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Engineered galactose oxidase variant enzymes

一种半乳糖氧化酶、工程化的技术，应用在产生GO酶,药物化合物和其他化合物的产生领域，能够解决氧化状态难以控制、物理伤害、危险等问题

Pending Publication Date: 2021-04-16
CODEXIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These oxidation methods are difficult to control in terms of oxidation state because some chemical reagents can over-oxidize the target alcohol
Furthermore, operation of the reaction under oxidizing conditions presents a hazardous situation that may result in explosion and severe physical damage to persons and property

Method used

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  • Engineered galactose oxidase variant enzymes
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  • Engineered galactose oxidase variant enzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0222] Improvement of GOA compared to SEQ ID NO: 2 for enantioselective production of EGA

[0223] The parental gene of the GOA (SEQ ID NO: 2) enzyme used to generate the variants of the present invention was codon optimized for expression in E. coli, synthesized and cloned into the pET-30a vector. Transform BL21(DE3) E. coli cells with the corresponding plasmid containing the GOA-encoding gene, and plate on Luria broth (LB) agar plates containing 1% glucose and 50 μg / mL kanamycin (KAN), and incubate at 37°C. Grow overnight. Pick a monoclonal colony and inoculate into 180 μL of 96-well shallow-well microtiter plate of LB containing 1% glucose and 50 μg / mL KAN. Use the plate with O 2 A permeable seal was sealed and the culture was grown overnight at 30°C, 200 rpm and 85% relative humidity (RH). Then, transfer 10 μL of each cell culture to a medium containing 390 μL TB, 50 μg / mL KAN, and 0.5 mM CuSO 4 wells of a 96-well deep-well plate. Use the deep well plate with O 2 Sea...

Embodiment 2

[0232] Preparation of Galactose Oxidase (GOA) Wet Cell Pellets

[0233] The parental gene of the GOA (SEQ ID NO:2) enzyme used to generate variants of the invention was codon optimized for expression in E. coli, and synthesized and cloned into the pCK900 vector (see, e.g., U.S. Patent No. 9,714,437, via incorporated herein by reference). W3110 E. coli cells were transformed with the corresponding plasmid containing the GOA-encoding gene, plated on LB agar plates containing 1% glucose and 30 μg / mL CAM, and grown overnight at 37 °C. Single clonal colonies were picked and inoculated into 180 µL of LB containing 1% glucose and 30 µg / mL CAM in a 96-well shallow-well microtiter plate. Use the plate with O 2A permeable seal was sealed and the culture was grown overnight at 30°C, 200 rpm and 85% RH. Then, transfer 10 µL of each cell culture to wells of a 96-well deep-well plate containing 390 µL TB and 30 µg / mL CAM. Use the deep well plate with O 2 Seal with permeable seal and in...

Embodiment 3

[0235] Preparation of cell lysates containing HTP GOA

[0236] The cryoprecipitate prepared as specified in Example 2 was lysed with 400 μL of lysis buffer containing 50 mM NaPi buffer, pH 7.4, 1 mg / mL lysozyme, 0.5 mg / mL PMBS. The lysis mixture was shaken for 2 hours at RT. Plates were then centrifuged at 4000 rpm and 4°C for 15 min. The supernatant was then used as a clarified lysate in the experiments described below for biocatalytic reactions to determine activity levels.

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Abstract

The present invention provides engineered galactose oxidase (GOase) enzymes, polypeptides having GOase activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GOase enzymes are also provided. The present invention further provides compositions comprising the GOase enzymes and methods of using the engineered GOase enzymes. The present invention finds particular use in the production of pharmaceutical and other compounds.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 695,570, filed July 9, 2018, and U.S. Provisional Patent Application Serial No. 62 / 822,286, filed March 22, 2019, which issued It is hereby incorporated by reference in its entirety for all purposes. [0002] field of invention [0003] The present invention provides engineered galactose oxidase (GO enzyme), polypeptides with GO enzyme activity and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing GO enzymes. The invention also provides compositions comprising GO enzymes, and methods of using engineered GO enzymes. The invention is especially useful in the production of pharmaceutical compounds and other compounds. [0004] References to Sequence Listings, Tables or Computer Programs [0005] The official copy of the sequence listing is submitted as an ASCII formatted te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P21/04
CPCC12N9/001C12N15/52C12Y101/03009C12P21/02C12N9/0006
Inventor 克里斯多佛·迈克尔·米克李斯奇奥斯卡·阿尔维左约瓦娜·纳佐尔哈尔文达·查格·马尼亚尔米凯拉·姜红霞·克拉夫奇克玛吉·塔布加·博拉-加尔斯克南希塔·苏布兰马尼安安娜·弗里斯科瓦斯卡尼古拉斯·M·马歇尔阿古斯蒂纳·罗德里格斯-格拉尼利奥迪帕克·维尔玛德万·安德鲁斯
Owner CODEXIS INC
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