244 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 relates to the technical field of biology, and discloses a carbonyl reductase mutant and application thereof in enzymatic synthesis of chiral alcohol. The amino acid sequence as shown in SEQ ID NO.2 is subjected to mutation that 36th lysine is mutated into any one of aspartic acid, threonine and valine, or / and 125th histidine is mutated into any one of lysine, serine and leucine, or 170th histidine is mutated into any one of lysine, cysteine and leucine. The carbonyl reductase mutants, especially single-site mutants Mut-T75K, Mut-T75D and Mut-E216M and a combined mutant Mut-T75K-E216M, with activity improved to different extents compared with that of a wild type are obtained, have excellent relative enzyme activity, thermal stability and substrate tolerance, have relatively high activity and stability for various chiral alcohols, and can be used for preparing the carbonyl reductase mutants. The catalyst has the advantages of high catalytic activity, high thermal stability, strong substrate tolerance, easiness in fermentation and the like.

The invention discloses a recyclable biochip stationary phase as well as a preparation method and application thereof, and belongs to the field of biochips. The preparation method of the biochip stationary phase comprises the following steps: (1) activating a glass slide by adopting plasma, and then putting the glass slide into a silane coupling agent solution to react, so that a silane coupling agent is covalently bound with the glass slide to obtain a functionally modified biochip; and (2) combining the 5'end modified single chain oligo with the functionally modified biochip to obtain the biochip stationary phase. The biochip stationary phase disclosed by the invention is low in cost, excellent in physicochemical property, good in stability and tolerance and capable of adapting to a complex environment; more importantly, the method can be recycled, the resource utilization rate is increased, the pollution is reduced, and the method has huge application potential in the leading technical field of DNA synthesis by an enzyme method and the like.

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.

The invention discloses a T7-RNA polymerase mutant and application thereof, and relates to the field of nucleic acid tool enzymes and nucleic acid biology. The T7-RNA polymerase mutant is obtained by mutating glycine at the 47th site from the N end of a full-length amino acid sequence of wild type T7-RNA polymerase into phenylalanine, tryptophan or tyrosine; or, the amino acid sequence of the T7-RNA polymerase mutant and the amino acid sequence of the wild type T7-RNA polymerase have at least 80% homology, and glycine in a peptide fragment as shown in SEQ ID NO.10 in the amino acid sequence is mutated into phenylalanine, tryptophan or tyrosine; the full-length amino acid sequence of the wild type T7-RNA polymerase is as shown in SEQ ID NO. 1. The T7-RNA polymerase mutant is suitable for enzymatic synthesis of RNA, and has strong application potential in the aspects of in-vitro transcription, RNA synthesis, RNA drug synthesis, RNA vaccine manufacturing, gene editing, in-vivo protein expression or in-vitro translation systems and the like.

2'-deoxycytidine-5'-monophosphate and an enzymatic synthesis method therefor, relating to the technical field of biosynthesis. The synthesis method comprises: constructing a first engineered strain and a second engineered strain; respectively carrying out fermentation culture and protein separation and purification on the first engineered strain and the second engineered strain to obtain high-purity deoxycytidine kinase and high-purity polyphosphate kinase; using 2'-deoxycytidine as a deoxycytidine substrate and adenosine triphosphate as a phosphate donor, cyclically feeding polyphosphate, and keeping the ratio of adenosine triphosphate to polyphosphate to a preset ratio to form an enzymatic reaction system; and performing an enzymatic reaction to obtain 2'-deoxycytidine-5'-monophosphate. High-concentration deoxycytidine monophosphate can be quickly and effectively produced, so that pollution hazards caused by a chemical synthesis method can be solved, and the input amount of adenosine triphosphate as a phosphate donor in a biosynthesis system can also be reduced, thereby reducing production costs.

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 relates to a method for synthesizing deoxynucleoside triphosphate by an enzymic method, which comprises the following steps: by taking deoxynucleoside monophosphate dNMP as a raw material and taking ATP (adenosine triphosphate) and acetyl phosphoric acid as phosphoric acid donors, synthesizing dNTP (deoxynucleoside triphosphate) by using nucleotide kinase and acetokinase through a one-pot enzymic method. The one-pot enzyme method is used for preparing deoxynucleoside triphosphate, and the method has the advantages of being simple in operation, short in production period, low in production cost, high in yield, small in three-waste discharge, suitable for large-scale industrial production and the like.

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 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 discloses an alpha-glucan phosphorylase (alpha GP) mutant. The amino acid sequence of the alpha-glucan phosphorylase mutant is as shown in SEQ ID NO. 3. The invention also discloses an application of the mutant in enzymatic synthesis of hexose, especially mannose. The alpha-glucan phosphorylase mutant provided by the invention has higher catalytic activity, can significantly improve the production efficiency of mannose, and can be used for industrial production of mannose.

The invention relates to the field of biosynthesis high polymer materials, in particular to a biosynthesis method and application of omega-hydroxy fatty acid polyester (omega-PHFA) based on a whole-cell catalytic system. The method comprises the following steps: constructing a recombinant microbial whole-cell catalysis system containing P450 monooxygenase, CoA ligase and acyltransferase, and synthesizing omega-PHFA with a C12-C18 long carbon chain skeleton by taking renewable fatty acid as a substrate. Compared with a traditional chemical / enzymatic synthesis method, the technology is greener, and the performance defects of existing biosynthetic PHA materials are overcome. The invention provides related genetically engineered bacteria and a method for producing omega-PHFA by using the genetically engineered bacteria, and the genetically engineered bacteria have a wide application prospect.

The invention discloses a new enzyme with acylsulfonate transferase activity or a mutant thereof and application thereof, and belongs to the field of enzyme engineering. On the basis of an NCBI (National Center of Biotechnology Information) database, a new enzyme sequence is mined through a bioinformatics means, a new enzyme is obtained through codon optimization, gene synthesis and cell expression, the activity of acylsulfonate transferase is measured, and a corresponding mutant is obtained by using a directed evolution method. Compared with reported mouse acyl sulfonate transferase (ASTIV), the enzyme sequence obtained in the invention has better catalytic activity in a regeneration system of PAPS, and provides a new choice for enzymatic synthesis of PAPS.

Provided is a method and a kit for synthesizing and retrieving a polynucleotide enzymatically, including an initiator containing having a free 3′-hydroxyl group; a polymerase for incorporating nucleotide monomers to the initiator to form a nucleic acid strand from the free 3′-hydroxyl group, wherein the nucleic acid strand includes a guiding nucleotide to be recognized by an endonuclease; and the endonuclease cleaving the nucleic acid strand to release a predetermine polynucleotide and newly form a free 3′-hydroxyl group at 3′ end of the remaining nucleic acid strand which serve as a reusable initiator. Also provided is a kit for synthesizing and retrieving a polynucleotide using the method.

The invention discloses acyl sulfonic acid transferase and application thereof, and belongs to the field of enzyme engineering. On the basis of an NCBI (National Center of Biotechnology Information) database, a new enzyme sequence is mined through a bioinformatics means to obtain the acyl sulfonate transferase derived from Podarcismuris, a new enzyme is obtained through codon optimization, gene synthesis and cell expression, and the activity of the acyl sulfonate transferase is measured. Compared with reported mouse acyl sulfonate transferase, the acyl sulfonate transferase provided by the invention has better catalytic activity in a regeneration system of PAPS, and a new choice is provided for enzymatic synthesis of PAPS.

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 invention relates to the technical field of biological pharmacy, in particular to a carbonyl reductase mutant and an enzyme catalytic synthesis method of a montelukast sodium intermediate, and the amino acid sequence of the carbonyl reductase mutant is shown as any one of SEQ ID NO: 6-8. According to the enzymatic synthesis method of the montelukast intermediate, a compound II is taken as a substrate, and in the presence of a carbonyl reductase mutant, a coenzyme, a coenzyme recycle hydrogen donor, a cosolvent and a buffer solution, a biological catalytic reaction is carried out to generate the montelukast intermediate, namely a compound I; the specific synthesis route is as follows: # imgabs0 #. The synthesis method is easy and convenient to operate and environmentally friendly, and efficient synthesis of the chiral intermediate is achieved.

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 present invention relates to the field of bioengineering, and specifically, to a biochip, a method for preparing the biochip, and a method for synthesizing DNA. The biochip comprises: an azido substrate; a DNA probe, wherein the probe is ssDNA, and its 5' end is modified with an alkynyl group; and the DNA probe is connected to the azido substrate through an azido and alkynyl addition reaction catalyzed by copper ions. The biochip and the preparation method provided by the present invention connect the probe and the substrate of the biochip through an azido click reaction, which can not only enhance the probe binding strength and keep the probe upright, but also reduce the non-specific binding of ssDNA to the glass slide, solve the technical problem that ssDNA is prone to lodging, and improve the efficiency of enzymatic DNA synthesis.