Title:
NITRILE HYDRATASE MUTANT, GENETICALLY ENGINEERED BACTERIUM CONTAINING SAME, AND USE THEREOF
Document Type and Number:
WIPO Patent Application WO/2020/147031
Kind Code:
A1
Abstract:
Disclosed are a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant, and the use thereof, falling within the technical field of enzyme engineering. According to the present invention, glycine at position 47 of a nitrile hydratase mutant αL6T/A19V/F126Y-βM46K/E108R/S212Y (disclosed in the inventive patent CN 102216455 A) is mutated to become asparagine, and the obtained new mutant enzyme has better temperature tolerance and product tolerance, and is thus conducive to industrial production in the future. Nicotinonitrile is used as a substrate, and a recombinant strain containing the nitrile hydratase mutant is fermented at a high density to perform a whole-cell catalytic reaction so as to prepare nicotinamide. Compared with a chemical production method, the method is safe and clean in terms of the production process and is free of environmental pollution; and compared with an enzymatic method, the method has a low-price substrate and a high catalytic efficiency, with the yield of the final product nicotinamide being at least 95% and the concentration being up to 680 g/L, thereby simplifying the separation and purification steps of the product.
Inventors:
ZHOU ZHEMIN (CN)
LIU ZHONGMEI (CN)
GUO JUNLING (CN)
ZHOU LI (CN)
CUI WENJING (CN)
LAN YAO (CN)
LIU ZHONGMEI (CN)
GUO JUNLING (CN)
ZHOU LI (CN)
CUI WENJING (CN)
LAN YAO (CN)
Application Number:
PCT/CN2019/071964
Publication Date:
July 23, 2020
Filing Date:
January 16, 2019
Export Citation:
Assignee:
UNIV JIANGNAN (CN)
International Classes:
C12N1/21; C12N9/88; C12N15/60; C12N15/70; C12P13/02; C12P17/12; C12R1/19
Foreign References:
| CN102216455A | 2011-10-12 | |||
| CN102899345A | 2013-01-30 | |||
| CN106544336A | 2017-03-29 | |||
| CN102492697A | 2012-06-13 | |||
| CN1584024A | 2005-02-23 | |||
| US5728556A | 1998-03-17 |
Other References:
SHI, YUE ET AL.: "High Level Expression of a Mutated Nitrile Hydratase Gene of Nocardia Sp. in Recombinant Escherichia Coli", JOURNAL OF TSINGHUA UNIVERSITY ( SCIENCE AND TECHNOLOGY, vol. 47, no. 12, 31 December 2007 (2007-12-31), pages 2176 - 2179, XP055419964, ISSN: 1000-0054
MIYANAGA, A ET AL.: "Mutational and Structural Analysis of Cobalt-Containing Nitrile Hydratase on Substrate and Metal Binding", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 271, 31 December 2003 (2003-12-31), pages 429 - 438, XP055096861, DOI: 10.1046/j.1432-1033.2003.03943.x
MIYANAGA, A ET AL.: "Mutational and Structural Analysis of Cobalt-Containing Nitrile Hydratase on Substrate and Metal Binding", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 271, 31 December 2003 (2003-12-31), pages 429 - 438, XP055096861, DOI: 10.1046/j.1432-1033.2003.03943.x
Attorney, Agent or Firm:
HARBIN SHINEIP INTELLECTUAL PROPERTY AGENCY CO., LTD. (CN)
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