公开(公告)号：US11332731B2

公开(公告)日：2022-05-17

申请号：US17208027

申请日：2021-03-22

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Zhongmei Liu ,  Junling Guo ,  Li Zhou ,  Wenjing Cui ,  Yao Lan

IPC分类号： C12N9/88 ,  C12N15/52 ,  C12N15/70 ,  C12N1/20 ,  C12P13/02

摘要： The disclosure discloses a nitrile hydratase mutant, a genetically engineered bacterium containing the mutant and applications thereof, and belongs to the technical field of enzyme engineering. In the disclosure, glycine at position 47 of a nitrile hydratase mutant αL6T/A19V/F126Y-βM46K/E108R/S212Y (disclosed in the patent of disclosure CN102216455A) is mutated to asparagine. The obtained new mutant enzyme has better temperature tolerance and tolerance to a product, and is conducive to future industrial production. The recombinant strain containing the nitrile hydratase mutant is fermented at high density, and 3-cyanopyridine is used as a substrate to carry out a whole-cell catalytic reaction to prepare nicotinamide. Compared with a chemical production method, the method has a safe and clean production process and no environmental pollution. Compared with an enzymatic method, the substrate price is cheap and the catalytic efficiency is high. The yield of the final product nicotinamide is over 95%, the concentration reaches 680 g/L, and the separation and purification steps of the product are simplified.

公开(公告)号：US12123007B2

公开(公告)日：2024-10-22

申请号：US17160561

申请日：2021-01-28

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Wenjing Cui ,  Laichuang Han ,  Zhongmei Liu ,  Li Zhou ,  Junling Guo ,  Yu Yan

IPC分类号： C12N15/75

CPC分类号： C12N15/75 ,  C12N2800/101 ,  C12N2840/105

摘要： The present disclosure discloses a method for constructing an efficient Bacillus subtilis promoter, and belongs to the technical field of gene engineering. According to the present disclosure, natural promoters identified by different sigma subunits are connected in series to obtain some double-series and triple-series promoters, the lengths of intervening sequences between core areas of the promoters are optimized on the basis of series connection of the promoters to further improve the activity of the promoters, finally, different RBS designs are performed on the promoters, and it is verified that this strategy can not only improve the compatibility between the promoters and other gene expression regulating and controlling elements, but also controllably regulate the expression of exogenous genes. Through the method provided by the present disclosure, people can obtain the promoters with higher activity and stronger designability and compatibility through simple and convenient promoter design and modification methods. The method is simple and easy to implement and has wide application prospects in the construction of an exogenous protein efficient expression system and synthetic biology research.

公开(公告)号：US12098373B2

公开(公告)日：2024-09-24

申请号：US17160582

申请日：2021-01-28

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Wenjing Cui ,  Laichuang Han ,  Wenliang Hao ,  Li Zhou ,  Zhongmei Liu ,  Yu Yan

IPC分类号： C12N15/75

CPC分类号： C12N15/75 ,  C12N2800/101 ,  C12N2830/002 ,  C12N2830/005 ,  C12N2830/006

摘要： The present disclosure discloses a Bacillus subtilis efficiently-induced expression system based on an artificial series promoter, and belongs to the technical field of gene engineering. According to the present disclosure, an efficient artificial series constitutive promoter is used, and by compositely designing the promoter and elements (a repressor and a binding site thereof) relevant to an operon, the activity of the constitutive promoter is regulated and controlled by an inducer to finally construct the B. subtilis efficiently-induced expression system induced by the inducer. The result indicates that compared with a P43 strongly constitutive promoter, the activity of the artificial series promoter in the system is about 15 times higher. The activity of the promoter can be accurately controlled by adding different concentrations of inducers. Therefore, the efficient expression system is simple in structure, high in activity and strict in regulating and controlling and has wide application prospects in heterologous protein efficient expression and synthetic biology research.

公开(公告)号：US12031165B2

公开(公告)日：2024-07-09

申请号：US17477668

申请日：2021-09-17

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Zhongmei Liu ,  Yufeng Liu ,  Li Zhou ,  Wenjing Cui ,  Junling Guo

IPC分类号： C12P13/04 ,  C12N9/88 ,  C12N9/90

CPC分类号： C12N9/88 ,  C12N9/90 ,  C12P13/04 ,  C12Y401/01012 ,  C12Y504/03009

摘要： Disclosed is a double enzyme tandem preparation method of L-2-aminobutyric acid, and belongs to the field of bioengineering. In the disclosure, recombinant Escherichia coli expressing L-glutamate mutase and recombinant Escherichia coli expressing L-aspartate-β-decarboxylase are separately cultured to obtain L-glutamate mutase and L-aspartate-β-decarboxylase. The two enzymes are added to a reaction system at a certain mass ratio, and L-glutamate is used as a substrate to carry out an enzyme reaction to prepare the L-2-aminobutyric acid. When the dosage of the L-aspartate-β-decarboxylase is 2 mg/mL, and the reaction time is 24 h, 8.5 mmol/L L-2-aminobutyric acid is produced by conversion, with a molar conversion rate of 85.00%. Compared with a chemical production method, the method disclosed by the disclosure has a safe production process and no environmental pollution. Compared with a multi-enzyme synthesis system with threonine as a substrate, the substrate is cheaper and the process is simpler.

公开(公告)号：US12104189B2

公开(公告)日：2024-10-01

申请号：US17399423

申请日：2021-08-11

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Zhongmei Liu ,  Ting Li ,  Dezhi Chen ,  Sailan Zhang ,  Li Zhou ,  Wenjing Cui ,  Zhongyi Cheng ,  Junling Guo

IPC分类号： C12N9/88 ,  C12N1/20

CPC分类号： C12N9/88 ,  C12N1/20 ,  C12Y402/01084

摘要： The disclosure discloses a mutant of nitrile hydratase derived from Caldalkalibacillus thermarum, and belongs to the technical field of enzyme engineering. The nitrile hydratase mutant Cal. t Nhase-A20V provided by the disclosure has a half-life of about 10 min at 70° C., which does not change much compared with the thermal stability of the wild enzyme. The specific enzyme activity of the mutant Cal. t Nhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better tolerance to a substrate and a product, and the final yield of nicotinamide produced by whole-cell catalysis reaches 598 g/L. Therefore, the nitrile hydratase mutant Cal. t Nhase-A20V provided by the disclosure has good enzymatic properties and is beneficial to future industrial production.

公开(公告)号：US11760988B2

公开(公告)日：2023-09-19

申请号：US17208066

申请日：2021-03-22

申请人： Jiangnan University

发明人： Zhemin Zhou ,  Zhongmei Liu ,  Li Zhou ,  Wenjing Cui ,  Wenqi Ye ,  Junling Guo ,  Chao Wang

IPC分类号： C12N9/88 ,  C12P13/06 ,  C12N1/21

CPC分类号： C12N9/88 ,  C12P13/06 ,  C12Y401/01011

摘要： The disclosure discloses an L-aspartate α-decarboxylase mutant and application thereof, and belongs to the technical field of enzyme engineering. In the disclosure, lysine at position 221 of L-aspartate α-decarboxylase is mutated to arginine, glycine at position 369 is mutated to alanine, and the obtained new mutant enzymes have better temperature tolerance and are beneficial to industrial production. The K221R and G369A recombinant strains are subjected to high-density fermentation, and with sodium L-aspartate as a substrate, a whole cell catalytic reaction is carried out to prepare β-alanine. Compared with a chemical production method, the method has the advantages that the production process is safe and clean, and has no environmental pollution. Compared with a pure enzyme catalysis method, the method has the advantages that the operation is simple and convenient. The yield of the final product β-alanine reaches 91% and 90% respectively, and the concentration reaches 162.15 g/L and 160.42 g/L respectively.