310 results about "Enzymatic synthesis" patented technology

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M and a synthesis method of rebaudioside M, and belongs to the technical field of gene engineering. The glycosyl transferase mutant for synthesizing the rebaudioside M is a glycosyl transferase UGT76G4 mutant and / or a glycosyl transferase UGT91C1 mutant, and the glycosyl transferase mutant is a glycosyl transferase UGT76G4 mutant. The glycosyl transferase UGT76G4 mutant is obtained by mutation of an amino acid sequence as shown in SEQ ID NO: 1: 85th amino acid is mutated into R from L, and 90th amino acid is mutated into G from I; the glycosyltransferase UGT91C1 mutant is obtained by mutating an amino acid sequence shown in SEQ ID NO: 3 as follows: the 204th amino acid is mutated into A from L, the 208th amino acid is mutated into A from F, and the 380th amino acid is mutated into D from G. The mutant can realize efficient catalytic synthesis of rebaudioside M, and the yield of rebaudioside M synthesized by an enzymic method is improved.

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 synthetic method of 2 '-fluoro-2'-deoxynucleosid-5 '-triphosphoric acid and application of the 2'-fluoro-2 '-deoxynucleosid-5'-triphosphoric acid. The synthesis method comprises the following steps: reacting in a system containing adenosine triphosphate, 2 '-fluoro-2'-deoxynucleoside, metal ions and deoxynucleoside kinase to obtain 2 '-fluoro-2'-deoxynucleoside-5 '-phosphoric acid; and reacting in a system containing polyphosphoric acid, 2 '-fluoro-2'-deoxynucleoside-5 '-phosphoric acid, metal ions and polyphosphate kinase to obtain 2'-fluoro-2 '-deoxynucleoside-5'-triphosphoric acid. The obtained 2 '-fluoro-2'-deoxynucleoside-5 '-triphosphoric acid can be used for preparing heterologous nucleic acid. The invention provides an economic and green new way for synthesis of 2 '-fluoro-2'-deoxynucleoside-5 '-triphosphate, solves the problems of complex steps, non-environment-friendly reagents and high cost in the existing chemical synthesis method, and provides a thought for preparation of nucleotide derivatives.

The invention discloses an adenosine kinase mutant with high thermal stability and application thereof, and relates to the technical field of enzyme engineering, and the adenosine kinase mutant with high thermal stability is obtained by performing point mutation on the 253rd site or the 82nd site of adenosine kinase as shown in SEQ ID NO: 1. According to the invention, an adenosine kinase mutant is used as a biocatalyst in an enzyme catalysis manner; the method is used for enzymatic synthesis of 5 '-adenylic acid, adenosine and adenosine triphosphate react to prepare adenylic acid, generation of by-products and impurities in the reaction is reduced to a certain extent, production efficiency and purity of adenylic acid are improved, reaction conditions are mild in preparation in an enzyme catalysis mode, pollution to the environment is avoided, and the method is suitable for industrial production. And the high-thermal-stability adenosine kinase mutant is applied to adenylic acid preparation, so that the preparation efficiency and yield of adenylic acid can be improved.

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 invention discloses a method for preparing multi-branch oligosaccharide. According to the method, an in-vitro enzymatic synthesis strategy is adopted, monosaccharide activation and glycosyl transferase catalytic reaction are coupled, operation is simple and safe, the reaction conversion rate and the product yield are greatly improved, and a foundation is laid for industrial production of multi-branched oligosaccharides, especially multi-branched human milk oligosaccharides. According to the invention, synthesis of human milk oligosaccharides with three-branch, four-branch and other multi-branch structures can be realized, and reference is provided for synthesis of oligosaccharides and glycoconjugates with more complex structures.

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 present application provides a method of incorporating omega-3 fatty acids, such as EPA / DHA, into polar lipid molecules present in lecithin, the method comprising: (a) performing an enzymatic exchange reaction between the fatty acids present in the polar lipids of lecithin and omega-3 fatty acids present in concentrated fish oil to obtain an oil having a high content of polar lipids and omega-3 fatty acids, and (b) a step of concentrating the polar lipid content of the oil obtained in step a by supercritical fractionation or molecular distillation.

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 discloses an enzymatic synthesis method of poly (deoxyribonucleotide). The invention provides a synthetic method of polydeoxyribonucleotide. The synthetic method comprises the following steps: 1) synthesizing a single-stranded DNA molecule at the 3'end of a base primer by using TdT enzyme; the base primer is a single-stranded DNA (deoxyribonucleic acid) molecule which is composed of random basic groups and has the length of 5-100nt; 2) amplifying the single-stranded DNA molecule to obtain an amplification product; and 3) breaking the amplification product to obtain the polydeoxyribonucleotide. When the TdT enzymatic ssDNA is synthesized, the length of the synthesized ssDNA can be controlled by accurately adjusting the reaction time. The generated ssDNA is used as a minimum structural unit, and can be broken into PDRN with a specific length after being subjected to single chain cyclization and rolling circle amplification.

The invention belongs to the technical field of enzyme engineering, and particularly relates to a terminal deoxynucleotidyl transferase mutant and application thereof, and the mutant is obtained by performing R189L / K191G mutation on any one of amino acid sequences as shown in SEQ ID NO.1-7. Starting from a truncated terminal deoxynucleotidyl transferase TdT derived from Zonotrichia albicola, 52 key residues located in a substrate binding pocket are fixed to obtain 7 terminal deoxynucleotidyl transferase mutant sequences, the unfolding temperature Tm of five of the 7 terminal deoxynucleotidyl transferase mutant sequences is 10 DEG C or above higher than that of wild type WT / LG, after the obtained mutants are subjected to heat treatment, the mutant sequences are separated from the wild type WT / LG, and the terminal deoxynucleotidyl transferase TdT is separated from the wild type WT / LG to obtain the 7 terminal deoxynucleotidyl transferase mutant sequences. According to activity characterization of the mutant, compared with a wild type, the mutant obtained by the invention can obviously enable a primer to extend, has higher product yield and can play an important role in DNA enzymatic synthesis.

The present invention relates to variant of Family A polymerases able to synthesize a nucleic acid fragment without template and to incorporate a reversible modified terminator nucleotide during the nucleic acid fragment synthesis. The present invention further relates to uses thereof for enzymatic synthesis of nucleic acid molecules.

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 relates to the technical field of medicines, and discloses a method for synthesizing L-7-methyltryptophan by an enzymic method, tryptophan synthetase is used as a biocatalyst, 7-methylindole and L-serine are used as substrates, the enzymic method synthesis of the L-7-methyltryptophan is realized through a one-step catalytic reaction, and after the reaction is finished, the L-7-methyltryptophan is obtained. A reaction solution is subjected to a series of separation and purification methods such as membrane filtration, decoloration and concentration, and a finished product of L-7-methyltryptophan is obtained. The method is mild in process reaction condition, high in substrate conversion rate, free of byproduct generation, high in chiral selectivity, environmentally friendly and safe, compared with a traditional chemical synthesis method, the yield is high, the cost is low, and the separation and purification process is easy to operate, short in production period, high in yield and suitable for large-scale industrial production.

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.