one or more substrates, and increased thermal stability. In a particular embodiment, the invention relates to improved expression of the galactose oxidase gene and galactose oxidase enzymes. GAO mutants that are highly active and/or thermostable are disclosed.
摘要翻译：本发明涉及编码真核生物酶，特别是氧化酶的改良的多核苷酸和多肽序列的表达。酶有利地在常规或简单的表达系统中产生。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。以这种方式产生的多肽或蛋白质的改进特征包括改进的表达，增强对一种或多种底物的活性和增加的热稳定性。在一个具体实施方案中，本发明涉及半乳糖氧化酶基因和半乳糖氧化酶的改进表达。公开了高活性和/或热稳定的GAO突变体。

8. 发明授权

US07098010B1 Directed evolution of oxidase enzymes 失效
标题翻译：氧化酶的定向进化
公开(公告)号：US07098010B1
公开(公告)日：2006-08-29
申请号：US09722602
申请日：2000-11-27
申请人： Frances H. Arnold , Ioanna P. Petrounia , Lianhong Sun
发明人： Frances H. Arnold , Ioanna P. Petrounia , Lianhong Sun
IPC分类号： C12N9/02 , C12N1/20 , C12N15/00 , C12Q1/68 , C12Q1/00 , C12P21/04 , C07H21/04
CPC分类号： C12N9/0006 , C12N15/102
摘要： This invention relates to the expression of improved polynucleotide and polypeptide sequences encoding for eukaryotic enzymes, particularly oxidase enzymes. The enzymes are advantagoeusly produced 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 manner include improved expression, enhanced activity toward one or more substrates, and increased thermal stability. In a particular embodiment, the invention relates to improved expression of the galactose oxidase gene and galactose oxidase enzymes. GAO mutants that are highly active and/or thermostable are disclosed.
摘要翻译：本发明涉及编码真核生物酶，特别是氧化酶的改良的多核苷酸和多肽序列的表达。这些酶在常规或简单的表达系统中有利地产生。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。以这种方式产生的多肽或蛋白质的改进特征包括改进的表达，增强对一种或多种底物的活性和增加的热稳定性。在一个具体实施方案中，本发明涉及半乳糖氧化酶基因和半乳糖氧化酶的改进表达。公开了高活性和/或热稳定的GAO突变体。

9. 发明授权

US5837202A Metal chelating lipids which are useful as sensors in fluorometric methods for the detection of metal ions 失效
标题翻译：金属螯合脂质，用作检测金属离子的荧光测定方法中的传感器
公开(公告)号：US5837202A
公开(公告)日：1998-11-17
申请号：US712193
申请日：1996-09-11
申请人： Frances H. Arnold , Darryl Y. Sasaki
发明人： Frances H. Arnold , Darryl Y. Sasaki
IPC分类号： C09B57/10 , G01N31/22 , G01N33/533 , G01N21/64 , C07C229/34 , C07D255/02
CPC分类号： G01N31/221 , C09B57/10 , G01N31/22 , G01N33/533
摘要： An fluorescent metal-chelating amphiphile having the structure: ##STR1## wherein A is a hydrophobic fluorophore, X and Y are aliphatic hydrocarbons having from 9 to 25 carbon atoms, B is a hydrophilic spacer, C is a metal chelator, and L is either an ether or ester linkage. The fluorescent metal-chelating amphiphile is combined with a matrix lipid to form lipid-based sensors which provide fluorometric detection of metal ions in liquids.
摘要翻译：具有以下结构的荧光金属螯合两亲物：其中A是疏水性荧光团，X和Y是具有9至25个碳原子的脂族烃，B是亲水间隔基，C是金属螯合剂， L是醚或酯键。荧光金属螯合两亲物与基质脂质组合以形成基于脂质的传感器，其提供液体中金属离子的荧光检测。

10. 发明授权

US5316935A Subtilisin variants suitable for hydrolysis and synthesis in organic media 失效
标题翻译：枯草杆菌蛋白酶变体适用于有机介质中的水解和合成
公开(公告)号：US5316935A
公开(公告)日：1994-05-31
申请号：US864298
申请日：1992-04-06
申请人： Frances H. Arnold , Keqin Chen
发明人： Frances H. Arnold , Keqin Chen
IPC分类号： C12N9/54 , C12N9/56 , C12N15/57 , C12N9/50 , C12P21/00
CPC分类号： C12N9/54
摘要： In accordance with the present invention, there are provided novel, modified subtilisin enzyme(s) having improved catalytic activity and/or stability in organic media.
摘要翻译：根据本发明，提供了在有机介质中具有改进的催化活性和/或稳定性的新型改性枯草杆菌蛋白酶。

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