121 results about "Enzymatic synthesis" patented technology

The invention discloses a high-stability carbonyl reductase mutant and application of the high-stability carbonyl reductase mutant in chiral alcohol synthesis. An amino acid sequence as shown in SEQ ID NO.2 is mutated as follows: 20th leucine is replaced by any one of lysine, threonine, aspartic acid or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine. Compared with a wild type enzyme, the stability of the obtained mutant is remarkably improved, particularly, single-point mutants Mut-L20I and Mut-L55I and a combined mutant Mut-L20I-L55I show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, has the advantages of high catalytic efficiency, high thermal stability, strong substrate tolerance, easiness in fermentation production and the like, and is suitable for industrial application of enzymatic synthesis of chiral alcohols.

The invention discloses an enzyme catalytic synthesis method of a luliconazole chiral intermediate, which comprises the following steps: adding a recombinant escherichia coli wet cell for expressing alcohol dehydrogenase, a recombinant escherichia coli wet cell for expressing formate dehydrogenase, a buffer solution, a substrate, a cosolvent, ammonium formate and a coenzyme into a reaction container, reacting at 25-35 DEG C for 2-24 hours, extracting, separating, and carrying out rotary evaporation, thereby obtaining the luliconazole chiral intermediate. The luliconazole chiral intermediate is obtained; the substrate is 2, 2 ', 4'-trichloroacetophenone; the alcohol dehydrogenase is a mutant of AKR3. The method is mild in reaction condition, environmentally friendly, high in regioselectivity and stereoselectivity, high in conversion rate, high in chiral purity, small in enzyme dosage, low in preparation cost and suitable for industrial production.

The present invention is directed to methods and device for template-free enzymatic synthesis of polynucleotides using a filtering barrier.

The invention provides a method for improving the oxidation resistance of phenylalanine based on N-nicotinoyl modification and application of the method. According to the method, N-nicotinoyl phenylalanine amide with antioxidant activity is obtained through chemical synthesis or enzymatic synthesis, and the antioxidant performance of N-nicotinoyl phenylalanine amide is evaluated by using an ABTS free radical scavenging method. Results show that the N-nicotinoyl phenylalanine amide can delay lipid oxidation in a food system, prolong the shelf life of the product and maintain the nutritional stability; in a cosmetic system, skin oxidative damage can be relieved, skin aging can be delayed, and skin color can be improved. The compound disclosed by the invention can show a remarkable antioxidant effect under the condition of a relatively low addition amount, so that a safe and effective antioxidant is provided for the fields of foods and cosmetics, and the compound has a good application value and a good popularization prospect.

The invention provides a high-protein feed raw material and a preparation method thereof, and belongs to the field of protein feeds.The preparation method comprises the steps that animal-derived organs are selected, an animal-derived organ enzyme solution is obtained through a pretreatment means, and natural protein components in the animal-derived organ enzyme solution are reserved to serve as crude protein nutritional ingredients; step 2, adding a substrate and a methyl donor into the animal-derived visceral organ enzyme liquid, and generating guanidinoacetic acid and creatine through an enzymatic synthesis reaction to obtain a mixed liquid; and step 3, carrying out enzyme system inactivation, centrifugal filtration, drying and crushing on the mixed solution to obtain a finished product. According to the invention, guanidinoacetic acid and creatine are synthesized by in-situ catalysis of animal-derived visceral organ natural enzyme systems, and can be naturally fused with protein components, so that the problem of compatibility is avoided, and growth functional factors do not need to be added from an external source; natural enzyme systems of animal livers and kidneys are directly utilized, industrial enzymes do not need to be purchased, and the natural enzyme systems are more adaptive to a raw material system; protein, natural enzyme systems and mineral substances in the raw materials are synchronously utilized, so that one material has multiple purposes.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing hybridization stringency and / or nuclease digestion for removing failure sequences.

The invention relates to a chemoenzymatic synthesis method of a high-glucose-type homogeneity mucoprotein core structural domain. The method comprises the following steps: firstly, catalytically synthesizing a key alpha-linked glycosylated amino acid block Fmoc-GalNacalpha-Ser / Thr-OH at a low temperature (-20 DEG C to 0 DEG C); then assembling the building blocks on a repeated polypeptide skeleton rich in proline, serine and threonine at accurate intervals (3-5 amino acid residues) through a solid-phase peptide synthesis technology to form a'sugar chain growth scaffold '; then, a series of high-specificity glycosyl transferases are used for carrying out sequential and directional sugar chain enzymatic extension on the scaffold, and a uniform core structure (such as a core type 3) is constructed; and finally, performing high-efficiency purification by a three-step series method of size exclusion chromatography, ion exchange chromatography and lectin affinity chromatography. The technical bottleneck of inhomogeneity (lt, 70%) of glycoforms in a traditional method is overcome, the mucoprotein core fragment with glycoform homogeneity larger than or equal to 95% and bioactivity highly similar to that of natural mucoprotein can be prepared on a large scale, and the method has wide application prospects in the biomedical fields of mucous membrane protective agents, drug delivery carriers and the like.

A method for producing amide-containing molecules of the formula:wherein: X1 and X2 are independently selected from NH2 and COOH; L is independently, in each instance, selected from hydrocarbon linkers containing 1-12 carbon atoms optionally containing one or more heteroatoms selected from O, N, S, and halogen atoms, and are the same or different; A independently represents an —NHC(O)— or —C(O)NH— linkage; w, x, y, and z are independently 0 or 1; the method comprising reacting at least two reactant molecules that form an amide bond with each other via X1 and X2 groups, in the presence of an amide synthetase enzyme, wherein the at least two reactant molecules are independently selected from the following formulas:wherein v is 0, 1, or 2.

The invention discloses a compound enzyme preparation-containing nutritional feed for regulating and controlling intramuscular fat of black pigs and a preparation method of the compound enzyme preparation-containing nutritional feed, and relates to the technical field of livestock and poultry breeding feeds. The invention discloses a black pig skeletal muscle cell fat directional transport feed which is prepared from the following components in parts by weight: 850-920 parts of basal feed, 5-12 parts of fat directional deposition compound enzyme group, 25-40 parts of amino acid-enzymatic synthesis substrate combination and 2-6 parts of lipolytic enzyme specific inhibitor. According to the black pig skeletal muscle cell fat directional transport feed, fat precursor directional transport is realized through the compounded fat directional deposition compound enzyme group, a fat synthesis pathway targeting black pig skeletal muscle cells and lipoprotein lipase; acetyl-coenzyme A carboxylase and fatty acid synthetase catalyze fat synthesis step by step, only intramuscular fat generation pathways are activated, subcutaneous and abdominal fat deposition paths are blocked, the technical defects that fat deposition of an existing single enzyme preparation is non-directional, and the body fat rate is caused to be abnormally increased are overcome, meanwhile, an exclusive amino acid-enzymatic synthesis substrate combination is matched, and the body fat synthesis efficiency is improved. Sufficient adaptive substrates are supplied to enzyme group catalytic reaction, and the problem that a single enzyme preparation is insufficient in catalytic efficiency is solved.

The invention is directed to methods and apparatus for parallel enzymatic synthesis of polynucleotides in an array of reaction chambers using a template-free polymerase that has sequence-dependent coupling efficiencies. Whenever sequences causing low efficiency coupling occur at a 3′ end of a growing chain of a polynucleotide being synthesized, one or more additional coupling cycles without de-protection steps are inserted into synthesis plans to provide additional time for completing the coupling reaction at that position of the polynucleotide.

This invention discloses a recombinant 2-O-sulfonyltransferase mutant and its preparation method, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (E28~N356) mutant of hamster-derived 2-O-sulfonyltransferase (L119V-R188E-A240I) with an N-terminal maltose-binding protein (MBP) tag. The recombinant 2-O-sulfonyltransferase mutant exhibits higher activity, 1.9 times that of the recombinant wild type, and greater stability, retaining 86.6% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant 2-O-sulfonyltransferase is increased by 5 times. This mutant, as a novel biocatalyst with high efficiency, stability, and controllable production costs, has significant application value and broad industrialization prospects in the industrial enzymatic synthesis of glycosaminoglycans such as heparin and heparan sulfate.

Disclosed is a method for enzymatic synthesis of a brivaracetam chiral intermediate, i.e., a method for synthesizing a brivaracetam chiral intermediate (R)-3-cyanohexanoic acid by catalyzing the hydrolysis of 3-cyanohexanitile using an enzyme with nitile hydrolysis activity, and the enzyme with nitrile hydrolysis activity is obtained by carrying out a single mutation or a double mutation on an amino acid at position 140 or an amino acid at position 175 in an amino acid sequence as set forth in SEQ ID NO.2. Compared with a wild type, the nitrilase mutant of the present invention has the activity increased by 10 times, an ee value increased to 300 or more from 39, a substrate conversion rate of 45%, and a product ee which can reach 98.5%, and the yield of (R)-3-aminomethyl-hexanoic acid by catalytic hydrogenation synthesis using (R)-3-cyanohexanoic acid reaches 85% or more. The present invention features a short synthesis route, mild reaction conditions, and high atom economy, and can be applied to the industrial synthesis of the brivaracetam intermediate.

The invention relates to the technical field of biological catalysis, and discloses a mutant enzyme for preparing rebaudioside I. The enzyme is a UGT76G1 mutant, and the mutation points of the mutant are L379V and L126A. The mutated enzyme realizes efficient in-vitro enzymatic synthesis of RI (the conversion rate is 45-60%), the problem of low efficiency (-7%) of an existing path is solved, and the production process has economic feasibility; secondly, the catalytic efficiency is improved by 6-9 times compared with the original level, and the fundamental improvement means that the raw material utilization rate and the production flux are greatly improved; and meanwhile, the seamless connection capability from a laboratory to large-scale production is realized, and a key technical foundation is laid for constructing a stable and controllable industrial production line.

The application discloses an enzyme method for synthesizing unnatural sugar and application thereof, and belongs to the technical field of organic chemical synthesis. The unnatural sugar comprises an unnatural sugar obtained by introducing an acyl group into a hydroxyl group at the 6th position of a bare sugar through an acylation reaction; and the bare sugar is obtained by performing derivatization modification on an amide site of a natural sugar. The synthesis method uses a SubC enzyme as a catalyst to realize selective acylation of the hydroxyl group at the 6th position of the bare sugar, and synthesizes the unnatural sugar with an ideal yield. The unnatural sugar can be applied to glycomics metabolic labeling and mass spectrometry analysis as a marker, has excellent biological safety, specificity and labeling efficiency, can avoid non-specific labeling, and has no obvious cytotoxicity.

The invention discloses an enzymatic synthesis method of a melogabalin intermediate and related alcohol dehydrogenase, racemic 3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone is used as a substrate, a genetically engineered bacterium for producing alcohol dehydrogenase is used as an enzyme catalyst, a genetically engineered bacterium for producing formate dehydrogenase, ammonium formate and coenzyme are matched, an enzymatic reaction is carried out under a stirring condition, and the melogabalin intermediate is obtained. And carrying out purification and separation on a reaction product to obtain the melogabalin intermediate (1R, 5S)-3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone. The method has the advantages of simple reaction, few steps, mild reaction, less pollution and the like, and has great industrial application potential and commercial value.

The invention relates to the technical field of biological medicine manufacturing, discloses an ancient herbal enzyme oral liquid for treating digestive system diseases and a preparation method, and aims to solve the problems that in the prior art, adaptability of strains and herbal fibers is insufficient, directional enzyme production induction is deficient, probiotic activity retention is poor, and a flora, fiber and enzyme cooperative closed loop cannot be constructed. The preparation method comprises the following steps: by taking genuine hemp seeds, Chinese herbaceous peony and the like as raw materials, matching with rhizosphere-separated saccharomyces cerevisiae Y-01, lactobacillus acidophilus L-03 and bacillus coagulans C-05, carrying out three-step directional fermentation of substrate pretreatment, flora colonization and enzymatic synthesis to produce CAZymes and SCFA, embedding probiotics through trehalose-chitosan, compounding a stabilizer adaptive preparation, and adjusting the ratio of the raw materials according to an IBS subtype. The fiber degradation efficiency and the functional product synthesis efficiency are improved, the gastric acid tolerance and the storage stability of the probiotics are enhanced, different IBS subtype requirements are met, and an effective means is provided for treatment of digestive system diseases.

The application discloses an alcohol dehydrogenase mutant and application thereof in enzymatic synthesis of a loratadine intermediate. A plurality of alcohol dehydrogenase mutants are developed, and a loratadine chiral intermediate is obtained through an enzyme catalytic synthesis method based on the mutants, wherein the reaction condition is mild, the environment is friendly, the reaction has high regional selectivity and stereoselectivity, the reaction conversion rate is high, the chiral purity of the product is high, the enzyme consumption is low, the preparation cost is low, the method is suitable for industrial production, the method avoids the problems of difficult chiral resolution and heavy metal residue in a product in a conventional method, and the method makes up for the deficiencies of a traditional chemical method.

The invention discloses a preparation method of all-trans-farnesol through a three-enzyme cascade reaction. The invention firstly provides a mutant of farnesyl pyrophosphate synthase, and the amino acid sequence of the mutant is shown as SEQ ID No.3. Compared with a wild type, the mutant is applied to enzyme catalytic synthesis of farnesol, so that the conversion rate of a gold product is remarkably improved. The invention also provides a three-enzyme cascade reaction preparation method of farnesol, which comprises the following steps: in the same reaction system, dimethyl allyl diphosphate is used as a substrate; according to the method, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase or a mutant thereof and farnesyl pyrophosphatase are used as catalytic enzymes for enzymatic reaction to prepare all-trans-farnesyl alcohol. According to the invention, in a single reaction system, three key enzymes are combined for application, so that continuous catalysis, high-efficiency and high-selectivity preparation of all-trans-farnesol is realized, process steps are simplified, and a new way is provided for green and efficient preparation of farnesol.

The invention discloses a method for synthesizing retinoic acid ceramide ester through enzyme catalysis and application of the retinoic acid ceramide ester, and belongs to the technical field of cosmetic raw material synthesis. According to the method, retinoic acid and ceramide NP serve as substrates, specific immobilized lipase serves as a catalyst, and in a non-aqueous phase organic solvent, the target product is efficiently synthesized through the steps of raw material preparation, anaerobic reaction system building, directional esterification reaction, aftertreatment and purification detection. The method solves the problems of low substrate solubility, more byproducts, difficulty in catalyst recovery and the like in the traditional process, has the advantages of mild reaction conditions, capability of retaining the activity of the raw materials, high product purity and anti-aging and barrier repair effects, and can be widely used for preparing raw materials of anti-wrinkle and repair cosmetics.

This invention discloses a fish-derived bioactive peptide with dual functions of enhancing saltiness and inhibiting adipocyte differentiation, and its applications, belonging to the field of biotechnology. The bioactive peptide is a γ-glutamylarginine peptide with the general formula γ-[Glu]n-Arg, where n is an integer from 1 to 4. This invention efficiently prepared a series of γ-[Glu]n-Arg peptides through a targeted enzymatic synthesis method, further elucidating the dual effects of this bioactive peptide at the molecular mechanism level. The bioactive peptide can effectively enhance saltiness perception by binding to the saltiness receptor TMC4, and inhibit adipocyte differentiation and reduce lipid accumulation through multiple targets by downregulating PPAR-γ, C / EBPα, and FAS genes, and upregulating AMPK and HSL genes. This invention provides a reliable scientific basis and technical support for developing innovative food ingredients with both flavoring and health functions.

The invention belongs to the technical field of enzyme engineering, and particularly relates to a guanylate kinase mutant with improved thermal stability and application and a product thereof. Aiming at the defects that wild guanylate kinase is insufficient in thermal stability and easy to inactivate in reaction, mutants (A24Q, S75P, K146P and L172I) are obtained by site-directed mutagenesis modification based on a wild guanylate kinase sequence. The mutant is replaced by specific amino acid residues, so that the thermal stability is remarkably improved, meanwhile, good catalytic activity is kept, wider temperature conditions can be tolerated, and the effective action time of enzyme is prolonged. The mutant can efficiently catalyze a phosphate group transfer reaction of guanylic acid and adenosine triphosphate, and is suitable for enzymatic synthesis of nucleoside compounds such as guanosine diphosphate. The application of the enzyme source can reduce the use amount and supplement frequency of enzyme preparations, reduce the production cost, improve the product yield and quality, and provide reliable enzyme source support for the industrial efficient production of nucleoside products.

The invention provides an S-adenosine homocysteine (SAH) analogue, an S-adenosine-L-methionine (SAM) analogue and application thereof. The preparation method comprises the following steps: synthesis of the SAH analogue and a fluoroalkyl SAM analogue, fluoromethylation modification of bioactive molecules by an HMT-MT cyclase cascade reaction system based on the fluoroalkyl SAM analogue, and construction of a fluoro derivative of a clinical drug synthesized by a one-pot enzymatic method. The SAH analogue adopted by the invention can effectively avoid spontaneous degradation of the corresponding SAM analogue, and is used for constructing a cascade reaction system based on HMT-MT cyclase, so that the yield of a fluoromethylation product is increased; it is confirmed that the fluoro-intermediate generated by MT can be accepted by downstream biosynthetase, so that a fluoro-derivative of a clinical drug synthesized by a one-pot enzymic method is constructed.

The present application provides a method for enzymatic synthesis of a nucleoside bearing a protecting group, and a composition. The method includes: using pyrimidine nucleoside phosphorylase or uracil nucleoside phosphorylase and purine nucleoside phosphorylase to catalyze a substrate to synthesize a nucleoside bearing a protecting group, where the substrate includes a substrate nucleoside, a substrate base and a substrate phosphate, and the substrate base bears a protecting group; the pyrimidine nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein PyNP shown in SEQ ID NO: 1; the uracil nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein UP shown in SEQ ID NO: 2; and the purine nucleoside phosphorylase includes a protein that is of more than 80% identity to a protein PNP shown in SEQ ID NO: 3, 7 or 8. The present application can solve the problem that there is no biosynthesis method to produce a nucleoside bearing a protecting group in the prior art, and thus is suitable for the field of enzyme catalysis.

The present invention relates to the technical fields of biocatalysis and biopharmaceuticals. Disclosed is an aldehyde / ketone-initiated enzymatic synthesis method for (1R,2R)-p-methylsulfonylphenylserinol. In the method provided in the present invention, using transaminase and transketolase as catalysts, in the presence of an amino donor and an aldehyde compound or a ketone compound, a substrate p-methylsulfonylbenzaldehyde is catalyzed to produce (1R,2R)-p-methylsulfonylphenylserinol. The method provided in the present invention reduces operation steps and can realize the replacement and recycling of expensive non-industrial starting material hydroxypyruvic acid, thereby reducing production costs and thus being suitable for industrial production.

The invention relates to a heparin 3-O-sulfuric acid transferase mutant with high enzyme activity and high thermal stability as well as a coding gene and application of the heparin 3-O-sulfuric acid transferase mutant. The amino acid sequence of the 3-O-sulfate transferase mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The 3-O-sulfuric acid group transferase mutant has remarkable and superior performance in the enzymatic synthesis of heparin glycosaminoglycans. Performance determination shows that the 3-O-sulfate transferase mutant SM1 realizes the synergistic improvement of the catalytic activity and the thermal stability. The total enzyme activity of the 3-O-sulfotransferase is improved by 1.5 times compared with that of wild chicken source 3-O-sulfotransferase. More prominently, the thermal stability of the 3-O-sulfo-transferase is fundamentally improved: the activity of the wild chicken source 3-O-sulfo-transferase is generally and rapidly attenuated within 24 hours at 37 DEG C, while the 3-O-sulfo-transferase mutant SM1 can still retain about 50% of initial activity after being subjected to heat preservation for 8 days under the same condition, so that the 3-O-sulfo-transferase mutant SM1 shows excellent operation stability.

The application discloses a polyfructose butyrate as well as preparation and application thereof, and is prepared by combining n-butyric acid or vinyl butyrate with polyfructose to form an ester bond by using an enzymatic synthesis method. The polyfructose butyrate can be well degraded and utilized by intestinal flora, and butyric acid is simultaneously generated, so that the polyfructose butyrate not only can promote richness and diversity of intestinal flora, improve intestinal flora, but also can promote growth of probiotics and inhibit growth of harmful bacteria. The polyfructose butyrate can significantly improve intestinal inflammation of mice and relieve symptoms of intestinal inflammation. The polyfructose butyrate can make polyfructose and butyric acid exert bioactivity at specific positions in the intestinal tract, and does not cause other burdens on the body.

This invention discloses a method for the enzymatic synthesis of L-aminobutyric acid (GABA), comprising the following steps: using threonine and ammonium formate as substrates, a three-enzyme combined catalytic reaction is carried out using leucine dehydrogenase, threonine deaminase, and formate dehydrogenase at a temperature of 40-55°C and a pH of 7.0-9.0 to obtain L-aminobutyric acid. The leucine dehydrogenase is the leucine dehydrogenase with the amino acid sequence shown in SEQ ID NO:1 or a mutant thereof. This invention can rapidly convert threonine to L-aminobutyric acid, yielding L-aminobutyric acid with high optical purity, thus improving economic efficiency.

The invention discloses a method for synthesizing beta-carboline alkaloid through enzyme catalysis, which comprises the following step of: reacting a compound shown in a formula (I) with a compound shown in a formula (II) in the presence of isoviscin synthetase to obtain the beta-carboline alkaloid shown in a formula (III). The method has the remarkable characteristics of mild conditions, no pollution, simple process route, high product conversion rate and the like.

In exemplary aspects, oligonucleotides are synthesized via a microfluidic reaction chamber integrating nanostructure / high-Q dielectric nanoantenna arrays of a metasurface device, the arrays providing synthesis sites. The method comprises; the chamber processing input reagents for producing oligonucleotides at outlets of the chamber; and optically exciting the synthesis sites by altering optical parameter(s) to cause at least one enzymatic synthesis cycle. In more specific examples, the method further includes effecting, after the cycle(s) occurs, PCR amplification of certain of the synthesized products by selecting and optically exciting one or more of the synthesis sites, and / or microfluidically segregating the one or more synthesized products, in response to the amplification step, into homogenous oligo pools.