Zhejiang University

Zhejiang Huakang Pharmaceutical Co., Ltd.

Zhejiang Chiral Medicine Chemicals Co. Ltd.

Zhejiang Yongtai Technology Co., Ltd.

Zhejiang Yongtai Pharmaceutical Co., Ltd.

Yuguo Zheng

Yaping Xue

Feng Cheng

Jianmiao Xu

Shuping Zou

Zhiqiang Liu

Renchao Zheng

Xiaojian Zhang

Qin Zhang

Zhe Xu

Neng Xiong

Mian Li

Youming Huang

Tianchun Liu

Weirong Fan

Zheming Wu

Jianfeng Weng

Feng Zhang

Jian Yang

Chengjun Liao

Jiaxing Luo

Changhui Hu

Yi Zheng

Renbao He

Hongming Shao

Yizhong Jin

Heng Li

Han Gao

Songtao Jiang

Guowei Luo

Qian Li

Yuan Zhou

Taogang Zhang

Weiwei Fan

Xiaoling Tang

Baoxing Mao

Xiafeng Lu

Deshui Chen

Jiamin Zhang

Dongyang Wu

Qinghua Li

Peijin Lv

Zhentao Jiang

Jumou Li

Liuyu Wang

Dongxu Jia

Jiaohua Lin

Zhiqian Liu

Xue Cai

Jing Wang

Xuhao Zeng

Jie Mao

Andi Dai

Dong Xie

NITRILASE MUTANT AND USE THEREOF IN CATALYTIC SYNTHESIS OF 2-CHLORONICOTINIC ACID

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.

Method of improving biomass high-solid enzymatic hydrolysis rate

The present invention discloses a method for preparing L-glufosinate ammonium powder. The method includes the following steps: (1) obtaining a transformation solution for performing biocatalytic transformation to prepare L-glufosinate ammonium, and filtering out bacteria to obtain a filtrate; (2) detecting the amount of ammonium sulfate in the filtrate obtained in step (1), adding calcium hydroxide or calcium oxide to react with the ammonium sulfate to produce calcium sulfate precipitates, and filtering out the calcium sulfate precipitates to obtain a filtrate; (3) detecting the amount of glufosinate ammonium in the filtrate obtained in step (2), adding zinc salt, adjusting pH to 5.5-6.8 to produce glufosinate ammonium zinc salt precipitates, and filtering and collecting the glufosinate ammonium zinc salt precipitates; (4) adding a solvent to dissolve the glufosinate ammonium zinc salt precipitates collected in step (3), and adjusting pH of a solution to 2-2.5 to produce L-glufosinate ammonium precipitates; and (5) recrystallizing the L-glufosinate ammonium precipitates obtained in step (4) to obtain purified L-glufosinate ammonium powder. The method provided by the present invention is simple to operate and low in cost, and has a better industrialization prospect.

Preparation method for L-glufosinate-ammonium powder

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 recombinantcontaining 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.

Nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid

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%.

Transaminase mutant and application thereof in preparation of sitagliptin intermediates

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 strainat one of the following sites: (1) M213S-N54V-F58E; (2) M213S-N54V-F58E-D207A; (3) M213S-N54V-F58E-D207A-S60T. According to the present invention, the D-amino acid oxidase mutant provides better catalytic efficiency, and when racemic D,L-phosphinothricin is used as a substrate for catalytic reaction, the conversion rate is much higher than that of the wild type enzyme, and the PPO yield is also greatly improved.

Method for asymmetrically preparing L-phosphinothricin by oxidation-reduction reaction through biological multi-enzyme coupling

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. 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. Compared with other catalysis processes such as the transaminase method, the method for preparing L-PPT of the present invention features relatively simple process, high conversion of raw materials of up to 100%, and high stereo selectivity.

Phosphinothricin dehydrogenase mutant, genetically engineered bacterium and one-pot multi-enzyme synchronous directed evolution method

A cofactor self-sufficientand 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, 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.

Cofactor self-sufficientand construction method and application thereof

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%.

Method for the production of glufosinate-ammonium

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 recombinantcontaining 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.

Nitrilase mutant and application thereof in the synthesis of an anti-epileptic drug intermediate

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.2 derived from Lysinibacillus composti is obtained through the gene mining, and then mutated, and an NADH-preferring phosphinothricin dehydrogenase mutant is screened out, which has a mutation site selected from one of the following: (1) A144G-V375F-M91A; (2) A144G-V345A-M91A; (3) A144G. This mutant enzyme can be used for catalytic reaction with an inexpensive coenzyme NAD.

Gene mining method combining functional sequence and structure simulation, NADH-preferring phosphinothricin dehydrogenase mutant and application thereof

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&#x2264;J&#x2264;6, 6&#x2264;K&#x2264;12 and 2&#x2264;L&#x2264;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. The present invention further discloses a control method of the device. The processes and equipment of the present invention are highly integrated to realize continuous integrated production of xylitol preparation with low energy consumption and high automation degree, and full utilization of raw materials.

Xylitol preparation device integrating evaporation, crystallization and centrifugal separation and control method thereof

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. The method provided by the present invention can also be used for the biocatalytic reaction of other pharmaceutical intermediates as the substrate catalyzed by the nitrilase, improving the activity of the whole cell catalyst in reaction, and also improving the solubility of other types of nitrilases and the activity of the corresponding whole cell catalysts.

Polypeptide tag, highly soluble recombinant nitrilase and application thereof in synthesis of pharmaceutical chemicals

Related to are a device and a method for performing continuous carbonation and impurity removal for xylose mother liquor. The device includes an alkali addition unit, a continuous carbonating unit, a discharge controlling unit, a COsupply station, a vapor station and an after-carbonation tank, wherein the alkali addition unit is configured to add Ca(OH)alkaline liquid into xylose mother liquor, the continuous carbonating unit is configured to introduce COsupplied from the COsupply station into the alkali-added xylose mother liquor to perform carbonation and mixing so as to remove impurities such as colloid and pigment in xylose mother liquor, the discharge controlling unit is configured to introduce COsupplied from the COsupply station and vapor transported from the vapor station into the carbonated xylose mother liquor so as to control and stabilize a pH value of the carbonated xylose mother liquor, and the after-carbonation tank is configured to collect and temporarily store the carbonated and impurity-removed xylose mother liquor so as to prepare for a next procedure. Further, a method using the device is disclosed. According to the device and the method, the pH of xylose mother liquor is continuously regulated and stabilized and continuous feeding and discharge are performed with highly automated device so as to achieve continuous and uninterrupted production, and thus facilitate improving the production efficiency.

Device and method for performing continuous carbonation and impurity removal for xylose mother liquor

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.

Machine learning gene mining method and phosphinothricin dehydrogenase mutant for amino translocation

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Zhejiang, China

Yuguo Zheng