Patents by Inventor Yuguo ZHENG

Yuguo ZHENG has filed for patents to protect the following inventions. This listing includes patent applications that are pending as well as patents that have already been granted by the United States Patent and Trademark Office (USPTO).

  • Patent number: 11535839
    Abstract: The present invention discloses encoding genes of nitrilase mutants 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, 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.
    Type: Grant
    Filed: February 2, 2021
    Date of Patent: December 27, 2022
    Assignee: ZHEJIANG UNIVERSITY OF TECHNOLOGY
    Inventors: Yaping Xue, Yuguo Zheng, Zhe Xu, Zhiqiang Liu
  • Publication number: 20220396816
    Abstract: The present invention discloses a transaminase mutant and application thereof in preparation of sitagliptin intermediates, the transaminase mutant is obtained by substitution of tyrosine with proline at position 74, substitution of glutamic acid with aspartic acid at position 228, substitution of leucine with alanine at position 254 and substitution of methionine with threonine at position 290 of the amino acid sequence shown in SEQ ID NO: 2. The present invention uses wet cells or a purified transaminase as a biocatalyst and a sitagliptin precursor ketone or a prochiral carbonyl compound as a substrate to prepare a sitagliptin intermediate or a sitagliptin ester intermediate; the total yield of the method reaches about 82%, and e.e. value of the product reaches 99%.
    Type: Application
    Filed: January 21, 2021
    Publication date: December 15, 2022
    Inventors: Zhiqiang LIU, Feng CHENG, Xiaojian ZHANG, Dongxu JIA, Yuguo ZHENG, Renbao HE, Yizhong JIN, Hongming SHAO, Jiaohua LIN, Feng ZHANG
  • Patent number: 11525131
    Abstract: 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.
    Type: Grant
    Filed: February 2, 2021
    Date of Patent: December 13, 2022
    Assignee: ZHEJIANG UNIVERSITY OF TECHNOLOGY
    Inventors: Yaping Xue, Yuguo Zheng, Zhe Xu, Zhiqiang Liu
  • Publication number: 20220372531
    Abstract: The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D/A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50° C.). 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.
    Type: Application
    Filed: December 11, 2020
    Publication date: November 24, 2022
    Inventors: Yaping XUE, Neng XIONG, Qian LI, Yuguo ZHENG
  • Patent number: 11492606
    Abstract: 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.
    Type: Grant
    Filed: January 31, 2019
    Date of Patent: November 8, 2022
    Assignee: ZHEJIANG UNIVERSITY OF TECHNOLOGY
    Inventors: Renchao Zheng, Yuguo Zheng, Qin Zhang, Zheming Wu, Xiaoling Tang, Xiafeng Lu
  • Publication number: 20220307061
    Abstract: Disclosed are a phosphinothricin dehydrogenase mutant, a recombinant bacterium and a one-pot multi-enzyme synchronous directed evolution method. The phosphinothricin dehydrogenase mutant, with an amino acid sequence as shown in SEQ ID No.1, is obtained by mutating alanine at position 164 to glycine, arginine at position 205 to lysine, and threonine at position 332 to alanine in a phosphinothricin dehydrogenase derived from Pseudomonas fluorescens. The recombinant bacterium is obtained by introducing a gene encoding the phosphinothricin dehydrogenase mutant into a host cell. The host cell can also incorporate a gene encoding a glucose dehydrogenase or a gene encoding a formate dehydrogenase to undergo synchronous directed evolution to achieve double gene overexpression. The one-pot multi-enzyme synchronous directed evolution method of the present invention can screen recombinant bacteria with greatly improved activity.
    Type: Application
    Filed: December 26, 2020
    Publication date: September 29, 2022
    Inventors: YAPING XUE, FENG CHENG, JUMOU LI, QINGHUA LI, YUGUO ZHENG, SHUPING ZOU, JIANMIAO XU
  • Patent number: 11408016
    Abstract: The present invention discloses an amino acid dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium, the amino acid dehydrogenase mutant is obtained by a single mutation or a multi-site mutation of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID No. 2. The amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the present invention has a specific enzyme activity that is 33 times higher than that of the original Aldo-keto reductase, and the concentration of the largest substrate, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reaches 500 mM, the amino acid dehydrogenase mutant has more industrial application prospects.
    Type: Grant
    Filed: June 28, 2019
    Date of Patent: August 9, 2022
    Assignee: ZHEJIANG UNIVERSITY OF TECHNOLOGY
    Inventors: Yaping Xue, Feng Cheng, Heng Li, Yuguo Zheng, Jianmiao Xu
  • Publication number: 20220220516
    Abstract: A method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multienzyme coupling, where D,L-phosphinothricin as a raw material is catalyzed by an enzyme catalysis system to obtain L-phosphinothricin, wherein the enzyme catalysis system comprises a D-amino acid oxidase mutant for catalyzing D-phosphinothricin in D,L-phosphinothricin into 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid and a transaminase for catalytic reduction of the 2-carbonyl-4-[hydroxy(methyl) phosphono]butyric acid into L-phosphinothricin; the D-amino acid oxidase mutant is obtained by mutation of D-amino acid oxidase in wild strain Rhodotorula taiwanensis at one of the following four sites: (1) M213S; (2) M213S-N54V-F58E; (3) M213S-N54V-F58E-D207A; (4) M213S-N54V-F58E-D207A-S60T.
    Type: Application
    Filed: June 8, 2020
    Publication date: July 14, 2022
    Inventors: YAPING XUE, FENG CHENG, LIUYU WANG, YUGUO ZHENG, JIANMIAO XU, SHUPING ZOU
  • Publication number: 20220204948
    Abstract: 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.
    Type: Application
    Filed: October 20, 2021
    Publication date: June 30, 2022
    Inventors: YAPING XUE, FENG CHENG, DONGYANG WU, SHUPING ZOU, JIANMIAO XU, YUGUO ZHENG
  • Publication number: 20220204949
    Abstract: Disclosed are a gene mining method combining functional sequence and structure simulation, an NADH-preferring phosphinothricin dehydrogenase mutant and an application thereof. The gene mining method comprises the following steps: (1) analyzing a characteristic sequence which an NADH-type glutamate dehydrogenase should have; (2) searching a gene library based on the characteristic sequence; (3) performing clustering analysis and protein structure simulation on genes obtained by the searching; (4) selecting genes that feature high gene aggregation and a protein structure similar to that of the known phosphinothricin dehydrogenase as candidate genes. A wild-type phosphinothricin dehydrogenase with an amino acid sequence as set forth in SEQ ID No.
    Type: Application
    Filed: December 30, 2021
    Publication date: June 30, 2022
    Inventors: YAPING XUE, FENG CHENG, JIAMIN ZHANG, SHUPING ZOU, JIANMIAO XU, YUGUO ZHENG
  • Publication number: 20220177868
    Abstract: 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 205 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.
    Type: Application
    Filed: December 11, 2020
    Publication date: June 9, 2022
    Inventors: Yaping XUE, Neng XIONG, Peijin LV, Yuguo ZHENG
  • Publication number: 20220135960
    Abstract: The present invention provides a polypeptide tag and its application in the synthesis of pharmaceutical chemicals, the recombinant nitrilase was obtained by connecting a polypeptide tag to the N-terminus of the amino acid sequence of the nitrilase; wherein amino acids at both ends of the polypeptide tag are uncharged glycine G, and the rest are a random combination of any one or more of glycine G, histidine H, glutamic acid E, aspartic acid D, lysine K and arginine R; The activity of the recombinant nitrilase in the preparation of 1-cyanocyclohexyl acetic acid is up to 3034.7 U/g dcw, the polypeptide tag significantly improves the soluble expression of nitrilase, and the whole cell catalyst hydrolyzes 1M substrate with the same concentration 30 minutes faster than the mother enzyme.
    Type: Application
    Filed: April 26, 2021
    Publication date: May 5, 2022
    Inventors: Yaping XUE, Dong XIE, Neng XIONG, Yuguo ZHENG
  • Publication number: 20220090029
    Abstract: 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%.
    Type: Application
    Filed: June 28, 2019
    Publication date: March 24, 2022
    Inventors: Yaping XUE, Feng CHENG, Qinghua LI, Yuguo ZHENG, Jianmiao XU
  • Publication number: 20210395174
    Abstract: The present invention relates to a xylitol preparation device integrating evaporation, crystallization and centrifugation, including a xylitol tank, a cleaning liquid tank, a recycling tank and a multiple distribution system, wherein the multiple distribution system includes J groups of evaporators for evaporation concentration, K groups of vacuum crystallization kettles for vacuum crystallization and L groups of centrifuges for centrifugation, wherein 2?J?6, 6?K?12 and 2?L?4; the evaporator, the vacuum crystallization kettle and the centrifuge in different groups are sequentially connected in series with one another through a pipeline and a valve respectively; by controlling on and off of each valve, a xylitol exchange liquid is switched and controlled between a series-connection mode and a parallel-connection mode in the multiple distribution system to enable evaporation, crystallization and separation processes to reach an optimal effect distribution so as to improve productivity.
    Type: Application
    Filed: March 13, 2020
    Publication date: December 23, 2021
    Inventors: Jian Yang, Yi ZHENG, Taogang ZHANG, Han GAO, Mian LI, Zhiqiang LIU, Yuguo ZHENG, Baoxing MAO, Xiaojian ZHANG, Weiwei FAN
  • Publication number: 20210381069
    Abstract: Related to are a device and a method for performing continuous carbonation and impurity removal for xylose mother liquor.
    Type: Application
    Filed: December 6, 2019
    Publication date: December 9, 2021
    Inventors: Jiaxing LUO, Guowei LUO, Chengjun LIAO, Songtao JIANG, Yuan ZHOU, Deshui CHEN, Zhiqiang LIU, Xiaojian ZHANG, Yuguo ZHENG, Mian LI
  • Publication number: 20210155919
    Abstract: The present invention discloses encoding genes of nitrilase mutants 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, 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.
    Type: Application
    Filed: February 2, 2021
    Publication date: May 27, 2021
    Inventors: Yaping XUE, Yuguo ZHENG, Zhe XU, Zhiqiang LIU
  • Publication number: 20210155920
    Abstract: 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 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.
    Type: Application
    Filed: February 2, 2021
    Publication date: May 27, 2021
    Inventors: Yaping XUE, Yuguo ZHENG, Zhe XU, Zhiqiang LIU
  • Patent number: 11001823
    Abstract: 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.
    Type: Grant
    Filed: January 24, 2019
    Date of Patent: May 11, 2021
    Assignee: ZHEJIANG UNIVERSITY OF TECHNOLOGY
    Inventors: Yaping Xue, Yuguo Zheng, Zhe Xu, Zhiqiang Liu
  • Publication number: 20210071215
    Abstract: The present invention discloses an amino acid dehydrogenase mutant and application thereof in synthesizing L-glufosinate-ammonium, the amino acid dehydrogenase mutant is obtained by a single mutation or a multi-site mutation of the amino acid at position 95, 108, 172, 303 of the amino acid sequence shown in SEQ ID No.2. The amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the present invention has a specific enzyme activity that is 33 times higher than that of the original Aldo-keto reductase, and the concentration of the largest substrate, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid reaches 500 mM, the amino acid dehydrogenase mutant has more industrial application prospects.
    Type: Application
    Filed: June 28, 2019
    Publication date: March 11, 2021
    Inventors: Yaping XUE, Feng CHENG, Heng LI, Yuguo ZHENG, Jianmiao XU
  • Publication number: 20210047609
    Abstract: The present invention discloses a method for promoting the fermentation of Gluconobacter oxydans to produce D-sorbitol dehydrogenase and pyrroloquinoline quinone. The method comprises: Gluconobacter oxydans is inoculated to a fermentation culture medium, fermented and cultured under the conditions of 28-32° C. and 150-180 rpm for 6-24 hours, the fermented solution is centrifuged, and wet bacteria are collected, thus acquiring bacteria cells containing D-sorbitol dehydrogenase and pyrroloquinoline quinone. The method promotes the synthesis of coenzyme pQQ and the enzyme activity of per unit volume D-sorbitol dehydrogenase, Gluconobacter oxydans cultured and acquired using the method is biotransformed to synthesize miglitol precursor 6-deoxy-6-amino(N-hydroxyethyl)-?-L-furan sorbose (6NSL), the conversion progress of the product 6NSL is increased by 21-35%, and a biotransformation step cycle is reduced from 48 hours to 36 hours.
    Type: Application
    Filed: January 24, 2019
    Publication date: February 18, 2021
    Inventors: Yuguo ZHENG, Xia KE, Yanghui LU, Zhongce HU, Yang WU