59 results about "Dehalogenase" patented technology

A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate and has at least two amino acid substitutions relative to the wild-type hydrolase. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.

The present invention relates to halocarbon and halohydrocarbon chemistry, including methods of dehalogenating halocarbons and halohydrocarbons to provide, inter alia, alcohols, polyols, and epoxides. In general, the methods involve reaction pathways catalyzed by altered hydrolase enzymes that can provide stereoselective or stereospecific reaction products. The invention also includes methods of providing altered nucleic acids that encode altered dehalogenase or other hydrolase enzymes. Additionally, the invention includes various reaction formats and kits.

The invention provides a gene engineering bacteria for producing dehalogenase and realizes the industrial production of dehalogenase industrialised by culturing the gene engineering bacteria via fermentation technology. The invention also provides the use of the gene engineering bacteria; and the halogenohydrin dehalogenase produced by the gene engineering bacteria can be applied to ring-opening reaction of ortho-halogenohydrin dehalogenation epoxidation and epoxide.

The invention belongs to the technical field of wastewater environmental-protection treatment, and particularly relates to a microorganism water treatment agent for heavy metal organic wastewater. The microorganism water treatment agent is prepared from the following raw materials: oyster shells, potassium feldspar, olivine, talc, serpentine, chlorite, attapulgite, ammonium molybdate modified bentonite, diatomaceous earth, aluminium polychlorid, magnesium hydrate, gram-negative bacteria, Bacillus mojavensis, polyacrylamide, tartaric acid, malic acid, citric acid, chitosan, ethylene glycol diglycidyl ether, epoxy chloropropane, thiosemicarbazide cellulose, corn starch, calcium alginate, hansenula polymorpha, candida, trailing plant, fagus longipetiolata sawdust, pinus sylvestris sawdust, walnut shell, lupulus, dehalogenase, horseradish peroxidase, urease and propionibacterium pentosaceum. A precipitate (alum floc) produced after treatment with the microorganism water treatment agent has good strippability, no solid particle is released into water basically, and the microorganism water treatment agent has rapid action speed and stable treatment effect, so that the adsorption time is usually not required to be prolonged during use.

A distal tip, and associated method of producing, of a biosensor ion sensing transducer for use in detecting an analyte comprising an halogenated organic compound in an environment such as soil or an aqueous environment. Features include: a biocomponent comprising at least one enzyme for carrying out a dehalogenation of the compound; the biocomponent is immobilized to a surface of the tip; a treatment of the biocomponent for maintaining a period of enzymatic efficacy; and the biocomponent stabilized by means preferably selected from the group consisting of crosslinking a surface of the immobilized biocomponent, crosslinking a polymer layer to the biocomponent, adding a gel-hardening agent to the biocomponent, adding a stabilizing agent to the biocomponent, and modifying a component of the immobilizing means. Immobilization can be carried out by: (a) entrapment within a hydrogel; (b) entrapment within a polymeric network; (c) (micro)encapsulation; (d) covalent-bonding; and (e) adsorption. The dehalogenase of the biocomponent may be selected from the group consisting of hydrolases, subclass EC 3.8, and lyases, subclass EC 4.5 as listed in Table 2A.

The present invention provides a new strain of bacillus circulans WZ-12 which can degrade methylene chloride and an application of the bacillus circulans WZ-12 for microorganism to decompose and dispose the methylene chloride and preparing for methylene chloride dehalogenase. The bacillus circulans WZ-12 is conserved in the China Center for Typical Culture Collection; the conserving date is 19th, January, 2007; the conserving serial number CCTCC No. is M207006. The beneficial effect of the present invention mainly lies in providing the new strain of the bacillus circulans WZ-12 which can degrade the methylene chloride, the bacillus can degrade the methylene chloride fast under the aerobic condition, which provides important base for researching into the biological degradation mechanism of halohydrocarbon organic pollutant represented by the methylene chloride, and the bacillus can also be applied to a biological disposal technology of organic waste gas directly. Besides, the strain is wild type with very clear genetic background, which is fit for genetic improvement and is hopeful to improve the capability of disposing the organic waste gas greatly.

The present invention relates to a near-universal non-corrosive, non-toxic, environmentally safe and user friendly decontaminant capable of detoxifying organophosphorus (OP)-based G-type, V-type neurotoxic chemical warfare, sulfur-mustard, and related OP based hazardous industrial materials in a dry powder form. The decontaminant contains OPH enzyme, OPAA enzyme, DFPase enzyme, dehalogenase enzyme, quaternary ammonium salt, a pH control reagent, a fire-fighting agent, and a foaming agent. The decontaminant is mixed with available water for use.

The invention relates to a new method of biologically synthesizing (R)-3-hydroxylglutarate monoester and more particularly provides a method of preparing the (R)-3-hydroxylglutarate monoester from (S)-4-chloro-3-hydroxybutyrate through halogenohydrin dehalogenase recombinant bacteria and nitrilase recombinant bacteria, or double-enzyme co-expression recombinant bacteria, in a one-pot manner. The (R)-3-hydroxylglutarate monoester is a key intermediate of statin medicines such as fluvastatin, rosuvastatin, pitavastatin and the like. The method is mild in reaction conditions, is free of pollution and is simple in process route.

The invention relates to a production method of a halide alcohol dehalogenase, which utilizes microbial fermentation to generate halide alcohol dehalogenase, and enhances the output and the activity of the halide alcohol dehalogenase trough optimal fermentation process and fermentation medium. The production method provided by the invention utilizes a genet engineering strain to express recombined escherichia coli of the halide alcohol dehalogenase, the fermentation medium is prepared from 9-15g / L of yeast powder, 4.5-7.5g / L of glycerol, 8-11g / L of NaCl, 1-2g / L of NaNO3, 2-3g / L of KH2PO4, 12-13g / L of K2HPO4 and 2-5mg / L of trace element, and the trace element comprises CoCl12.6H2O, MnCl2.4H2O, CuCl2.4H2O, biotin and the like. The production method provided by the invention can obviously enhance the output of the halide alcohol dehalogenase, and the enzymatic activity can be enhanced to 900U / ml.

Provided is a microorganism including a gene encoding a protein having a dehalogenase activity, a composition for using in reducing a concentration of fluorinated methane in a sample, the composition including the microorganism including the gene encoding the protein having the dehalogenase activity, and a method of reducing the concentration of fluorinated methane in the sample.

The embodiment of the invention provides a biological preparation method of (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate and belongs to the field of biological pharmacy. The preparation method comprises the following steps: by taking (5R)-6-chloro-5-hydroxy-3-tert-butyloxohexanoate as a raw material in a triethanolamine hydrochloride buffer solution, maintaining the pH value to be 6-8 under the actions of a complex enzyme of recombinant ketoreductase and glucose dehydrogenase, dehalogenase, a cofactor and glucose by using a cyaniding reagent, and reacting to obtain the (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate at room temperature. According to the preparation method provided in the invention, the (5R)-6-chloro-5-hydroxy-3-tert-butyloxohexanoate is taken as the raw material, a reduction reaction and a cyano substitution reaction are finished to obtain the product through a one-pot method under the catalysis of the complex enzyme of recombinant ketoreductase and glucose dehydrogenase and the dehalogenase, and the method is simple in process and low in production cost.

The invention discloses a dehalogenase DhmB, an encoding genes and application thereof. The dehalogenase DhmB is a protein in the following (a) or (b); (a) an amino acid sequence is a protein of a sequence 2 in a sequence table; (b) a protein associated with dehalogenation is obtained by the amino acid sequence, which is shown in the sequence 2 of the sequence table and subjected to replacement and / or missing and / or adding of one or a plurality of amino acid residues. The dehalogenase DhmB is easy in purification, good in stability, resistant to high-medium temperature, applicable to the optimum temperature of 65 DEG C, resistant to organic solvents and capable of degrading organohalogen compounds with low-carbon chains, classic pesticides and chemical toxicant and the like and has wide industrial application prospects.

The invention discloses a separation and purification method of dehalogenase with stereoselectivity from a strain of marine bacteria and an application of the dehalogenase in biological resolution. According to the method, two types of 2-haloacid dehalogenase with stereoselectivity, namely L-2-haloacid dehalogenase and D-2-haloacid dehalogenase, are obtained from the strain of the marine bacteria, namely pseudomonas stutzeri by separation and purification. The apparent molecular weight of the enzyme of an enzyme subunit of the L-2-haloacid dehalogenase is about 26kDa, and the L-2-haloacid dehalogenase further has great reaction activity in the pH range of 8-11. D-hydroxy halogenated acid with configuration conversion is generated by selectively catalyzing the conversion of chiral L-2-haloacid through the enzyme. The D-2-haloacid dehalogenase can catalyze the conversion of D-2-haloacid and generate L-2-hydroxy acid, and further has good application prospects as a chiral resolution reagent.

The invention is directed to novel reductive dehalogenase genes encoding for reductive dehalogenases, which are capable of dehalogenating halogenated organic compounds and may be useful for environmental assessment and monitoring, and in the bioremediation of pollutants. In particular, the invention provides isolated polynucleotides of novel reductive dehalogenase genes dcpA and dcpB and fragments thereof as well as isolated polypeptides encoding the DcpA and DcpB proteins or fragments thereof. The invention is also directed to methods of identifying and / or quantifying dechlorinating bacterial organisms or polynucleotides encoding a reductive dehalogenase, such as the dcpA or dcpB polynucleotides or fragments thereof, in a sample.

The invention provides a dehalogenase gene LinB, dehalogenase, dehalogenase genetically engineered bacteria and a construction method and application method of the dehalogenase genetically engineeredbacteria. The dehalogenase gene LinB comprises (a) a nucleotide sequence as shown in SEQ ID NO.1, (b) a nucleotide sequence for coding an amino acid sequence as shown in SEQID NO.2, or (c), a variantof (a) or(b). The dehalogenase comprises polypeptide coded by a nucleotide sequence as shown in SEQID NO.1, an amino acid sequence as shown in SEQ ID NO.2, or a variant of the amino acid sequence. Theconstruction method of the dehalogenase genetically engineered bacteria comprises the steps of cloning the dehalogenase gene LinB into a recombinant expression carrier, and performing guiding into host cells so as to obtain the dehalogenase genetically engineered bacteria capable of expressing the dehalogenase.

The invention discloses an enzyme electrode, which is prepared by the following steps of: preparing recombinant engineering bacteria by using a DCM degrading bacterium Methylobacterium H13, and obtaining DCM dehalogenase pure enzyme solution through induction culture expression and purification; mixing the DCM dehalogenase pure enzyme solution with sodium alginate to obtain an embedding material;spreading the embedding material on the surface of an electrode matrix, and fixing the embedding material by Ca (NO3)2 and the like to obtain the enzyme electrode. The invention also provides a methodfor regenerating coenzyme-reduced glutathione based on electrochemistry by using the enzyme electrode, which comprises the following steps of: injecting DCM-containing wastewater into a cathode electrolytic tank, and performing constant current electrolysis on the reduced glutathione GSH serving as coenzyme. The method utilizes electrochemical technology and a medium of the enzyme electrode to provide sufficient electrons for regenerating coenzyme GSH and promote GSSG to regenerate GSH.

The invention discloses a method for degrading 1,2,3-TCP (trichloropropane) by engineered strain bacillus subtilis. According to the method, firstly, alkyl halide dehalogenase genes, halogenohydrin dehalogenase genes and epoxide hydrolase genes which can degrade 1,2,3-TCP and intermediate products of the 1,2,3-TCP are synthesized; the gene sequence and the codase amino acid sequence are respectively shown as SEQ ID NO.1 to 6; then, the bacillus subtilis integrating carrier containing the gene is built; the carrier converts the bacillus subtilis; by a gene ectopic integration and resistance gene knock-out method, the gene is integrated into the bacillus subtilis genome; the engineered strain bacillus subtilis capable of secreting and expressing alkyl halide dehalogenase, halogenohydrin dehalogenase and epoxide hydrolase is obtained. The engineered strain bacillus subtilis has a good degradation effect on TCP and TCP intermediate products; the complete TCP degradation can be realized; the characteristics of high degradation efficiency, high TCP tolerance, stable inheritance and the like are realized; the application prospects are good.

The invention discloses a fluoroacetic acid dehalogenase mutant. The sequence of the fluoroacetic acid dehalogenase mutant comprises a sequence obtained by mutating the 155th amino acid residue H shown in SEQ ID NO.1 and / or the 156th amino acid residue W shown in SEQ ID NO.1. The fluoroacetic acid dehalogenase mutant has the activity of catalyzing a bromination substrate, particularly a 2-bromobutyric acid substrate. The invention also provides an application of the fluoroacetic acid dehalogenase mutant in preparation of (R)-2-bromobutyric acid and / or (R)-2-hydroxybutyric acid. When the fluoroacetic acid dehalogenase mutant is used for preparing (R)-2-bromobutyric acid, the production cost is low, the stereoselectivity is high, and industrial production is facilitated.

The invention discloses a dehalogenase HadD14 as well as a coding gene and application thereof. The protein provided by the invention is a protein shown as a sequence 2 in a sequence table. Experiments prove that the soluble expression quantity of the dehalogenase HadD14 (sequence 2) can be remarkably increased through prokaryotic expression of escherichia coli by adopting the modified gene sequence (sequence 1), and the obtained dehalogenase HadD14 has high activity, has thermal stability under the treatment condition of not higher than 70 DEG C, and has pH stability under the condition of pH 5.0-8.2; and under the treatment conditions of not higher than 10 percent of methanol, 10 percent of ethanol and 30 percent of DMSO (Dimethyl Sulfoxide), relatively strong organic solvent stability is achieved.

The invention discloses a preparation method of a linezolid intermediate. According to the method, the linezolid intermediate is obtained by reaction of an epoxide and a cyanate in water under the catalysis of dehalogenase. The reaction temperature is 40-60 DEG C, the reaction pH is 7.0-9.0, the content in percentage by mass-water volume of the epoxide is 1%-15%, and the amount of the dehalogenase is 50000U of the epoxide per gram-2 million U of the epoxide per gram. According to the preparation method disclosed by the invention, the dehalogenase is used for a catalytic reaction, the yield can achieve 90%, and in comparison with the yield without the use of the dehalogenase, the yield is obviously greatly improved; and furthermore, chiral separation is not required, high-temperature and high-pressure conditions are not required, the reaction is mild, the safety is high, the preparation method is environment-friendly, the raw materials are low in cost and easy to obtain, the reaction yield is high, and the preparation method is further suitable for industrial production.

The invention discloses dehalogenase HldD1, an encoding gene thereof and application of the dehalogenase HldD1. The invention provides a protein represented by a sequence 2 of a sequence list. Experiments prove that the dehalogenase HldD1 obtained through subjecting a gene sequence (sequence 1) disclosed by the invention to prokaryotic expression through Escherichia coli has high activity and has relatively high organic solvent stability separately under the treatment conditions of not higher than 20% of ethanol, not higher than 20% of acetonitrile and not higher than 40% of DMSO.

Disclosed is a process in which a recombinant organism, such as a yeast, expressing a heterologous S-adenosylmethionme (SAM)-dependent methyl halide transferase (MHT) protein is combined with a halide and a carbon source in a cultivation medium under conditions in which methyl formate is produced. The cell may genetically modified to express methyl formate synthase, methanol dehydrogenase and / or hydrolytic dehalogenase at levels higher than a cell of the same species that is not genetically modified. The methyl formate may be collected and used in a variety of applications. The halide may be chlorine, bromine or iodine.

The invention belongs to the technical field of wastewater environmental-protection treatment, and particularly relates to a microorganism water treatment agent for heavy metal organic wastewater. The microorganism water treatment agent is prepared from the following raw materials: oyster shells, potassium feldspar, olivine, talc, serpentine, chlorite, attapulgite, ammonium molybdate modified bentonite, diatomaceous earth, aluminium polychlorid, magnesium hydrate, gram-negative bacteria, Bacillus mojavensis, polyacrylamide, tartaric acid, malic acid, citric acid, chitosan, ethylene glycol diglycidyl ether, epoxy chloropropane, thiosemicarbazide cellulose, corn starch, calcium alginate, hansenula polymorpha, candida, trailing plant, fagus longipetiolata sawdust, pinus sylvestris sawdust, walnut shell, lupulus, dehalogenase, horseradish peroxidase, urease and propionibacterium pentosaceum. A precipitate (alum floc) produced after treatment with the microorganism water treatment agent has good strippability, no solid particle is released into water basically, and the microorganism water treatment agent has rapid action speed and stable treatment effect, so that the adsorption time is usually not required to be prolonged during use.

The invention discloses a protein one-step directional immobilization method based on a Halo-tag specific dehalogenation reaction. According to the method, a Halo-tag gene is recombined to an inactiveC terminal of target receptor protein, the surface of a separation medium is modified with halogenated alkanoic acid through an acylation reaction, induced expression is performed, and then by utilizing a specific dehalogenation reaction between C-terminal dehalogenase of a recombinant receptor and haloalkane on the surface of the separation medium, one-step directional covalent immobilization ofthe Halo-tag recombinant receptor in a cell lysis solution is realized. The novel protein one-step directional immobilization method is established. Due to the fact that one-step high-specificity immobilization of the protein can be achieved through the cell lysis solution, the problems that the protein immobilization period is long and the specificity of a protein immobilization method is low are effectively solved.

The invention is directed to novel reductive dehalogenase genes encoding for reductive dehalogenases which are capable of dehalogenating halogenated organic compounds and may be useful in the bioremediation of pollutants. In particular, the invention provides an isolated polynucleotide of a novel vinyl chloride dehalogenase gene (bvcA). The novel vinyl chloride dehalogenase gene encodes a reductive dehalogenase that is capable of the complete reduction of vinyl chloride to ethene.

The invention relates to a process for enzymatic synthesis of (R)-4-cyano-3-hydroxybutyrate from sodium alginate immobilized halogenohydrin dehalogenase. The process comprises the following steps: firstly, mixing sodium alginate with HHDH, cross-linking with glutaraldehyde, embedding by using calcium chloride, immobilizing for 1 hour, after the immobilization rate of HHDH is greater than 46%, enabling immobilized enzyme and a substrate (S)-4-chlorine-3-hydroxybutyrate (ATS-4) to react for 4 hours till the generation rate of ATS-5 is not less than 70%, recycling HHDH, and performing secondary catalytic reaction, wherein the generation rate of ATS-5 is not less than 35%. By adopting the method of sodium alginate immobilized halogenohydrin dehalogenase, the immobilization velocity of sodium alginate to halogenohydrin dehalogenase is rapid, the immobilization rate is high, and after the sodium alginate immobilized halogenohydrin dehalogenase is recycled after first enzymatic synthesis of (R)-4-cyano-3-hydroxybutyrate, relatively high catalytic efficiency can be stilled maintained, and the immobilization effect is good.