137 results about "Isomerase Gene" patented technology

The invention discloses novel N-acetylneuraminic acid-producing escherichia coli engineering bacteria as well as a construction method and application thereof. The engineering bacterial is constructed by introducing an encoded 6-glucosamine phosphate acetylase gene, an N-acetyl glucosamine-2-isomerase gene and an N-acetylneuraminic acid synthetase gene into escherichia coli to express, carrying out strengthened expression on 6-glucosamine phosphate deaminase gene contained in the escherichia coli per se and knocking off genes, for decomposing and utilizing enzyme in metabolic pathways, of the N-acetylneuraminic acid in the engineering bacteria. The engineering bacteria disclosed by the invention can be used for fermentation culture to synthesize the N-acetylneuraminic acid by using glucose or glycerinum as a substrate.

The invention provides a specific isomerase produced from lactic acid bacteria, wherein biological isomerization is employed for preparing conjugated aliphatic acid, the enzyme isomerizes 9c, 12c unsaturated fatty acid to form 9c, 11t conjugated aliphatic acid, such as 9c, 11t conjugated linolic acid, 9c, 11t conjugated linolenic acid. The invention also discloses the process for preparing conjugated aliphatic acid by conducting mutagenesis, modification, isomerase gene cloning to lactic acid bacteria, and various products of the prepared conjugated aliphatic acid and lactic acid bacteria product related with conjugated aliphatic acid.

The invention discloses an E.tenella protein disulfide linkage isomerase gene EtPDI (Clone ID is BW1-E06,and the Genbank accession number of is EF552214). The gene is connected with a procaryon expression vector pGEX-4T-2; a procaryon expression recombination plasmid pGEX-4T-EtPDI is constructed and is expressed in a colibacillus system; and most of the expressed recombining protein exists in a soluble form. The recombining protein 4T-EtPDI is purified to carry out SPS-PAGE and is transferred to a PVDF film; and antiserum of E.tenella oocyst oral immunized chicken is used as first resistance and goat anti-chicken IgG is used as second resistance to carry out Western-blot analysis, thereby indicating that the gene has certain antigen. The gene is used for preparing an anti-chicken coccidiosis drug and an anti-chicken coccidiosis vaccine.

The invention discloses a method for preparing tagatose by using whole-cell catalysis. The method comprises the following steps of transferring plasmid containing an alpha-glucan phosphorylase gene (or cellulosic polysaccharide phosphorylase and cellobiose phosphorylase genes, or a sucrose phosphorylase gene), a phosphoglucomutase gene, a glucose phosphate isomerase gene, a 6-phosphate tagatose epimerase gene, and a 6-phosphate tagatose phosphatase gene into engineered escherichia coli, so as to obtain converted bacterial strain; inducing the converted bacterial strain to produce enzyme, and permeabilizing; mixing the permeabilized bacterial strain, establishing a whole-cell enzyme catalysis reaction system with starch or starch derivative (or mixture of cellulase and cellulose or cellulose derivative, or sugarcane), and performing the whole-cell enzyme catalysis reaction, so as to obtain the tagatose. The method has the advantages that the tagatose is prepared by the whole-cell catalysis; the cost is low, the pollution is low, the yield rate is high, and the like; the method is suitable for preparing the tagatose in a scale way.

The invention relates to a wolfberry lycopene epsilon-cyclase gene and a recombinant vector comprising the gene. The gene is selected from one of the following nucleotide sequences: 1) a nucleotide sequence shown in a sequence SEQ ID NO.1; and 2) a homologous sequence or an allelic gene thereof and a derivative nucleotide sequence thereof in which 70% of one or more nucleotides are added, replaced, inserted or deleted in the nucleotide sequence shown in the sequence SEQ ID NO. 1. By extracting the total RNA of fresh wolfberry leaves, a wolfberry lycopene epsilon-cyclase gene Lm LcyE is cloned by using a 3'RACE technology, and an obtained complete genome sequence is 1569. An escherichia coli expression vector pET28a-LmLcyE is constructed, the enzymatic activity of cloned wolfberry isopentenyl pyrophosphate isomerase gene Lm LcyE encoding is identified by using an escherichia coli heterologous expression system, and the wolfberry lycopene epsilon-cyclase gene Lm LcyE can catalyze lycopene for cyclizing the lycopene into delta carotene, so that metabolism flows downstream, thereby improving the content of alpha-carotenoid.

The invention discloses an immobilization method for cells containing glucose isomerase. The immobilization method comprises the following steps of adding wet bacteria obtained by performing fermented culture on a recombinant genetic engineering strain containing glucose isomerase genes in buffer liquor so as to obtain bacterial suspension; adding a carrier in the bacterial suspension; uniformly stirring the bacterial suspension; adding polyethyleneimine and glutaraldehyde to perform crosslinking; filtering the bacterial suspension after performing stirring crosslinking on the bacterial suspension for 1-2 hours at the temperature of 0-30 DEG C; washing filter cakes by using distilled water; extruding the filter cakes into long strips by using an axial extruder; airing the long strips at room temperature; and smashing the long strips into granules so as to obtain immobilized glucose isomerase cells containing the glucose isomerase. The immobilization method has the advantages that the cost of an immobilization material is low, the method is easy to operate and high in mechanical strength, the immobilization material is stable at high temperature, prepared recombinant escherichia coli whole cells for producing the glucose isomerase in an immobilization manner can be used for catalyzing D-glucose to produce D-fructose at high temperature, the conversion rate is 54% at the temperature of 85 DEG C, after being repeatedly used ten times, the cells still have enzyme activity which is higher than 90%, and the industrial application prospect is high.

The invention discloses dracaena cambodiana chalcone isomerase DcCHIL1, an encoding gene thereof and an application. The nucleotide sequence of the gene comprises: (a) a nucleotide sequence as shown in SEQ ID NO: 1; or (b) a nucleotide sequence formed by substituting, deleting and/or increasing one or a plurality of proteins expressing the same function of the nucleotide sequence as shown in SEQ ID NO: 1; or (c) a nucleotide sequence with 90% or more homologous proteins expressing the same function of the nucleotide sequence (a) or (b). The amino acid sequence of the dracaena cambodiana chalcone isomerase DcCHIL1 is as shown in SEQ ID NO: 2, or is an amino acid sequence formed by substituting, deleting or increasing one or a plurality of amino acids of SEQ ID NO: 2 and has the same function. The dracaena cambodiana chalcone isomerase DcCHIL1 gene is obtained by cloning for the first time, the obtained DcCHIL1 proteins have chalcone isomerase activity, the gene is transferred into dracaena cambodiana by biotechnology, so that dracaena cambodiana flavone accumulation and the yield of dragon's blood can be improved, and the gene has a wide application prospect and great economic values.

The invention relates to engineered Escherichia coli capable of high-yield production of fumaric acid; specifically, fumarase coded genes fumA and fumC of Escherichia coli are knocked off, and maleic cis-trans isomerase gene from Serratia marcescens is converted to obtain the engineered Escherichia coli. The invention also discloses a method of synthesis of catalyzing fumaric acid from maleic acid by using the engineered Escherichia coli. The method includes culturing the engineered Escherichia coli by fermenting, subjecting the engineered Escherichia coli to high-density inductive expression when OD600 reaches 40-80, and applying the obtained fermentation broth to biochemical catalysis of maleic acid in presence of Engineered Escherichia coli to obtain fumaric acid. The genes fumA and fumC in the Escherichia coli are knocked off by using Red homologous recombination, a metabolic pathway of its fumaric acid to L-malic acid is cut off, the maleic cis-trans isomerase from Serratia marcescens is then expressed, and the obtained genetically-engineered Escherichia coli can efficiently convert maleic matrix to synthesize high-purity fumaric acid, there is nearly no L-malic acid byproduct synthesized, and basis is provided for the industrialized production of fumaric acid by whole-cell process catalysis of maleic acid.

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 discloses thermostability transformation of maleic acid cis-trans isomerase and application thereof, and belongs to the field of enzyme engineering. The maleic acid cis-trans isomerase gene maiA from serratia marcescens is subjected to thermostability transformation by a semi-rational design method. Compared with the wild maleic acid cis-trans isomerase, the obtained mutant has the advantages that the half-life period of the mutant G27A-G171A at 55 DEG C is 2.9 times of that of the wild type, and the enzyme activity is 1.96 times of that of the wild type; the half-life period of the mutant G27A-K104R is 4.18 times of the wild type at 55 DEG C, and the enzyme activity is 1.59 times of that of the wild type.

The invention discloses a gene and its protein of phosphomannose isomerase, and its application. The purpose is that providing a gene and its protein of phoshomannose isomerase, and applying the gene and protein in selecting trans-genic plant. The gene has one of nucleotide sequences as follow: 1) the SEQ ID NO: 1 DNA sequence in sequence table; 2) coding the DNA sequence of SEQ ID NO: 2 in the sequence table; 3) the nucleotide sequence that can hybrid with restrict DNA sequence of SEQ ID NO: 1 in the sequence table at high strict condition. Mannose has no harm for human and animal, degradation-liable in environment and selected marker system has the advantages of product yield safety, the selecting regent price is cheap, the selecting procedure is simplicity and the selected marker system don't impact the metabolism balance of transformation plant, the trans-genic plant grow vigorous, significance effect selection. The selected marker system has potential and extensive application outlook in trans-genic plant study and its market process; it may be the leading selected marker system in plant transformation.

The invention discloses escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and a preparation method and application of the escherichia coli genetically engineered bacteria, and belongs to the technical field of genetic engineering. The escherichia coli genetically engineered bacteria have the isoprene synthesizing approach, and meanwhile methanol dehydrogenase gene MDH, 3-hexulose-6-P synthase gene RmpA, 3-hexulose-6-P isomerase gene RmpB and methanol dehydrogenase activated protein gene Act are overexpressed. Meanwhile, the invention further provides a method and application method for establishing the escherichia coli genetically engineered bacteria. The escherichia coli genetically engineered bacteria can biologically synthesize isoprene with methanol as the substrate and has the advantages of being high in conversion efficiency, low in production cost and the like; the conversion rate of methanol-isoprene can reach 21.83%.

The invention discloses a method for synthesizing lacto-N-neotetraose based on balanced UDP-sugar supply. Recombinant bacillus subtilis is obtained by integrating two beta-1,4-galactosyltransferases,integrating a glucose-6-phosphate isomerase gene, a glutamine-fructose-6-phosphate aminotransferase gene, a UTP-glucose-1-phosphate uridyl transferase gene and a UDP-glucose-epimerase gene, knocking out a UDP-glucose 6-dehydrogenase gene and exogenously expressing a beta-1,3-N-glucosamine transferase gene on a genome of a host bacterium bacillus subtilis 168 delta amyE: P43-lacY, P43-lgtB and PxylA-comK. The recombinant bacillus subtilis synthesizes the lacto-N-neotetraose, and the yield on a shake flask can reach 1800 mg/L, and the efficient synthesis of the lacto-N-tetraose is achieved.

The invention discloses glucose isomerase genes, encoding enzymes, recombinant vectors and genetic engineering bacteria and application of glucose isomerase gene engineering bacteria in aspects of preparation of D-fructose by virtue of catalysis of D-glucose isomerization. According to the recombinant Escherichia coli for efficiently expressing high-temperature-resistant glucose isomerase, the problem that general glucose isomerase is low in optimal reaction temperature is solved; the glucose isomerase produced by using the strain has the advantages of high yield, simple process, convenient industrial application and the like, the strain can be directly used for producing high fructose syrup, and cell disruption is not needed; and moreover, after fermentation is ended, the total enzyme activity of D-glucose reaches 715U/L under the condition of 85 DEG C, and the conversion rate of the D-fructose is 55 percent, so that a good technical support is provided for performing large-scale production of the high fructose syrup and taking the high fructose syrup as an important food additive.

The invention discloses a glucose isomerase mutant and application thereof, belonging to the technical field of enzyme genetic engineering and enzyme engineering. According to the invention, a high temperature glucose isomerase gene (NCBI coding: CP000088) is obtained from total DNA of thermobifida fusca, and after site-directed mutagenesis, high efficiency expression of the glucose isomerase gene with a high conversion rate is realized, with the plasmid pT7-7 or a vector that can express glucose isomerase as an expression vector and E. coli BL21 (DE3) or a bacterial strain that can express glucose isomerase as an expression host; the glucose isomerase gene has altogether 1158 nucleotide and encodes 385 amino acids; expression plasmid is constructed in the invention, glucose isomerase is expressed through conversion of bacteria or yeast, and an obtained recombinase mutant has activity of glucose isomerase and has a conversion rate of 60% at a temperature of 70 DEG C, 7% higher than the conversion rate of a parent; optimum temperature of the recombinant glucose isomerase is 80 DEG C, an optimum pH is 10, and the half life of the recombinant glucose isomerase at a temperature of 70 DEG C is no less than 30 h. The recombinant glucose isomerase is particularly applicable to production of F55 high-fructose syrup in the industry of foodstuffs.

The invention relates to a construction method for streptomycete expression plasmids and a production method for keratinase. The construction method for streptomycete expression plasmids includes the following steps: a promoter Xi of actinoplanes missouriensis xylose isomerase and a terminator of streptomycete avermitilis amylase are sleeved, and the promoter-shine-dalgarno (SD) sequence comes from 90 to 269bp of an actinoplanes missouriensis xylose isomerase gene; a cloned src family kinases (sfks) gene is inserted in a construction expression frame structure of a synthetic Xi promoter-SD-amyA2 terminator fragment, and then a conventional method is used for constructing the streptomycete expression plasmids. The production method for keratinase includes: leading the streptomycete expression plasmids into an expression host of streptomycete lividans TK24 through conjugal transfer, performing recombinant expression and generating the keratinase. Specific activity of a crude enzyme solution is 1700 U/mg after expression of the expression plasmids in the streptomycete lividans TK24 and is improved by 50 times compared with specific activity of starting strain streptomycete fradiae varieties S-221, and yield of the keratinase is much higher than starting strains after the expression.

The invention discloses a disulfide bond isomerase gene Trpdi2 from Trichoderma reesei and application thereof. The disulfide bond isomerase gene separated from Trichoderma reesei has a polynucleotide sequence of (a) (b) or (c): (a) a polynucleotide sequence shown as SEQ ID No.1; (b) a polynucleotide sequence complementary with the polynucleotide sequence in (a) according to the principle of complementary base pairing; and (c) a cDNA sequence of the polynucleotide sequence (a)shown in the SEQ ID NO.1 or (b) polynucleotide sequence: a polynucleotide sequence shown in SEQ ID NO.2. The invention also obtains disulfide isomerase the from the encoding of the disulfide bond isomerase gene separated from the Trichoderma reesei and verifies the application of disulfide isomerase.

The invention belongs to the technical field of genetic engineering and particularly relates to construction and application of a genetic engineering strain for xylose-induced production of N-acetylneuraminic acid. According to the construction and the application, a N-acetylglucosamine synthesis way is integrated with a genome, a N-acetylglucosamine 2-epimerase gene bAGE and a N-acetylneuraminicacid lyase gene shNAL are introduced, a N-acetylneuraminic acid synthesis way is constructed, and a key gene nanTEK of a catabolism way of the N-acetylneuraminic acid synthesis way is knocked out. Meanwhile, metabolic ways of precursor substances required for synthesizing the N-acetylneuraminic acid are subjected to multicopy strengthening, and part of bypath metabolic ways are subjected to knockout, so that the N-acetylneuraminic acid high yielding strain is obtained. In case of shake-flask fermentation for 36h, the yield of the N-acetylneuraminic acid can reach 10.8g/L to the maximum, the production intensity can reach 0.3g/(L*h) which is the maximum value reported at present, and thus, the engineering strain has an important industrial application value.

The invention provides recombinant mortierella alpina oPAI for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes and provides a method for constructing a recombinant mortierella alpina strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes, and the invention further provides a use of the recombinant mortierella alpina strain for producing fatty acid, namely biological synthesizing conjugated linoleic acid. With mortierella alpina uracil auxotroph strain MAU1 as a material, a recombinant strain for heterologous expression of linoleic acid isomerase gene from propionibacterium acnes is successfully constructed, and a target product conjugated linoleic acid is detected in the recombinant strain added with long-chain acyl-coenzyme A for synthesizing an enzyme inhibitor.

The invention discloses a high-temperature-resistant glucose isomerase mutant and application of the mutant in catalyzing the isomerization of D-glucose to prepare D-fructose. The mutant is obtained through single or multiple mutations of one or more of a 89th position, a 136th position and a 352th position of an amino acid sequence shown in SEQ ID NO.1. By means of the mutant, the efficient expression of high-conversion-rate glucose isomerase genes is achieved, and the enzyme activity is 7.23 U/mg. The optimal reaction temperature of the ToGI mutant is 110 DEG C, and in high fructose syrup prepared by using catalysts to catalyze 400 g/L D-glucose at the high temperature of 100 DEG C, the concentration of D-fructose is as high as 61.3%.