231 results about "Glucose dehydrogenase (pyrroloquinoline-quinone)" patented technology

The present invention relates to a biosensor which comprises an electrode system including at least one pair of electrodes, at least one insulating base plate for supporting the electrode system, a first reaction layer provided at least on a working electrode of the electrode system, including an organic compound having a functional group capable of bonding or being adsorbed to an electrode and a hydrophobic hydrocarbon group, a second reaction layer provided on the first reaction layer, including an amphiphilic lipid capable of bonding or being adsorbed to a hydrophobic portion of the first reaction layer, and a reagent system carried in a two-component membrane composed of the first and second reaction layers, including at least membrane-binding type pyrroquinoline quinone-dependent glucose dehydrogenase and an electron mediator.

An object of the present invention is to provide: a novel gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase having excellent properties that it has excellent reactivity to glucose, excellent thermal stability, and excellent substrate-recognition performance and also has a low activity for maltose; a process for the production of the enzyme using a transformant cell transfected with the gene; and a method for the determination of glucose, a reagent composition for use in the determination of glucose, a biosensor for use in the determination of glucose and others, each characterized by using the enzyme obtained. The invention relates to a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide containing an amino acid sequence: X1-X2-X3-X4-X5-X6 (wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue); and others.

The invention discloses an alcohol dehydrogenase, a gene thereof, a recombinant expression vector and a recombinant expression transformant respectively containing the gene, a recombinase of the alcohol dehydrogenase, and an application of the alcohol dehydrogenase in asymmetric reduction synthesis of chiral diaryl secondary alcohol as a catalyst, and belongs to the technical field of bioengineering. The alcohol dehydrogenase is from Kluyveromyces sp. CCTCCM2011385, has a carbonyl group reduction function, and also has a hydroxy group oxidation function. Extra addition of glucose dehydrogenase and other enzymes used for cofactor circulation is not needed when the alcohol dehydrogenase is used in the reduction of diaryl ketone into the chiral diaryl secondary alcohol as a biocatalyst, and the alcohol dehydrogenase has the advantages of high catalysis efficiency, mild reaction conditions, easy product recovery and low cost, so the alcohol dehydrogenase has very good application and exploitation prospect in the production of antihistamine medicines.

A flavin-binding glucose dehydrogenase with a high substrate specificity for D-glucose. The flavin-binding glucose dehydrogenase which is derived from a microorganism belonging to the genus Mucor. The flavin-binding glucose dehydrogenase has a low reactivity for maltose, D-galactose and D-xylose compared to its reactivity for D-glucose, and therefore is relatively unaffected by these saccharide compounds. The flavin-binding glucose dehydrogenase is also relatively unaffected by dissolved oxygen, and allows accurate measurement of glucose amounts even in the presence of saccharide compounds other than glucose in samples.

A fusion protein of pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) and a cytochrome is disclosed. PQQGDH is, for example, a water-soluble PQQGDH derived from Acinetobacter calcoaceticus. The cytochrome is, for example, an electron transfer domain of quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni. The fusion protein of the present invention shows intramolecular electron transfer from PQQ, a redox center, to the cytochrome, which allow construction of a direct electron transfer-type glucose sensor which requires no electron mediators.

The invention discloses a method for biologically preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester with recombinant escherichia coli expressed ketoreductase and application thereof in preparing (S)-4-chloro-3-hydroxy butyric acid ethyl ester through asymmetric reduction of 4-chloroacetoacetic acid ethyl ester. The recombinant escherichia coli for expressing the gene of the ketoreductase (KRED) is coupled with glucose dehydrogenase (GDH) so that the escherichia coli cell is recombined into a catalyst; efficient preparation of (S)-4-chloro-3-hydroxy butyric acid ethyl ester through asymmetric reduction of 4-chloroacetoacetic acid ethyl ester in a single water phase is realized no matter in the presence of or in the absence of coenzyme NAD(P)H; and the molar transformation rate is higher than 92% and the enantiomer excess (e.e.) of the product is 100%.

The invention belongs to the technical field of gene engineering and particularly relates to carbonyl reductase AcCR and an encoding gene and application thereof. The amino acid sequence of the carbonyl reductase AcCR is shown as SEQ ID NO.1. The nucleotide sequence of the gene for encoding the carbonyl reductase AcCR is shown as SEQ ID NO.2. The carbonyl reductase AcCR comes from bacillus aceticus XZY003 and can catalyze 13 carbonyl compounds to generate corresponding chiral alcohol of single enantiomers. The carbonyl reductase AcCR and glucose dehydrogenase GDH are co-expressed in a prokaryotic expression system or a eukaryotic expression system, the biological reaction process of biological catalytic conversion is conducted through the carbonyl reductase, in-situ regeneration of coenzyme is achieved, production cost is greatly reduced, the optical purity of an obtained product is high, the reaction process is efficient, and conditions are moderate.

An object of the present invention is to provide a novel glucose dehydrogenase, a method for producing the glucose dehydrogenase, and applications of the glucose dehydrogenase. The flavin-binding glucose dehydrogenase of the invention has the following characteristics (1) and (4): (1) Molecular weight: the molecular weight of a polypeptide moiety in the enzyme is about 68 kDa as measured by SDS-polyacrylamide electrophoresis; (2) Km value: the Km value for D-glucose is about 15 mM or less; (3) Temperature stability: stable at a temperature of 55° C. or less; and (4) pH stability: stable at a pH range of 3.0 to 8.5.

The invention discloses a method for preparing (R)-phenylglycol from SD-AS sequence coupled (R)-carbonyl reductase and glucose dehydrogenase and belongs to the technical field of biological catalytic asymmetric transformation. The invention provides recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G. The recombinant escherichia coli E. coli RIL/pET-R-SD-AS-G is preserved in the China center for type culture collection (CCTCC) and has a preservation number of CCTCC NO: M2015170. R-carbonyl reductase and glucose dehydrogenase are coupled by a coexpression way, and the coupled enzyme, a cultured recombinant strain and 2-hydroxyacetophenone as a substrate undergo a catalytic asymmetric transformation reaction under optimized biotransformation reaction conditions to produce (R)-phenylglycol with optical purity of 100% and a yield of 99.9%. The method utilizes a SD-AS sequence high efficiency coupling double-enzyme technology, solves the problem of limitation of chiral catalytic reaction coenzyme regeneration cycle and provides an effective approach for high efficiency bio-preparation of the (R)-phenylglycol.

The invention relates to a preparation method of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The preparation method is characterized in that tert-butyl (S)-6-chloro-5-hydroxy-3-carbonylhexanoate is taken as a substrate and the substrate is subjected to reduction reaction in the presence of a biocatalyst, a coenzyme and coenzyme regeneration systems to generate tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate; the biocatalyst is ketoreductase; the coenzyme is NADP (nicotinamide adenine dinucleotide phosphate); the coenzyme regeneration systems are glucose and glucose dehydrogenase; the reduction reaction is carried out in a phosphate buffer solution at 25-30 DEG C in the presence of an enzyme protection reagent; the pH value of a reaction system is controlled to be 6.0-8.0 by adding a sodium carbonate solution. The preparation method has the effects of substantially increasing the concentration of the substrate and reducing the usage amount of the enzyme, so that the product has good production environment friendliness and the commercial value of industrialization is prominent.

It is intended to highly efficiently produce a large amount of novel FAD-GDH capable of more accurately measuring a glucose level. Provided is a transformant in which a DNA encoding a flavin adenine dinucleotide-binding glucose dehydrogenase selected from the group consisting of (a) a DNA encoding the amino acid sequence of SEQ ID NO: 20; (b) a DNA consisting of the base sequence of SEQ ID NO: 19; (c) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity; (d) a DNA encoding the amino acid sequence of SEQ ID NO: 34; (e) a DNA consisting of the base sequence of SEQ ID NO: 33; and (f) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity has been introduced. Further, a method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase using the transformant is provided.

The invention belongs to the technical fields of biological pharmacy and biological engineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of (R)-4-chloro-3-hydroxy-butyrate. The carbonyl reductase mutant disclosed by the invention is obtained by replacing phenylalanine at the 85th site of a sequence shown as SEQ ID NO.2 with methionine, or is obtained by replacing one or more sites except the 85th site with amino acid residues on the basis that phenylalanine at the 85th site of the sequence shown as SEQ ID NO.2 is replaced with methionine.The invention also relates to a recombinant expression vector containing the carbonyl reductase mutant gene, an engineering bacterium containing the carbonyl reductase mutant gene and a glucose dehydrogenase gene, and application of the genetic engineering bacterium in preparation of (R)-4-chloro-3-hydroxy-butyrate by asymmetric reduction of chloroacetoacetate. Compared with a wild enzyme, the carbonyl reductase mutant disclosed by the invention has the advantages that the reducing capability on chloroacetoacetate is obviously improved and the carbonyl reductase mutant has good industrial application prospect.

The invention discloses a fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH, and belongs to the technical field of genetic engineering and biological catalysis. Fusion expression is carried out on carbonyl reductase and glucose dehydrogenase to achieve coupling between the two enzymes, and a separated and purified fusion protein in a successfully constructed recombinant bacteria package is utilized to carry out biotransformation COBE to obtain S-CHBE. By optimizing reaction conditions, a substrate of the fusion protein can be totally converted, the optical purity of a product is above 99%, and the TTN reaches up to 1200. The final concentration of the substrate is 305 mmol/L through a substrate batch replenishing strategy, the yield of the product reaches up to 53%, the product of 162 mmol/L is obtained, and the e.e. value remains above 99%. The fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH provide a novel research thought for coupling a coenzyme regeneration cycle approach into a biotransformation approach of the S-CHBE and have great significance for simplifying the a chiral transformed way.

The invention provides an alcohol dehydrogenase LC3 and a gene and application thereof. An amino acid sequence of the alcohol dehydrogenase LC3 is shown in SEQ ID NO:2. An alcohol dehydrogenase gene lc 3 is extracted from lactobacillus curieae, a gene engineering strain is established, the heterologous expression of the alcohol dehydrogenase gene lc 3 is realized, and the enzymatic characteristic of the produced alcohol dehydrogenase LC3 is excellent. A co-expression gene engineering strain containing a glucose dehydrogenase gdh gene and the alcohol dehydrogenase gene lc3 is also successfully established, so as to realize the co-expression of glucose dehydrogenase and alcohol dehydrogenase. The alcohol dehydrogenase LC3 has the characteristics that the alcohol dehydrogenase LC3 and the gene engineering strain are applied to the production of chiral alcohol(R)-4-chloride-3hydroxy ethyl butyrate and other chiral alcohol products by a biological enzyme synthesizing method; the substrate concentration is high, the reaction rate is high, and the convenience is realized.

A mutant glucose dehydrogenase having an amino acid sequence at least 80% identical to SEQ ID NO:3 and having glucose dehydrogenase activity, wherein amino acid residues corresponding to positions 326, 365 and 472 of said amino acid sequence are replaced with glutamine, tyrosine and tyrosine, respectively, and wherein said mutant glucose dehydrogenase shows an improved substrate specificity to glucose and a reduced reactivity to disaccharides.

The invention discloses recombinant escherichia coli for synthesizing lactoyl N-neotetraose and a construction method and application of the recombinant escherichia coli. The recombinant escherichia coli are obtained by knocking out a beta-galactosidase lacZ gene, a glucosamine-6-phosphodeaminase nagB gene, a UDP-acetylglucosamine epitope isomerase wecB gene and a UDP-glucose dehydrogenase ugd gene in the host genome of escherichia coli and overexpressing a lactose transportase lacY gene, beta-1, 3-N-glucosaminidase lgtA gene and a beta-1, 4 galactosyltransferase lgtB gene on the genome. The yield of lactoyl-N-neotetraose synthesized by recombinant escherichia coli reaches 985 mg/L, and a foundation is laid for further metabolic engineering transformation of escherichia coli to produce lactoyl-N-neotetraose.

The invention relates to a production method of glucose dehydrogenase, which mainly uses flow feeding technology in microbial fermentation and genetically engineered microorganisms to ferment to produce glucose dehydrogenase. The production method specifically includes: culturing recombinant escherichia coli containing glucose dehydrogenase gene at the culture temperature of 37 DEG C so as to express glucose dehydrogenase; inoculating the recombinant escherichia coli to culture medium for fermentation, adding supplementary medium into fermented broth containing the recombinant escherichia coli during fermentation by the flow feeding technology, and increasingly the flow feeding speed in a gradient form with prolonging of the fermentation time; and adding inducer when initial growth phase of the bacterium is over, reducing the temperature from 37 DEG C to about 28 DEG C, and maintaining constant temperature until fermentation is over. By the method, fermentation conditions for fermentation process meet actual needs of the bacterium as far as possible, the density of the bacterium can be increased to a high level, the yield of glucose dehydrogenase is increased, and great industrialapplication value is achieved.

The invention discloses a kit for detecting glucose by using a glucose dehydrogenase method, and belongs to the kit for detecting glucose containing enzymes. The kit disclosed by the invention comprises 4 bottles of glucose dehydrogenase method reagents and a glucose standard solution. The kit disclosed by the invention comprises 8.0mmol/L of sodium oxalate dissolved in the phosphate buffer solution of which the concentration is 120.0mmol/L, 6000U/L of glucose dehydrogenase, 180U/L of mutarotase, 4.0 mmol/L of NAD + (Nicotinamide Adenine Dinucleotide), and 0.2% of ProClin300 preservative. In the determination, the sodium oxalate inhibits activities of endogenous lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase to control interfere reaction, so as to increase the specificity of the reaction, and improve the accuracy of detection results. The kit disclosed by the invention is less affected by a sample/reagent volume ratio during a detecting process, and the linear range is up to 35.0mmol/L. The use method of the kit is identical with the original glucose dehydrogenase method, so that the kit does not increase the burden on an experimenter, and almost does not increase the cost of the reagent, is economical, convenient and easy and is the kit for determining glucose by using glucose dehydrogenase method with high accuracy.