38 results about "Sucrose phosphorylase" patented technology

The invention discloses a method for biosynthesizing epigallocatechin gallate glucoside, and belongs to the technical field of biocatalytic synthesis. According to the method, a sucrose phosphorylase mutant is adopted as a catalyst, the sucrose phosphorylase mutant is subjected to single-point mutation or combined mutation on the basis of an amino acid sequence SEQ ID NO.1, and the enzyme activity of the sucrose phosphorylase is improved through mutation; the mutant is utilized to realize efficient catalytic synthesis of epigallocatechin gallate glucoside, and in the whole catalytic process, the synthesis method is mild in condition, simple to operate, short in reaction time and high in catalytic efficiency, and has a good application prospect.

The invention discloses recombinant escherichia coli for producing sucrose phosphorylase as well as a construction method and application of the recombinant escherichia coli, and belongs to the technical field of biological engineering. The sucrose phosphorylase is constructed, the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO.11, a recombinant escherichia coli BdSP-L341V / L343F / V346P for expressing the sucrose phosphorylase is constructed, the sucrose phosphorylase expressed by recombinant bacteria or whole cells of the recombinant bacteria are used for carrying out catalytic reaction, and efficient production of ascorbyl glucoside can be realized. The invention also provides a method for producing ascorbic acid glucoside, the catalytic reaction is carried out at normal temperature and normal pressure, toxic and harmful organic solvents are not used, and the pollution to the environment is reduced; the whole catalytic process is a one-step reaction, has the advantages of high conversion rate, simple purification step and simple process, can better save the production cost, and has wide industrial application prospects.

The invention relates to the technical field of enzyme engineering, and particularly discloses a preparation method of immobilized sucrose phosphorylase (SPase) with low cost and high stability. According to the method, epoxy resin is synergistically modified through gallic acid (GA) and a polyethyleneimine-chitosan copolymer (PEI-CS), a covalent bond-hydrogen bond-ionic bond ternary cross-linked network is formed, and efficient immobilization of SPase is achieved. The preparation method comprises the following steps: preparation of a PEI-CS copolymer, preparation of a GA-PEI-CS modification liquid, modification treatment of epoxy resin, immobilization of SPase and determination of enzyme activity. The prepared immobilized enzyme has high enzyme activity retention rate (96.5%), wide temperature and wide pH stability (high activity is kept at 60 DEG C and pH 4.0-10.0), long reuse life (82.1% of enzyme activity is kept after 50 times of reuse) and low cost (26.7 yuan / kg), and can be widely applied to the fields of food industry, cosmetic industry and biological pharmacy.

The invention discloses a method for synthesizing alpha-arbutin through biotransformation, and belongs to the technical field of biochemistry. A gene cloning technology is utilized, sucrose phosphorylase Spase in leuconostoc mesenteroides and sucrose phosphorylase gtfa in bifidobacterium adolescentis are screened and found through a Deep Molecules and DLKcat algorithm on a Uniprot database, novopro is utilized for codon optimization, then the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa are expressed on a plasmid vector PET-28a and are converted into an escherichia coli BL21 (DE3) strain, and the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa in the bifidobacterium adolescentis are obtained. An engineering strain for heterologous expression of the gene is constructed by taking the gene as a chassis cell. According to the process, sucrose and hydroquinone are taken as co-substrates, and bioconversion is carried out under the action of sucrose phosphorylase from different sources to obtain alpha-arbutin.

The present invention provides engineered sucrose phosphorylase (SP) enzymes, polypeptides having SP activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing SP enzymes are also provided. The present invention further provides compositions comprising the SP enzymes and methods of using the engineered SP enzymes. The present invention finds particular use in the production of pharmaceutical compounds.

The invention belongs to the field of gene engineering and the technical field of molecular biology, and particularly relates to double promoters for expressing sucrose phosphorylase and application of the double promoters. The double promoters comprise any one of the following sequences: a nucleotide sequence as shown in SEQ ID NO.1 or a complementary sequence of the nucleotide sequence; 1, and a nucleotide sequence as shown in SEQ ID NO. 2 or a complementary sequence thereof. The sucrose phosphorylase is synthesized in bacillus subtilis by adopting a double-promoter expression system, the effect of the sucrose phosphorylase is obviously superior to the expression effect of a single promoter P43, the extracellular enzyme activity is improved by 403%, and efficient production is realized. Meanwhile, the DNA fragment with the promoter and RBS functions provided by the invention can realize secretory expression of protein genes under the condition of not adding an inducer, provides an effective means for secretory expression of protein by bacillus subtilis by applying the DNA fragment to expression of sucrose phosphorylase, and has a very good development prospect.

The application provides a non-inducible engineering strain for producing alpha-matrix and a construction method and application thereof. The engineering strain of the application is a recombinant Escherichia coli, which contains an expression regulatory element and a sucrose phosphorylase gene, and the expression regulatory element includes a PgapA-infC tandem promoter, a Plpp-infC tandem promoter or a PalsR-infC tandem promoter. The engineering strain can greatly shorten the protein expression period without adding an inducer, and the bacterial body after fermentation culture is ended is used as a whole cell catalyst to synthesize alpha-matrix by glycosylation reaction of a substrate hydroquinone and a sugar donor (such as sucrose), and the molar conversion rate of the substrate hydroquinone is high, and the engineering strain has an industrial application prospect.

The present disclosure relates to an engineered organism comprising at least one sucrose phosphorylase encoding gene; at least one sucrose permease encoding gene; and at least one phosphoglucomutase encoding gene, such that said engineered organism utilizes sucrose, wherein said engineered organism has ≥ 90% homology to Cupriavidus genus The present disclosure further relates to a method for carrying out fermentation using the engineered organism in the presence of sucrose, such that sucrose is utilized.

The application relates to the technical field of genetic engineering, in particular to a sucrose phosphorylase mutant and application thereof in synthesis of alpha-maronia. Streptococcus parasanguinis The application is characterized in that the histidine at the 152th position of the wild-type sucrose phosphorylase from Streptococcus sanguis is mutated into threonine, and the isoleucine at the 336th position is mutated into proline, so that the sucrose phosphorylase mutant is obtained, the catalytic activity and stability of the sucrose phosphorylase are significantly improved. When sucrose and hydroquinone are used as substrates, the yield of alpha-maronia is increased by more than 40% compared with the wild type, and the catalytic reaction time can be greatly shortened.

The invention relates to the technical field of gene engineering, in particular to a sucrose phosphorylase mutant and application thereof in alpha-arbutin synthesis. Specifically, a sucrose phosphorylase mutant is obtained by simultaneously mutating multiple sites of amino acids at the 13th site, the 206th site and the 243rd site of wild sucrose phosphorylase derived from streptococcus parahaemolyticus, recombinant escherichia coli is constructed, and the recombinant sucrose phosphorylase is produced by fermentation of the recombinant escherichia coli. According to the present invention, the efficiency of the alpha-arbutin production using sucrose and hydroquinone as the raw materials is significantly improved, and the test results show that the conversion rate of the alpha-arbutin production using sucrose and hydroquinone as the raw materials can achieve 90%, and the product concentration achieves 123 g / L, such that the strong industrial application potential is provided.

The invention relates to the technical field of enzyme engineering, in particular to recombinant sucrose phosphorylase or a functional fragment thereof, a preparation method and application thereof. The recombinant sucrose phosphorylase has at least one amino acid sequence as shown in SEQ ID NO: 1. The recombinant sucrose phosphorylase has the characteristics of high activity and high specificity, has specificity on catalysis of glucosidic bonds of polyphenol and glucoside, and can specifically catalyze 4'and 4 ''sites of polyphenol to form glucosidic bonds with glucose.

The invention discloses a construction method and application of escherichia coli for producing a heparin precursor by using cane sugar. The invention provides a method for constructing a recombinant strain, which comprises the following steps: overexpressing a heterologous sucrose-6-phosphohydrolase gene sacA and a sucrose phosphorylase gene spI in escherichia coli to obtain an engineering strain which can grow by using sucrose as a carbon source and can effectively synthesize a heparin precursor; a sucrose specific enzyme IIBC component gene sacP of a phosphotransferase system and a sucrose-6-phosphohydrolase gene sacA are co-expressed, and recombinant bacteria can more efficiently synthesize a heparin precursor in shake-flask culture, so that the yield of the heparin precursor is increased. The recombinant strain constructed by the invention has a good application prospect.

The application discloses a kind of added Co 2+ The application discloses a method for improving the yield of D-allulose produced by multi-enzyme catalysis, belonging to the field of biotechnology. The application provides a method for producing D-allulose by multi-enzyme catalysis, which comprises adding sucrose phosphorylase from Bifidobacterium adolescentis, fructokinase from Clostridium acetobutylicum, D-allulose-6-phosphate phosphatase from Clostridium thermocellum, and D-allulose-6-phosphate-3-epimerase from Pantoea sp to a reaction system containing sucrose and ATP, and then adding a metal ion solution to the reaction system to produce D-allulose. The application synthesizes an important low-calorie functional rare sugar D-allulose using inexpensive sucrose as a substrate, and experiments show that the addition of cobalt ions can increase the yield of D-allulose by five times.

The application discloses application of high-efficiency sucrose phosphorylase in production of glycerol glucoside or preparation of a glycerol glucoside product. A preparation method of the glycerol glucoside comprises generating glycerol glucoside from glycerol and sucrose by using sucrose phosphorylase for catalysis; and an amino acid sequence of the sucrose phosphorylase is shown as SEQ ID No. 1. The sucrose phosphorylase provided by the application has high catalytic activity, and a whole-cell biological catalyst can perform catalytic reaction to produce glycerol glucoside under the conditions of 30 DEG C to 50 DEG C and pH 4.5 to 9.0, the substrate conversion rate is high, the yield of GG can reach more than 400 g / L, the proportion of 2-alpha GG is high, and the sucrose phosphorylase has high industrial application potential.

The invention discloses a sucrose phosphorylase mutant and application thereof in preparation of L-ascorbyl glucoside, and belongs to the field of enzyme engineering modification. According to the invention, sucrose phosphorylase is semi-rationally modified, homologous modeling is used for mutant prediction, a small mutant library is constructed, and a mutant with improved thermal stability is obtained after screening. The mutants are used for synthesizing the L-ascorbyl glucoside, reaction conditions are optimized, the yield of the L-ascorbyl glucoside is improved, and a foundation is laid for improving stability and large-scale application of industrial production.

This invention relates to the field of enzyme engineering technology, and particularly to a recombinant sucrose phosphorylase or its functional fragment, its preparation method, and its applications. The recombinant sucrose phosphorylase has at least one amino acid sequence as described in SEQ ID NO:1. The recombinant sucrose phosphorylase exhibits high activity and high specificity, specifically catalyzing the formation of glycosidic bonds between polyphenols and glucosides, and can specifically catalyze the formation of glycosidic bonds between polyphenols and glucose at the 4' and 4'' positions.

The invention discloses an anti-atherosclerosis myricetin glycoside and a preparation method and application thereof, and the preparation method comprises the following steps: by taking sucrose as a glycosyl donor and myricetin as a glycosyl receptor, carrying out enzymatic reaction under the catalysis of sucrose phosphorylase, and transferring glucosyl generated by decomposition of the sucrose to C4-site hydroxyl of myricetin to obtain the myricetin glycoside, the sucrose phosphorylase is derived from leuconostoc mesenteroides. According to the method, oriented synthesis of myricetin glucoside can be realized, the product is single and does not have redundant byproducts, and the subsequent separation and purification process is greatly simplified. The myricetin glucoside is prepared through enzymatic catalysis, the reaction can be efficiently carried out under mild conditions, high temperature, high pressure and other harsh processes are not needed, energy consumption and equipment cost are reduced, impurity generation in the reaction process is reduced, and the production cost is reduced from the source.

The invention discloses a sucrose phosphorylase mutant, an engineering bacterium and application of the sucrose phosphorylase mutant in catalytic synthesis of alpha-arbutin. The sucrose phosphorylase CbSP mutant is obtained by performing site-saturation mutation or combined mutation on the 53rd site, the 60th site, the 135th site, the 196th site, the 197th site or the 234th site of an amino acid sequence shown as SEQ ID No.3. The invention further discloses a preparation method of the sucrose phosphorylase CbSP mutant. The catalytic activity and the thermal stability of the mutant constructed by the invention are remarkably improved, the specific enzyme activity of the sucrose phosphorylase CbSPM2 mutant is improved by 47.39 times compared with that of a wild type, the maximum substrate (hydroquinone) feeding amount can reach 55g / L, the product concentration is gradually increased along with the extension of time, most substrates can be converted within 24h, the molar conversion rate of the substrates is 90.23%, and the product concentration is 122.7 g / L. The CbSP mutant disclosed by the invention has a good industrial application prospect.

The present invention relates to the field of enzyme catalysis technology, in particular to a sucrose phosphorylase with enhanced heat resistance and its application in the preparation of α-arbutin by high-temperature catalysis. Its amino acid sequence is shown in SEQ ID NO: 3, and it has stronger heat resistance compared with the wild-type sucrose phosphorylase. The sucrose phosphorylase with enhanced heat resistance of the present invention realizes industrial biocatalysis at high temperature, can improve the catalytic efficiency, reduce the risk of microbial contamination, and at the same time helps to reduce the substrate viscosity and increase the transfer rate, which is of great significance for shortening the reaction time and improving the production efficiency.

The present application relates to the technical field of genetic engineering, and particularly relates to a sucrose phosphorylase mutant and application thereof in synthesis of alpha-mogroside. Streptococcus parasanguinis The present application is obtained by simultaneously mutating amino acid multiple sites of 13th, 206th and 243rd of wild-type sucrose phosphorylase derived from Parahaemolyticus (Parahaemolyticus), and constructing recombinant Escherichia coli by using the sucrose phosphorylase mutant, and then fermenting the recombinant sucrose phosphorylase to significantly improve the efficiency of producing alpha-mogroside by using sucrose and hydroquinone as raw materials. The conversion rate of producing alpha-mogroside by using sucrose and hydroquinone as raw materials can reach 90%, and the product concentration reaches 123 g / L, so the present application has strong industrial application potential.

The invention relates to the technical field of bioengineering, in particular to a sucrose phosphorylase mutant BaSP-P134A and an application of the sucrose phosphorylase mutant BaSP-P134A in production of glucose-1-phosphoric acid. The sucrose phosphorylase mutant BaSP-P134A is obtained by carrying out site-specific mutagenesis on amino acid at the 134 position of an amino acid sequence as shown in SEQ ID NO.2; the site-specific mutagenesis is that proline at the 134 site of the amino acid sequence as shown in SEQ ID NO. 2 is mutated into alanine. The sucrose phosphorylase mutant BaSP-P134A can be applied to the field of industrial production, G-1-P can be efficiently prepared, the production intensity is high, the catalytic effect on a high-concentration substrate is good, the enzyme shows better stability, efficient conversion of sucrose can be achieved, and a favorable basis is further provided for industrial application of sucrose phosphorylase.

The application discloses a fisetin-4'-O-alpha-D-glucopyranoside for resisting lung cancer and a preparation method and application thereof, and the preparation method comprises the following steps: taking sucrose as a glycosyl donor, taking fisetin as a glycosyl acceptor, and performing an enzymatic transglycosylation reaction under the catalysis of sucrose phosphorylase to direct the glucose group generated by decomposing the sucrose to be transferred to a hydroxyl group at a C4' position of the fisetin, so that a target product is obtained; and the sucrose phosphorylase is derived from Bifidobacterium adolescentis. The application retains the original core biological activities of the fisetin, such as antioxidation and anti-inflammation, provides high-quality raw materials for the application of the fisetin in the fields of innovative drug development for resisting lung cancer and preparation of medical intermediates, and expands the clinical transformation potential of fisetin compounds.

The invention belongs to the field of genetic engineering and the technical field of molecular biology, and relates to a DNA fragment, in particular to a DNA fragment, with a promoter function, of bacillus subtilis and application of the DNA fragment in production of sucrose phosphorylase. According to the method for screening the single promoter, efficient secretory expression of BbrSP-VF and BadSP-QL is achieved in bacillus subtilis, the effect of the method is remarkably superior to the expression effect of a single promoter P43, and the extracellular enzyme activity is improved by 247% and 236% respectively. According to the DNA fragment with the promoter, secretory expression of protein genes can be achieved under the condition that an inducer does not need to be added, the DNA fragment with the promoter is applied to expression of sucrose phosphorylase, an effective means is provided for secretory expression of protein by bacillus subtilis, and the DNA fragment with the promoter has a good development prospect.

The invention discloses a method for selectively producing EGCG-4 '-O-alpha-D glucopyranoside by using sucrose phosphorylase, and belongs to the technical field of biological catalytic synthesis. An engineered mutant derived from leuconostoc mesenteroides sucrose phosphorylase (LmSPase) is used as a catalyst. The mutant is constructed through single-point or combined mutation on the basis of a wild type with the sequence of SEQ ID NO.1, and the enzyme activity, the stability and the substrate specificity of the mutant are all remarkably improved. By utilizing the high-performance mutant, a method for efficiently synthesizing (-)-epigallocatechin gallate 4 '-O-alpha-D glucopyranoside is established. The method is mild in reaction condition, simple and convenient to operate, short in reaction time and high in catalytic efficiency, and shows good application potential.

The present invention relates to the field of enzyme catalysis technology, and in particular to a sucrose phosphorylase with enhanced catalytic activity and its application. The amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO: 3, and the sucrose phosphorylase has stronger catalytic activity than the wild-type sucrose phosphorylase. The sucrose phosphorylase with enhanced catalytic activity of the present invention can improve reaction efficiency, significantly reduce the activation energy of the chemical reaction, and accelerate the reaction process; it also reduces energy consumption and environmental impact, reduces production costs, enhances adaptability, improves production flexibility, and can promote the development of the green chemical industry.

Antibiotic-free systems for maintaining plasmids in host cells and methods of using same. The plasmids can comprise one or more plasmid-maintenance genes and a gene of interest configured to express in the host cell. The one or more plasmid-maintenance genes can comprise one or more of a prophage repressor gene, a sucrose phosphorylase gene, and an alcohol / aldehyde dehydrogenase gene. The methods can comprise growing the host cell with the plasmid comprised within the host cell, wherein the plasmid can be maintained during growth in the host cell without the use of antibiotics. In systems with a plasmid comprising a sucrose phosphorylase gene, the methods can comprise growing the host cell in the presence of sucrose as a sole carbon source. In systems with a plasmid comprising an alcohol / aldehyde dehydrogenase gene, the methods can comprise growing the host cell in the presence of glucose as a sole carbon source.

The invention relates to the technical field of gene engineering, in particular to a sucrose phosphorylase mutant and application thereof in synthesis of alpha-arbutin. The preparation method comprises the following steps: mutating histidine at the 152nd site of wild type sucrose phosphorylase from streptococcus sanguineus into threonine and mutating isoleucine at the 336th site of the wild type sucrose phosphorylase from streptococcus sanguineus into proline, so as to obtain a sucrose phosphorylase mutant; the catalytic activity and the stability of the sucrose phosphorylase are obviously improved. When alpha-arbutin is synthesized by taking sucrose and hydroquinone as substrates through detection, the yield is increased by more than 40% compared with that of a wild type, and the catalytic reaction time can be greatly shortened.

The invention discloses a sucrose phosphorylase mutant and application of the sucrose phosphorylase mutant in synthesis of epigallocatechin gallate glucoside, and belongs to the technical field of enzyme engineering and biological catalysis. The sucrose phosphorylase mutant is obtained by mutating a plurality of amino acid sites on the basis of an amino acid sequence of SEQ ID NO.1. The mutant not only remarkably improves the enzyme activity, but also more importantly changes the regioselectivity of the mutant; by utilizing the mutant, efficient catalytic synthesis of (-)-epigallocatechin gallate 4 ''-O-alpha-D glucopyranoside is realized, and a key foundation is laid for stable industrial production and large-scale application.

The invention belongs to the technical field of biology, and discloses a sucrose phosphorylase mutant and application thereof. Compared with wild sucrose phosphorylase, the sucrose phosphorylase mutant obtained by rational design and enzyme evolution screening on the basis of an amino acid sequence as shown in SEQ ID NO: 1 has the advantages that the protein structure and function are changed, and the catalytic activity of the sucrose phosphorylase mutant is obviously improved; higher conversion rate and conversion rate from ascorbic acid to ascorbic acid glucoside can be obtained. A sucrose phosphorylase mutant gene is introduced into escherichia coli for expression, and ascorbic acid can be more efficiently catalyzed to produce ascorbyl glucoside. The method for producing ascorbyl glucoside has the characteristics of simplicity in operation, low cost, high product synthesis efficiency and reduction of environmental pollution, and has a good industrialization prospect.

The invention discloses a sucrose phosphorylase mutant, the amino acid sequence of the sucrose phosphorylase mutant is SEQ ID NO: 3 or SEQ ID NO: 5, and the sucrose phosphorylase mutant is obtained by site-directed mutagenesis on the basis of sucrose phosphorylase from microorganism Bifidobacterium thermophilum. Compared with sucrose phosphorylase from other sources, the sucrose phosphorylase mutant provided by the invention can catalyze a high-concentration substrate, has higher catalytic activity, wider substrate range, higher yield and higher-purity product 2-alpha-GG, and can be used for industrial production of glycerol glucoside.