118 results about "Cofactor" patented technology

The invention relates to the technical field of synthetic biology, in particular to a squalene high-yield saccharomyces cerevisiae engineering bacterium and a construction method and application thereof.Saccharomyces cerevisiae CEN.PK.2-1D is used as an original strain, pentose phosphate pathway genes ZWF1, GND1, TAL1 and TKL1 and IDP1 genes of TCA circulation are integrated in a genome of the original strain through homologous recombination, and the squalene high-yield saccharomyces cerevisiae engineering bacterium is obtained. Constructing a dual-channel cofactor supply system to improve the intracellular NADPH level and the NADPH / NADP + ratio; according to the saccharomyces cerevisiae genetic engineering strain Sc-Sq04 constructed by the method disclosed by the invention, the yield of squalene obtained by shake flask fermentation reaches 741.08 + / -28.9 mg / L by further integrating all genes of an MVA way, ERG20 and ERG9 genes, and a new thought is provided for transforming saccharomyces cerevisiae to synthesize terpene compounds and promoting industrial application.

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and / or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

This invention discloses an enzyme preparation for catalytic synthesis of epiinositol and a method for synthesizing epiinositol, belonging to the field of genetic engineering technology. The enzyme preparation includes squalinositol dehydrogenase, NADH oxidase, formate dehydrogenase, and muscle inositol dehydrogenase; the amino acid sequence of squalinositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of muscle inositol dehydrogenase is shown in SEQ ID NO. 8. The enzyme activities of the squalinositol dehydrogenase mutant and the muscle inositol dehydrogenase mutant obtained by mutation in this invention are significantly increased, thus further improving the yield and conversion rate of epiinositol during catalytic synthesis. Furthermore, the enzyme preparation of this invention also contains NADH oxidase and formate dehydrogenase, achieving in-situ regeneration of NADH, thereby effectively reducing the amount of cofactors and NADH used in the catalytic synthesis of epiinositol, and thus reducing production costs.

The invention discloses a difunctional alcohol dehydrogenase derived from Geobacillus thermodenitrifis, the difunctional alcohol dehydrogenase takes NAD < + > as a cofactor, can specifically catalyze a continuous oxidation reaction of omega-hydroxy fatty acid, and catalyzes the omega-hydroxy fatty acid to synthesize alpha, omega-binary fatty acid through single enzyme catalysis without an aldehyde intermediate. The alcohol dehydrogenase has dual catalytic functions of alcohol oxidation and aldehyde oxidation, the oxidation process from hydroxymethylene to carboxyl can be completed in one step, the reaction steps are simplified, accumulation and separation of intermediate aldehyde are avoided, the conversion efficiency is improved, and the alcohol dehydrogenase is alpha, omega-dibasic fatty acid and is a novel alcohol dehydrogenase. The invention provides a simple and efficient biocatalysis way for biosynthesis of dodecanedioic acid, especially dodecanedioic acid, and has important industrial application value.

The invention discloses a Rogowski eutrophy alkali-producing engineering strain for producing p-coumaric acid as well as a construction method and application of the Rogowski eutrophy alkali-producing engineering strain. The preparation method comprises the following steps: by taking a chemoautotrophic microorganism Ropriavidus alcaligenes H16 as an original strain, firstly, by knocking out a synthetic route of a strain poly-3-hydroxybutyrate (PHB), introducing a tyrosine ammonia lyase gene from Rhodotorula glutinis to construct a basic engineering strain capable of producing p-coumaric acid, and then, carrying out fermentation on the basic engineering strain to obtain the p-coumaric acid. Then, endogenous 3-deoxy-7-phosphoheptanone acid synthase gene aroG1 and chorismate synthase gene aroC are overexpressed, and the total copy is increased, so that the p-coumaric acid synthetic pathway flux of the engineering strain is enhanced; and introducing NAD (P) transhydrogenase gene pntAB from Escherichia coli to improve the supply of cofactors, so as to finally obtain the eutrophy alcaligenes Rosei engineering strain capable of synthesizing p-coumaric acid by using CO2.

The present invention relates to a biocatalytic synthesis of esters that can also be implemented on an industrial scale. In particular, the invention relates to a process for the production of esters, via a hemiacetal intermediate, consisting of the following steps: – a step of bringing into contact, in a reaction medium, • a mixture of a C1– Cn alcohol and a C1– Cn aldehyde with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal and the starting aldehyde, or alternatively • a C1– Cn alcohol with an enzymatic catalytic system comprising an oxidized cofactor selected from NAD(P)+ and NAD+ and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the formed hemiacetal, the starting alcohol, and the formed aldehyde, or alternatively • a C1– Cn aldehyde with an enzymatic catalytic system comprising a reduced cofactor selected from NAD(P)H or NADH and a polypeptide of the oxidoreductase group having preferential dehydrogenase activity toward the starting aldehyde and the formed hemiacetal, where n may be any number, and – a step of isolating the corresponding ester.

Provided are engineered polypeptides having dehydrogenase activity that exhibit increased catalytic efficiency for nicotinamide-based unnatural cofactors relative to wild-type or parent polypeptides that encode a dehydrogenase that utilizes nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. Also provided are cell-free and whole cell biotransformation systems for converting a substrate into a product using a redox reaction with a re-cycled unnatural cofactor that utilizes an engineered polypeptide that has dehydrogenase activity and which exhibits increased catalytic efficiency for nicotinamide-based unnatural cofactor.

The invention relates to gene modification, in particular to a method for improving the catalytic capacity of engineering P450 peroxygenase hydrogen peroxide and application of the method. The invention relates to a method for improving hydrogen peroxide catalytic capacity of P450 enzyme, which comprises the following steps of: identifying a channel of the P450 enzyme by using Caver Web 1.0 software by taking a heme cofactor of the P450 enzyme as a starting point; the method comprises the following steps: selecting a target channel with the channel length of 10-30, the curvature of 0-2 and the diameter of 0-3 as a target channel, then selecting amino acid of which the side chain faces the channel at an inlet or a bottleneck of the target channel as key amino acid, and then performing specific mutation on one or more key amino acids, further, at least one channel capable of improving the hydrogen peroxide catalytic capability of the P450 enzyme is obtained, so that the catalytic capability of the P450 enzyme is improved under the catalysis of hydrogen peroxide. The catalytic activity of a plurality of mutants obtained by the invention is compared with that of a wild enzyme, and the channel with high catalytic activity is an H2O2 channel. The application of the method can improve the hydrogen peroxide utilization capability of the P450 enzyme, and further improves the activity of the catalytic substrate of the P450 enzyme.

This application discloses a recombinant bacterial strain that highly expresses nonspecific peroxygenase, its construction method, and its applications, relating to the field of microbial engineering technology. The strain of this invention uses *Escherichia coli* BL21(DE3) as the host. First, the key gene *hemA* for heme synthesis is integrated into the genome using the CRISPR-transposon system. Then, a chassis engineered strain A2 with 2 copies of optimal integration is screened and co-transformed with the rDcaUPO-A161C expression plasmid and the molecular chaperone plasmid pG-KJE8. The intracellular heme level of this strain is increased by 2.84 times compared to the original strain, and the total enzyme activity of rDcaUPO-A161C is cumulatively increased by 8.9 times and the specific enzyme activity is increased by 6.21 times compared to the wild type. This fundamentally solves the problems of insufficient cofactors, low folding efficiency, and poor activity in heterologous expression of nonspecific peroxygenases, and can be used for the efficient production of nonspecific peroxygenases.

Bispecific antibodies whose FIX activation-inhibiting activity is not elevated and whose FVIII cofactor function-substituting activity is elevated have been successfully discovered.

The present invention provides light chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of ACE910 (Emicizumab), novel light chains showing FVIII cofactor function-substituting activity, and heavy chain amino acid substitutions that improve the FVIII cofactor function-substituting activity of novel light chain-containing bispecific antibodies.

The invention provides a mutant R41D / S193N of 7beta-hydroxysteroid oxidative dehydrogenase, a gene sequence of the mutant enzyme, a recombinant expression vector containing the gene sequence of the enzyme and an engineering strain containing the vector. The invention also provides a construction method of a recombinant expression vector and an engineering strain, a method for preparing the mutant enzyme by using the engineering strain, and application of the mutant enzyme in in-vitro enzyme catalysis of 7-ketolithocholic acid to produce ursodeoxycholic acid. Compared with a wild type, the R41D / S193N mutant provided by the invention shows stronger preference on a cofactor NADH, is more economical than the traditional NADPH utilization, is simple and convenient in preparation process, high in yield and simple in downstream treatment process, does not involve other solvents except water in the preparation process, belongs to a green biosynthesis method, and is suitable for industrial production. Meanwhile, the method has relatively high atom economy and step economy.

This invention belongs to the field of biocatalysis technology and discloses a method and application for the preparation of 1,2-phenylethylene glycol using a dual-enzyme pathway with normalized catalysis. This invention uses racemic epoxide phenylethane as the starting substrate and first utilizes epoxide hydrolase-engineered E. coli / pveh2 cells. G191A / N195Q Alternatively, selective hydrolysis can be performed using E. coli / sfeh3, followed by normalization transformation using E. coli / smeh engineered bacteria expressing SmEH epoxide hydrolase as a catalyst, directly generating high-optical-purity (R)-1,2-phenylethylene glycol or (S)-1,2-phenylethylene glycol. The reaction conditions of this invention are mild, require no expensive cofactors or heavy metal catalysts, and have a substrate utilization rate close to 100%, significantly reducing production costs and environmental pollution.

The present invention relates to a method for producing disaccharides using beta-glucosidase and its cofactors, and a composition for inducing enzyme production in a Trichoderma strain, which contains the produced disaccharides. Specifically, the method comprises inducing beta-glucosidase and a divalent metal ion, manganese ion (Mn ion), as a cofactor, on a glucose substrate. 2+ ), magnesium ion (Mg 2+ ), zinc ion (Zn 2+ ) or copper ions (Cu 2+ ), the disaccharide production rate can be increased and disaccharides can be obtained at a high concentration. In addition, since the composition with a high content of disaccharides produced by the production method of the present invention has an excellent effect of inducing enzyme production in Trichoderma strains, it is possible to improve the enzyme productivity of Trichoderma by applying a fermentation process that utilizes the produced disaccharides at a high concentration.

The invention relates to the technical field of biochemical engineering and medicinal chemistry, and provides a method for improving the resolution efficiency of a praziquantel racemoid by an enzyme method, which comprises the following steps: S1, preparing oxidase, selectively oxidizing dextropraziquantel by the oxidase, and basically not oxidizing levo praziquantel; s2, contacting oxidase with the racemic praziquantel mixture in a Tris-HCl buffer system with the pH value of 7.5-8.5, adding Fe as a cofactor, and reacting at the temperature of 35-39 DEG C for 4-6 hours; and S3, separating and purifying the reaction mixture obtained in the step S2 to obtain the high-purity levo-praziquantel. The problem of low resolution efficiency of the praziquantel racemate is successfully solved through an enzymatic resolution technology with high efficiency, strong specificity and mild conditions.

The application relates to the technical field of biological detection, in particular to a method for in-situ quantitative detection of specific glycosylation of target protein based on double probes. The method provided by the application comprises the following steps: a protein probe and a glycan probe are used to combine with target protein and target glycan respectively, and the spatial proximity relationship after combination triggers hybridization between the probes on the same target protein to form hybrid double strands; the DNA enzyme of the protein probe is used to enzymatically cut the glycan probe on the hybrid double strands under the action of a metal cofactor; and the report peptide released after enzymatic cutting of the glycan probe is subjected to mass spectrometric quantitative analysis, so that the signal of protein-specific glycoform is converted into a mass spectrometric signal.

This application relates to a hybrid of glutamate synthase, a host cell, and a method for preparing a compound, the hybrid comprising any one of the following: (1) an α-GS subunit of NADPH-dependent glutamate synthase and a β-domain of NADH-dependent glutamate synthase; and (2) an α-domain of NADH-dependent glutamate synthase and a β-GS subunit of NADPH-dependent glutamate synthase. The process of generating a specific product can regulate the balance of cofactors, maintain the redox balance throughout the cell, and thereby enhance the production capacity of the strain.

The application discloses genetically engineered bacteria for synthesizing L-tryptophan and application thereof. The application realizes the following through two key gene regulation strategies (serA down-regulation and serB up-regulation): the accumulation amount of glutamic acid by-products is significantly reduced from 8-20 g / L to below 1.14 g / L, and the reduction ranges are 87.74% and 90.3% respectively; the sugar acid conversion rate (glucose / L-tryptophan) of L-tryptophan is increased from 19.2% to 20.9% and 21.3% respectively; without additional addition of expensive cofactors or inducers, the cost-effectiveness is significant.

Described herein is a method of producing a reduced labelled cofactor comprising one or more xH atom, wherein x is 2 or 3. Described herein is also a method of producing a reduced labelled reaction product comprising one or more xH atom, wherein x is 2 or 3, wherein the method comprises producing a reduced labelled cofactor according to the invention. Described herein are also systems for performing such methods.

Provided are a genetically engineered bacterium for high-yield production of D-pantothenic acid (D-PA), and a preparation method therefor and a use thereof. The D-PA production yield of the engineered bacterium is significantly improved by means of the following actions: overexpressing acetolactate synthases IlvB and IlvN for pyruvate diversion while deleting a transcription attenuation region and mutating a valine feedback inhibition site; overexpressing ketol-acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and altering the coenzyme preference of IlvC from NADPH to NADH to unlock the upstream pathway for D-PA synthesis; overexpressing PanB and overexpressing a serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; at the same time, enhancing the expression of ketopantoic acid reductase PanE; and finally overexpressing pantothenate synthetase PanC and increasing the content of cofactor ATP required by PanC. By means of the integrated enhancement of the above multiple modules, the provided engineered bacterium achieves improved cell growth due to balanced cofactors and increased D-PA.

Treatment with epimerase in vitro forms D-psicose from D-fructose dissolved in aqueous solution, and then D-psicose is converted into the oxidized cofactor NAD(P). + A method for preparing an aqueous solution containing allose by reducing allitol to allitol in vitro with NAD(P)H-dependent oxidoreductase, which is accompanied by the formation of a compound, and then enzymatically oxidizing allitol to allose. (Figure 1)

The present application relates to the field of animal nutrition and microbiology, and in particular to a nutrition intervention method for regulating eggshell mineralization by egg chicken intestinal flora-metabolites, a preparation, a sustained-release granule and an application thereof, comprising the following steps: A. intestinal flora homeostasis establishment period: adding a bile acid precursor complex, a flora regulating complex and an intestinal environment regulator in the feed of 24-week-old laying hens at a proportion of 0.5%, continuously for 14 days; B. metabolite production enhancement period: adding a metabolite precursor complex and a metabolic activity regulator to the feed of laying hens at the peak of egg production at a proportion of 0.8%; C. shell gland mineralization efficiency improvement period: adding a calcium transporter activating complex, a carbonic anhydrase cofactor and an ion channel regulator to the feed of laying hens at the later stage of egg production at a proportion of 1.0%, to promote the production of key metabolites and activate the whole process molecular mechanism of shell gland mineralization.

This invention discloses a genetically engineered bacterium producing high levels of L-homoserine, its construction method, and its applications, belonging to the field of biotechnology. The genetically engineered bacterium uses *Escherichia coli* W3110 as the substrate cell. By knocking out the metA, lysA, thrBC, pflB, ldhA, poxB, adhE, iclR, sthA, tdcC, lacI, and ptsG genes, and replacing the promoters of ppc, aspC, and gdhA genes with the inducible strong promoter Ptrc, and by performing site-directed mutagenesis on the thrA and asd genes, and simultaneously overexpressing the thrA*, pnhsd, asd, asd*, and rhtA genes using free plasmids, a recombinant strain HOM20 was constructed. This strain, fermented in a 5 L fermenter for 48 h, achieved an L-homoserine yield of 135.4 g / L and a sugar-acid conversion rate of 44%, significantly superior to existing technologies. This invention achieves directional carbon flow allocation, cofactor balance, feedback inhibition removal, and enhanced product efflux through multi-gene synergistic modification, and has broad prospects for industrial application.

The invention discloses a method for crystallizing BM3 protein in bacillus megaterium. The method comprises the following steps: constructing a gene for coding the BM3 protein into an expression vector, carrying out induced expression in escherichia coli, carrying out affinity chromatography purification, excising a fusion tag, carrying out gel filtration purification, incubating with cofactors FAD, FMN and NADPH to form a compound, and crystallizing under a specific pool liquid condition through a seated drop gas phase diffusion method. According to the method, the high-quality BM3 protein crystal suitable for X-ray diffraction analysis can be obtained, and a basis is provided for analyzing the three-dimensional structure of the BM3 protein crystal and researching an electron transfer mechanism.

The invention discloses an omega-transaminase mutant with high thermal stability and application thereof, relates to the technical field of biology, and discloses plasmids and recombinant cells of the omega-transaminase mutant, and the nucleotide sequence of a coding gene of the mutant is shown as SEQ ID No.1. According to the application of the mutant of the [alpha], [omega]-transaminase in synthesis of the Litinib intermediate, a buffer solution, a substrate, a cofactor, an amino donor and a catalyst are sequentially added into a reaction system for reaction, and the Litinib intermediate (2S, 5R)-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester is generated. The activity and stereoselectivity of the omega-transaminase mutant obtained through enzyme engineering modification are obviously improved. The alpha, omega-transaminase has high stability and catalytic efficiency and can catalyze synthesis of (2S, 5R)-5-amino-2-methylpiperidine-1-carboxylic acid tert-butyl ester under the substrate concentration of 100 mM, the reaction conversion rate can reach 42%, and the diastereoselectivity of the product can reach 95% or above.

The invention relates to a recombinant bacterium for high yield of lactose-N-fucopentasaccharide 1 and application of the recombinant bacterium, and belongs to the technical field of fermentation. According to the method disclosed by the invention, UTP regenerated protein is recruited by utilizing the specificity of the phase separation protein PodJ, and a highly-ordered micro-region structure is formed in a cell, so that efficient in-situ regeneration of UTP is realized. According to the strategy, the substrate supply of key enzyme in the synthetic route of the lactosyl-N-fucopentasaccharide 1 is obviously enhanced, and the synthetic efficiency of the saccharide is effectively improved. On the basis, a synthetic route of the lactose-N-fucopentasaccharide 1 is further optimized, a recombinant strain for high yield of the lactose-N-fucopentasaccharide 1 is successfully constructed, the strain is subjected to system optimization in genetic background and metabolic pathways, efficient concentration and cyclic utilization of cofactors can be realized in a subcellular level micro interval, and the yield of the lactose-N-fucopentasaccharide 1 is increased. Dependence of exogenous cofactor addition is greatly reduced, the overall economical efficiency and stability of the system are improved, and the method has good application prospects.

The present disclosure provides luciferases that have a compact size, robust stability, cofactor independence, efficient cellular expression, higher catalytic efficiency, and / or unique substrate orthogonality. The present disclosure provides luciferase-fluorescent protein FRET fusions that can be used in multi-parametric imaging, e.g., in cellulo, in vivo, or both.

The disclosure provides a metabolic pathway for producing a metabolite, the metabolic pathway having a co-factor purge valve system for recycling a cofactor used in the metabolic pathway.

The invention discloses a method for synthesizing spermidine through a double-bacterium co-culture system, and relates to the technical field of bioengineering. The method comprises the following steps: constructing a first strain for synthesizing and secreting ornithine; a second strain is constructed and used for synthesizing aspartic semialdehyde and butanediamine, the second strain comprises carboxyl spermidine dehydrogenase and carboxyl spermidine decarboxylase, the carboxyl spermidine dehydrogenase is a mutant enzyme obtained by performing G13A and / or K159R mutation on an amino acid sequence as shown in SEQ ID NO: 1, and the carboxyl spermidine decarboxylase is a mutant enzyme obtained by performing G13A and / or K159R mutation on an amino acid sequence as shown in SEQ ID NO: 2. The carboxyl spermidine decarboxylase is a mutant enzyme obtained by carrying out D82E single-point mutation on an amino acid sequence as shown in SEQ ID NO: 2; and co-culturing and fermenting the first strain and the second strain to synthesize spermidine. According to the present invention, the metabolism division and the efficient synergy can be achieved, the metabolism load and the butanediamine toxicity accumulation can be reduced, and the catalytic efficiency of the key reaction and the cofactor utilization efficiency can be improved so as to significantly improve the spermidine yield and the conversion efficiency.