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.

Abstract: 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.

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.

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.

Abstract: Related to are a device and a method for performing continuous carbonation and impurity removal for xylose mother liquor.

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.

Abstract: 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.

Abstract: The present invention discloses a polyphosphate kinase mutant, engineered strain and application thereof, wherein the polyphosphate kinase mutant is obtained by single- or multi-site mutations of the amino acid at position 79, 106, 108, 111 or 285 of the amino acid sequence shown in SEQ ID No. 2. The present invention provides a variety of polyphosphate kinase mutants derived from Cytophaga hutchinsonii, and the specific enzyme activity of these mutants is 2.7-17.9 times higher than that of the parent polyphosphate kinase, more than 70% of the amount of adenosine triphosphate (ATP) consumption in ATP-dependent biocatalytic synthesis reactions may be reduced by the ATP regeneration system constituted by the mutants, which has broad industrial application prospects.

Abstract: 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.

Abstract: The present invention discloses a method for preparing L-glufosinate ammonium by biological enzymatic de-racemization, a glufosinate ammonium dehydrogenase mutant and a use thereof. The method for preparing L-glufosinate ammonium by biological enzymatic de-racemization includes catalyzing D,L-glufosinate ammonium as a raw material by a multi-enzyme catalysis system to obtain L-glufosinate ammonium. The enzyme catalysis system includes D-amino acid oxidase for catalyzing D-glufosinate ammonium in the D,L-glufosinate ammonium to 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid, and a glufosinate ammonium dehydrogenase mutant for catalytically reducing 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butanoic acid to L-glufosinate ammonium. The glufosinate ammonium dehydrogenase mutant is obtained by mutation of glufosinate-ammonium dehydrogenase in wild fungi Thiopseudomonas denitrificans at a mutation site of V377S.

Abstract: 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 glutamate dehydrogenase by 2-fold, resulting in a significant increase in the spatiotemporal yield of the-glufosinate synthesis reaction.

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.

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%.

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.

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.

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.

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.

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.

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.

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.