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1. 发明授权

US11001823B2 Nitrilase mutants and application thereof 有权
公开(公告)号：US11001823B2
公开(公告)日：2021-05-11
申请号：US16619344
申请日：2019-01-24
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Yuguo Zheng , Zhe Xu , Zhiqiang Liu
IPC分类号： C12N9/78 , C12P13/00 , C12N15/00 , C12N1/20 , C12N15/63
摘要： The present invention discloses a nitrilase mutant and application thereof. The mutant is obtained by mutating the amino acid at position 201 or replacing one or more amino acids at region 324-381 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NIT5-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid. In the present invention, by protein molecular modification, thermal stability of pure nitrilase LNIT5 at 45° C. is increased up to 4.5 times, and while 1-cyanocyclohexylacetonitrile is hydrolyzed using recombinant Escherichia coli containing nitrilase mutant at high temperature (45° C.), the product yield is increased. Therefore, the mutants obtained in the present invention have a good application prospect in highly efficiently catalyzing 1-cyanocyclohexylacetonitrile to 1-cyanocyclohexyl acetic acid, the gabapentin intermediate.

2. 发明授权

US10260059B2 Nitrilase from arabis alpina, its encoding gene, vector, recombinant bacterial strain and uses thereof 有权
公开(公告)号：US10260059B2
公开(公告)日：2019-04-16
申请号：US15687800
申请日：2017-08-28
申请人： Zhejiang University of Technology , Zhejiang Chiral Medicine Chemicals Co. Ltd.
发明人： Renchao Zheng , Yuguo Zheng , Qin Zhang , Youming Huang , Jianfeng Weng , Tianchun Liu , Weirong Fan
IPC分类号： C12N9/78 , C12P13/00
摘要： The disclosure provides a nitrilase from Arabis alpina, which belongs to genus Arabis, family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and >99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

3. 发明授权

US12104191B2 Cofactor self-sufficient 有权
公开(公告)号：US12104191B2
公开(公告)日：2024-10-01
申请号：US17823853
申请日：2022-08-31
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Shuping Zou , Zhentao Jiang , Jianmiao Xu , Feng Cheng , Yuguo Zheng
IPC分类号： C12P13/00 , C07K14/245 , C12N1/21 , C12N9/00 , C12N9/04 , C12N9/06 , C12N9/10 , C12N9/12 , C12N15/10 , C12N15/52 , C12N15/70
CPC分类号： C12P13/001 , C07K14/245 , C12N9/0006 , C12N9/0016 , C12N9/1077 , C12N9/1205 , C12N9/1241 , C12N9/93 , C12N15/1082 , C12N15/52 , C12N15/70 , C12Y101/01119 , C12Y104/01002 , C12Y204/02011 , C12Y207/01086 , C12Y207/07018 , C12Y603/01005 , C12N2800/101
摘要： A cofactor self-sufficient Escherichia coli and its construction method and application in the synthesis of L-glufosinate are provided. The present invention expresses a NADH kinase and key enzymes of the cofactor synthesis pathway in E. coli, and knocks out the genes of enzymes that catabolizes cofactor, and with the addition of co-metabolic intermediates during cell incubation, the intracellular NADP(H) concentration is increased by at least 50% and the catalytic activity of glufosinate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the glufosinate synthesis reaction.

4. 发明授权

US12031160B2 Method for the production of glufosinate-ammonium 有权
公开(公告)号：US12031160B2
公开(公告)日：2024-07-09
申请号：US17419048
申请日：2019-06-28
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Feng Cheng , Qinghua Li , Yuguo Zheng , Jianmiao Xu
IPC分类号： C12P13/04 , C12N9/06
CPC分类号： C12N9/0016 , C12P13/04
摘要： The present invention provides a glufosinate-ammonium dehydrogenase mutant and application in synthesis of L-glufosinate-ammonium thereof, the method uses 2-carbonyl-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid or its salts as a substrate and the glufosinate-ammonium dehydrogenase or cells containing the glufosinate-ammonium dehydrogenase as a biocatalyst to carry out reductive amination, thereby obtaining L-glufosinate-ammonium. The method has features of high conversion rate of raw materials, high yield, easy separation and purification of the product, and high chiral purity; compared with other catalytic processes, the method in the present invention has features of relatively simple process and a conversion rate of raw materials up to 100%.

5. 发明授权

US11492606B2 Nitrilase mutant, construction method therefor, and application thereof 有权
公开(公告)号：US11492606B2
公开(公告)日：2022-11-08
申请号：US16968808
申请日：2019-01-31
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Renchao Zheng , Yuguo Zheng , Qin Zhang , Zheming Wu , Xiaoling Tang , Xiafeng Lu
IPC分类号： C12N9/78 , C12P13/00 , C12N15/70
摘要： The present invention discloses a nitrilase mutant and its construction method and its application in the synthesis of chiral intermediate of pregabalin in the technical field of bioengineering. The present invention, respectively, takes turnip nitrilase BrNIT and arabidopsis nitrilase AtNIT as parent, using peptide fragment displacement method, displaces the sites 226-286 of BrNIT amino acid sequence and sites 225-285 of AtNIT amino acid sequence with sites 225-285 of Arabis alpina L. nitrilase AaNIT, obtain nitrilase mutants BrNIT225-285 and AtNIT225-285 of which the amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3. Compared with wild type nitrilase, the activity of the nitrilase mutant provided by the present invention in catalyzing and hydrolyzing racemic IBSN and the stereoselectivity of the product show substantial improvement, it can satisfy the requirements of industrial application, and has good application prospect in efficient catalysis of racemic IBSN to synthesize 3-cyano-5-methylhexanoic Acid.

6. 发明授权

US11781117B2 Machine learning gene mining method and phosphinothricin dehydrogenase mutant for amino translocation 有权
公开(公告)号：US11781117B2
公开(公告)日：2023-10-10
申请号：US17505945
申请日：2021-10-20
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Feng Cheng , Dongyang Wu , Shuping Zou , Jianmiao Xu , Yuguo Zheng
IPC分类号： C12N9/06 , C12P13/04 , G16B30/10 , C12N9/04 , C12N1/21 , C12N15/53
CPC分类号： C12N9/0016 , C12N9/0006 , C12P13/04 , G16B30/10 , C12Y104/01002 , C12Y104/01003 , C12Y104/01004
摘要： Disclosed are a machine learning gene mining method and a phosphinothricin dehydrogenase mutant for amino translocation. The phosphinothricin dehydrogenase mutant for amino translocation is obtained by mutation of a wild-type phosphinothricin dehydrogenase with an amino acid sequence as shown in SEQ ID No.2 at one of the following sites: (1) E263D-K134R-H96A-R290V; (2) E263D-K134R-H96A; (3) E263D-K134R; (4) E263D; (5) E263N; (6) E263C; and (7) E263G. The present invention utilizes the site-saturation mutagenesis technology to mutate a phosphinothricin dehydrogenase gene as shown in SEQ ID No. 1, finds that the 263rd, 134th, 290th and 290th positions are the key sites affecting enzyme activity and stereoselectivity, and obtains a mutant with enzyme activity and ee value much higher than those of the parent phosphinothricin dehydrogenase.

7. 发明公开

US20230295677A1 METHOD OF IMPROVING BIOMASS HIGH-SOLID ENZYMATIC HYDROLYSIS RATE 审中-公开
公开(公告)号：US20230295677A1
公开(公告)日：2023-09-21
申请号：US18023067
申请日：2021-11-30
申请人： ZHEJIANG HUAKANG PHARMACEUTICAL CO., LTD. , ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Mian Li , Zhiqian Liu , Xuhao Zeng , Changhui Hu , Jing Wang , Xue Cai , Xiaojian Zhang , Yuguo Zheng
IPC分类号： C12P19/14 , C12N9/42
CPC分类号： C12P19/14 , C12N9/2437 , C12Y302/01004 , C12P2201/00
摘要： The present invention provides a method of improving a biomass high-solid enzymatic hydrolysis rate. By screening particle sizes of the biomass substrate, different water content controls are performed on coarse and fine particle biomass substrates and then an entire water content of the mixture of the coarse and fine particle biomass substrates is controlled to achieve a reduced initial viscosity of the enzymatic hydrolysis and improve liquefaction speed, and finally improve the biomass high-solid enzymatic hydrolysis rate. The enzymatic hydrolysis system of the present invention can be easily constructed with lower equipment requirements, lower energy consumption, higher enzymatic hydrolysis efficiency and simpler operation process, helping the industrial production and applications.

8. 发明授权

US11525131B2 Recombinant vector constructed from an encoding gene of a nitrilase mutant, a recombinant genetic engineered strain and application thereof 有权
公开(公告)号：US11525131B2
公开(公告)日：2022-12-13
申请号：US17165319
申请日：2021-02-02
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Yuguo Zheng , Zhe Xu , Zhiqiang Liu
IPC分类号： C12N9/78 , C12P13/00 , C12N15/63
摘要： The present invention discloses a recombinant vector constructed from an encoding gene of a nitrilase mutant, a recombinant genetic engineered strain and application thereof the nucleotide sequence of the gene is shown in SEQ ID No. 5, and the amino acid sequence of the mutant is shown in SEQ ID No. 6. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NITS-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

9. 发明授权

US11987826B2 Nitrilase mutant and application thereof in the synthesis of an anti-epileptic drug intermediate 有权
公开(公告)号：US11987826B2
公开(公告)日：2024-05-21
申请号：US17604623
申请日：2020-12-11
申请人： ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Yaping Xue , Neng Xiong , Peijin Lv , Yuguo Zheng
IPC分类号： C12N9/78 , C12P7/40 , C12P7/54 , C12P13/00
CPC分类号： C12N9/78 , C12P13/002 , C12Y305/05001
摘要： The present invention provides a nitrilase mutant protein with increased thermal stability and its application in the synthesis of an anti-epileptic drug intermediate, wherein the mutant is obtained by mutating one or two of the amino acids at position 151, 223 and 250 of the amino acid sequence shown in SEQ ID No. 2. the thermal stability of the nitrilase mutant AcN-T151V/C223A/C250G was increased by up to 1.73 folds. The yield of the final product was up to 95% using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1M 1-cyanocyclohexylacetonitrile to produce 1-cyanocyclohexyl acetic acid at 35° C. And the yield of the final product was up to 97% when hydrolyzing 1.2M 1-cyanocyclohexylacetonitrile at 35° C. The final yield was up to 80% when using the nitrilase mutants obtained by the present invention to synthesize gabapentin.

10. 发明公开

US20240011057A1 METHOD FOR PREPARING XYLITOL BY USING XYLOSE SECONDARY MOTHER LIQUOR THROUGH FERMENTATION 审中-公开
公开(公告)号：US20240011057A1
公开(公告)日：2024-01-11
申请号：US18023496
申请日：2021-11-30
申请人： ZHEJIANG HUAKANG PHARMACEUTICAL CO., LTD. , ZHEJIANG UNIVERSITY OF TECHNOLOGY
发明人： Xuhao Zeng , Jiaxing Luo , Jing Wang , Changhui Hu , Shuncheng Fang , Zhiqian Liu , Xue Cai , Xiaojian Zhang , Yuguo Zheng , Mian Li
IPC分类号： C12P7/18 , C12N1/20
CPC分类号： C12P7/18 , C12N1/205 , C12N2500/34 , C12N2500/74 , C12N2500/32 , C12N2500/76 , C12N2500/14 , C12R2001/19
摘要： The present invention relates to a method of preparing xylitol by using a xylose secondary mother liquor through fermentation. In the method, fermentation treatment is performed on a mixed liquid of the xylose secondary mother liquor and a xylose concentrate, and the xylose secondary mother liquor is subsequently batch-fed instead of glucose in the fermentation process. Thus, a glucose content in the fermentation system is stabilized, and some xylose is provided at the same time. In this way, the use amount of glucose and costs are reduced, and the system xylose can be relatively stabilized, thus improving the entire conversion rate. In the present invention, adding the xylose secondary mother liquor by mixed fermentation and batch feeding and the like enables the contents of heterosaccharides such as arabinose and mannose in the fermentation system to be always within a range of metabolization by fermenting strains, facilitating subsequent purification and improving the xylitol purity.

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