190 results about "Enzymatic synthesis" patented technology

A method for separating amoxicillin and phenylacetic acid from reaction solution in one-step enzymatic synthesis of amoxicillin is provided. The method employs immobilized penicillin acylase mutant to catalyze the one-step synthesis of amoxicillin from penicillin potassium, and develops a separation process for the resulting reaction mixture. The technical scheme mainly comprises: Firstly separating the immobilized penicillin acylase mutant from the reaction solution through filtration; subsequently isolating amoxicillin via crystallization; followed by separating and recovering phenylacetic acid through toluene extraction and back extraction. This separation method enables rapid and efficient isolation of amoxicillin with high production yield, achieving an average crystallization rate of 93.22%. Concurrently, it demonstrates effective separation and recovery of phenylacetic acid while allowing recyclable use of the toluene extractant.

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 present invention is directed to methods, compositions and kits for template-free enzymatic synthesis of polynucleotides having sequences capable of forming G-quadruplex (G4) structures. In accordance with the invention elongation reactions affected by G4 formation are carried out in the presence of polyC oligonucleotides, such as polyC initiators, that inhibit or prevent formation of either intra-strand or inter-strand G4 structures.

The invention provides a 7beta-hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodesoxycholic acid by a biological enzyme method, through a genetic engineering technology and computer-aided design, phosphoric acid coordination residues of protein are modified, the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant with higher activity under an NADH (Nicotinamide Adenine Dinucleotide Hydroxide) system is constructed, and the 7beta-hydroxysteroid dehydrogenase (7beta-HSDH) mutant can be used for synthesizing ursodesoxycholic acid. The modification strategy is suitable for other proteins for modifying cofactor preference and improving enzyme activity, and has remarkable industrial value when being used for efficiently catalyzing CDCA to be converted into UDCA through a one-pot one-step method.

A method of synthesising a 1N-alkyl derivative of pseudouridine comprises contacting a substrate with a methyltransferase in the presence of a cofactor to produce the 1N-alkyl derivative of pseudouridine. The substrate has a structure of: Formula (I). The 1N-methyl derivative of pseudouridine has a structure of: Formula (II). R1 is a hydroxyl group, a monophosphate group, a diphosphate group, or a triphosphate group. R2 is H or a hydroxyl group. R3 is a methyl group or an ethyl group. The cofactor is a source of a methyl group or an ethyl group. The methyltransferase is configured to transfer the methyl group or ethyl group from the cofactor to the substrate. Enzymatic synthesis of 1N-methyl derivatives of pseudouridine may be more efficient and produce less waste than chemical syntheses. Also provided are methyltransferases and buffers useful in the method; and polynucleotides, expression vectors, and microorganisms useful for producing the methyltransferases.

The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

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 belongs to the technical field of product separation in enzymatic synthesis of antibiotics, and particularly relates to a method for separating and purifying amoxicillin and phenylacetic acid in the production process of amoxicillin. An immobilized penicillin acylase mutant is used for catalyzing penicillin potassium to synthesize amoxicillin in one step, a set of method for separating amoxicillin and phenylacetic acid is developed for an obtained reaction mixture, and the technical scheme mainly comprises the following steps: firstly, separating the immobilized penicillin acylase mutant from a reaction solution through filtration; then, amoxicillin is separated out through crystallization; and then crystallizing, separating and recovering phenylacetic acid through toluene extraction, back extraction and the like. According to the separation method, the amoxicillin can be rapidly and efficiently separated, the yield of the amoxicillin is relatively high, and the average crystallization rate reaches 93.22%; meanwhile, the separation and recovery effects of phenylacetic acid are good; and the extraction agent toluene can be recycled.

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 discloses the use of an oxidoreductase and its mutants in the biosynthesis of naringol, belonging to the field of bioengineering technology. The present invention, for the first time, obtains an oxidoreductase with high conversion capacity for naringol from the marine killer yeast (Wickerhamomyces anomalus M15), and obtains an oxidoreductase mutant with even higher capacity through site-directed mutagenesis. Compared with existing oxidoreductases capable of catalyzing the conversion of naringol, the oxidoreductase and its mutants have good substrate tolerance, high conversion rate, and high salt tolerance. The oxidoreductase and its mutants provided by the present invention provide conditions for the in vitro enzymatic synthesis of naringol, achieving an environmentally friendly and efficient catalytic conversion of naringol to the naringol product. The present invention will provide an important tool enzyme for the synthesis of naringol and bring significant economic benefits to the naringol synthesis industry.

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 an enzymatic reaction catalyst and application thereof in synthesis of quercetin 3, 4 '-di-O-beta-d-glucoside, and relates to the technical field of biological catalysis. The enzymatic reaction catalyst comprises glycosyl transferase and sucrose synthase, and the glycosyl transferase comprises glycosyl transferase I and glycosyl transferase II; the amino acid sequence of the glycosyl transferase I is as shown in SEQ ID NO: 4, the amino acid sequence of the glycosyl transferase II is as shown in SEQ ID NO: 5, and the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO: 6. According to the invention, glycosylation reaction of hydroxyls at 3 and 4'sites of quercetin is established, and high-efficiency and low-cost enzymatic synthesis of the natural product quercetin 3, 4 '-di-O-beta-d-glucoside is realized.

The invention relates to a high-flux enzymatic DNA synthesis method based on framework nucleic acid. Specifically, the invention provides a TdT-framework nucleic acid compound which can accurately regulate and control the position and distance of terminal deoxynucleotidyl transferase (TdT) so as to achieve the purpose of high-flux enzymatic DNA synthesis. The invention also provides a method for preparing the TdT-framework nucleic acid compound, and a method for synthesizing DNA (Deoxyribonucleic Acid) by using the TdT-framework nucleic acid compound. When the TdT-framework nucleic acid compound is used for synthesizing DNA, high-flux enzymatic DNA synthesis can be realized, and the TdT-framework nucleic acid compound has important significance for developing a high-flux DNA synthesis technology with mild conditions.

The invention provides a method for enzymatic synthesis of sucrose laurate in a deep eutectic solvent. DL-menthol is used as a hydrogen bond donor, lauric acid is used as a hydrogen bond acceptor, and the DL-menthol and the lauric acid are heated and stirred according to the molar ratio of 1: 2, so that the eutectic solvent required by an experiment is generated. After the eutectic solvent prepared by the method is compounded with pyridine, not only can the enzyme activity be maintained, but also the solvent performance is relatively good, and the eutectic solvent can be used for enzymatic synthesis of sucrose ester. The method comprises the following steps: adding sucrose and vinyl laurate into a 15mL reaction tube, then adding Novozymes 435 and a DL-menthol: lauric acid (1: 2)-pyridine solvent, and carrying out a stirring reaction under the conditions of 50 DEG C and 300r / min. After the reaction is carried out for 72 hours, the reaction yield is monitored by a liquid phase, and the yield of the sucrose laurate is 97.65%. The invention provides a new thought for enzymatic synthesis of sucrose ester.

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.

The invention provides a preparation method of liver-targeted drug-loaded liposome, and belongs to the field of biological medicine. The hepatic targeting carbohydrate chain is constructed through a multi-step enzymatic reaction, and the accuracy of a modification site is ensured. The synthesized galactose ligand is connected to the surface of the liposome through a covalent bond, so that the targeting property and the stability are enhanced. The synthesis of the galactose ligand provides targeting functional groups with clear structures for the liposome, and the groups are integrated to the surface of the liposome through physical entrapment and chemical coupling in the preparation of the liposome, so that active targeting delivery is finally realized. The collaborative design of the two is based on an ASGPR-mediated liver targeting mechanism, and the delivery efficiency is optimized through enzymatic synthesis and a nanotechnology. In in vivo experiments, plasma and liver distributions of liposomes are determined. In an in-vitro experiment, the uptake efficiency of the lipidosome in liver cancer cells is determined. The experiments aim at verifying that the advantages of the lipidosome in liver cancer cell delivery are improved by optimizing the PEG chain length and the exposure of residues.

This invention concerns a covalent conjugate comprising a glycan being covalently linked through a pyrophosphate (PP) linker to a lipid carrier molecule which is phytol, as well as a cell-free in vitro method and a cell free in vitro system for forming of a glycosylated peptide or polypeptide using said conjugate. The invention further concerns an enzymatic synthesis system for the formation of a glycan-conjugate of this invention. The invention further concerns a peptide or polypeptide with a glycosyl-moiety acceptor residue having an exogenous N-linked glycosylation sequence X1X2NYT, wherein X1 is any one of alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), cysteine (C), tryptophan (W) and methionine (M), and wherein X2 is either alanine (A) or valine (V), as well as an N-glycosylated conjugate of said peptide or polypeptide.

The invention discloses a biosynthesis method and application of oligo pool. The biosynthesis method comprises the following steps: 1) fixing a probe, and combining a solid-phase carrier with a primer chain; (2) the solid-phase carrier is distributed as required, the solid-phase carrier or a deprotection product is distributed into M tubes as required according to the volume, and M is any natural number selected from 1, 2, 3 and 4; 3) chain extension: adding the reaction liquid into an enzymatic DNA synthesis system; 4) tube combination; (5) performing deprotection; (6) the steps (2)-(5) are repeated, the step (5) is executed N times in an accumulated mode, and N is selected from any one non-zero natural number; and 7) sampling, quality control and application. On the basis of a DNA (deoxyribonucleic acid) enzymatic synthesis technology, microspheres are used as solid-phase carriers to regulate and control the synthesis types of basic groups in each round so as to control the sequence diversity of the final oligo pool.

The invention belongs to the technical field of biochemistry, and particularly relates to an efficient enzymatic synthesis method of holothurian glycosaminoglycan. According to the invention, a high-efficiency enzymatic synthesis approach of the sea cucumber glycosaminoglycan is established, sulfated fucose and chondroitin sulfate are used as raw materials, and the sea cucumber glycosaminoglycan with anticoagulant activity is efficiently prepared by using the alpha 1, 3-fucosyltransferase mutant and L-fucose kinase / GDP-fucose pyrophosphorylase and adopting a one-pot multi-enzyme synthesis strategy. And important technical support is provided for development of novel anticoagulant drugs.

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 invention belongs to the technical field of product separation of antibiotics synthesized by an enzyme method, and particularly relates to a product separation and recovery process of amoxicillin synthesized by a one-step method. An immobilized penicillin acylase mutant is used for catalyzing penicillin potassium to synthesize amoxicillin in one step, a set of method for separating amoxicillin and phenylacetic acid is developed for an obtained reaction mixture, and the technical scheme mainly comprises the following steps: firstly, separating the immobilized penicillin acylase mutant from a reaction solution through filtration; then, amoxicillin is separated out through crystallization; and then crystallizing, separating and recovering phenylacetic acid through toluene extraction, back extraction and the like. According to the separation method, the amoxicillin can be rapidly and efficiently separated, the yield of the amoxicillin is relatively high, and the average crystallization rate reaches 93.22%; meanwhile, the separation and recovery effects of phenylacetic acid are good; and the extraction agent toluene can be recycled.

The invention provides a one-step enzymatic synthesis technology of (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester. Specifically, the invention provides a method for preparing (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester by a one-step method, and the method comprises the step of obtaining (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester from p-methylsulfonyl benzaldehyde and L-threonine ethyl ester or a salt thereof under the action of transaldolase in the presence of phosphopyridoxal as a coenzyme.

The present invention relates to the field of biopharmaceutical technology, and in particular to a method for the enzymatic synthesis of a carbonyl reductase mutant and a montelukast sodium intermediate. The amino acid sequence of the carbonyl reductase mutant is shown in any one of SEQ ID NOs: 6-8. The enzymatic synthesis method of the montelukast sodium intermediate of the present invention uses compound II as a substrate, and in the presence of the carbonyl reductase mutant, a coenzyme, a coenzyme cyclic hydrogen donor, a cosolvent, and a buffer, a biocatalytic reaction is carried out to produce the montelukast sodium intermediate, namely, compound I. The specific synthetic route is as follows: #imgabs0#. The synthetic method of the present invention is simple to operate, environmentally friendly, and achieves efficient synthesis of chiral intermediates.