77results about How to "Good enzymatic properties" patented technology

The invention provides glutamine transaminase with improved enzymatic activity, wherein the amino acid sequence is shown as SEQ ID NO.1. According to the invention, amino acid at the N end of MTG (Microbial Transglutaminase) maturase is subjected to deficiency and saturation mutation by taking the efficient expression of MTG in colibacillus as an improvement platform, and a mutant strain with good enzymatic property is obtained. The enzyme activity is improved by 1.85 times, and the thermal stability is improved by 2.7 times. The modified enzyme is more suitable for industrial applications, the production cost can be lowered, and the production efficiency can be improved.

The present invention relates to an isolated mutant acid phosphatase / phytase with improved enzymatic properties. The mutant acid phosphatase / phytase composition is particularly useful in animal feed compositions.

The invention belongs to the technical field of microorganism gene engineering, and particularly relates to deacetylase capable of producing high-yield glucosamine and a coding gene thereof. The gene CsnagA is separated from the natural strain cronobacter sakazakii 0360. Genomic DNA is separated from cronobacter sakazakii with the preservation number of CCTCC NO:M 2016162. The nucleotide sequence of the gene is shown in SEQ ID NO:1. The sequence of protein coded by the gene is shown in SEQ ID NO:2. By means of genetic recombination, the gene is transferred into escherichia coli to construct genetically engineered bacteria, so as to obtain an efficient glucosamine fermentation production capacity, and glucosamine can be produced with the enzymatic biosynthesis method. Compared with the prior art, the deacetylase has the advantages that the production efficiency is improved, and the production cost is reduced.

The invention discloses a mutant of nitrile hydratase derived from caldalkalibacillus thermarum, and belongs to the technical field of enzyme engineering. The half-life period of the nitrile hydratasemutant Cal.t Nhase-A20V at 70 DEG C is about 10 min, the thermal stability of the mutant Cal.t Nhase-A20V has no too large change compared with that of a wild enzyme, and the specific enzyme activityof the mutant Cal.t Nhase-A20V is 128% of that of the wild enzyme. At the same time, the mutant also has better substrate tolerance and product tolerance, the final yield of nicotinamide produced bywhole-cell catalysis reaches 598 g / L; therefore, the nitrile hydratase mutant Cal.t Nhase-A20V provided by the invention has very good enzymatic properties, and is beneficial to industrial productionin future.

The invention discloses a catalase mutant with improved enzyme activity and heat stability, and belongs to the field of biological engineering. Catalase derived from bacillus subtilis (B. subtilis WSHDZ-01) is subjected to site-specific mutagenesis, so that a lysine of an amino acid sequence coded by the catalase is respectively mutated into tyrosine Y, valine V, methionine M or isoleucine I at the site 114. The expression quantity of the catalase mutant is respectively 2.23, 2.41, 1.46 and 1.38 times of the expression quantity before mutation, and the half-life period of the catalase after mutation is respectively 2.17, 1.5, 2 and 1.67 times of the half-life period before mutation in the terms of thermal tolerance. The catalase mutant is suitable for industrialization production requirements and has wide application prospect.

The invention provides a new esterase gene named Est_p1 and a recombination expression system thereof. The gene is cloned from the metagenomic library of the marginal mud 100 meters under the South China Sea. The gene is an 891bp long amino acid numbered 296, which successfully constructs a recombination expression system of a colon bacillus named pET28a-Est_p1 / BL21. The molecular weight of a target protein is 33.5 kD. With pNP-butyric ester as catalyzing substrates, the Est_p1 is proved to be a mildly alkaline medium temperature esterase. The Est_p1 has strong catalytic activity for short esters and is applicable to esterification and transesterification industries.

The invention discloses alkaline thermal-stability SGNH family esterase EstD1. The amino acid sequence of the alkaline thermal-stability SGNH family esterase EstD1 is one of the amino acid sequence (1) shown in SEQ ID NO.1 and the amino acid sequence (2), wherein one or more pieces of amino acid are added or replaced or lacked or inserted, shown in SEQ ID NO.1. The invention further discloses a gene EstD1 of the alkaline thermal-stability SGNH family esterase EstD1, the nucleotide sequence of the gene EstD1 is one of the nucleotide sequence (1) shown in SEQ ID NO.2 and the nucleotide sequence (2), wherein one or more pieces of nucleotide are added, replaced, lacked or inserted, shown in SEQ ID NO.2, the nucleotide sequence (3) formed through hybridization with the nucleotide sequence (1) or nucleotide sequence (2) under strict conditions and the nucleotide sequence (4) which is different from the nucleotide sequence (1), the nucleotide sequence (2) and the nucleotide sequence (3) due to the degeneracy of a genetic codon. The alkaline thermal-stability SGNH family esterase EstD1 can be used for deacetylation of 7-ACA.

The invention discloses a Beta-N-acetylglucosaminidase of Paenibacillus barungensis, an encode gene thereof and an application of beta-N-acetylglucosaminidase. The provided Beta-N-acetylglucosaminidase PbNag39 is excellent in the enzymatic property, and has a specific activity against chitosan is 28.3 U / mg, and the final products of hydrolysis of chitosan oligosaccharides are N-Acetyl glucosamine.The Beta-N-Acetylglucosaminidase and chitinase can hydrolyze chitin powder synergistically and obtain N-Acetyl glucosamine. The Beta-N-acetylglucosaminidase PbNag39 has the characteristics of high specific activity, good thermal stability and excellent hydrolysis characteristics. It can stabilize and play a catalytic role in a wide range of pH, and has important application value in chitin conversion.

The invention belongs to the technical field of a material, and discloses a penioillin acylase preparation and an immobilization method. The immobilization method comprises the following steps of (1)under the conditions of stirring at room temperature, dripping a potassium hydroxide water solution into a tetrakis hydroxymethyl phosphonium chloride solution to obtain a mixed solution containing trisphosphine oxide; (2) adding magnetic cellulose nanometer crystals into the mixed solution containing trisphosphine oxide in the step (1); performing stirring for 1 to 30min at room temperature; performing centrifugal washing to obtain the treated magnetic cellulose nanometer crystals; (3) diluting the penioillin acylase into a buffer solution with the pH being 6 to 11 to obtain an enzyme solution; (4) adding the treated magnetic cellulose nanometer crystals into the enzyme solution; performing curing at room temperature to obtain the immobilized penioillin acylase, i.e., the penioillin acylase preparation. The method is simple; the cost is low; the enzyme activity recovery rate of the prepared immobilized penioillin acylase preparation is high; the enzyme loading rate is high; meanwhile,the stability is high.

The invention provides an alpha-amylase truncated body and application thereof, and relates to meaningful alpha-amylase variant which simultaneously improves thermal endurance and catalytic activity, which is characterized in that 45-467 amino acid peptide fragment of bacillus subtilis Cn7alpha-amylase is cut off, and a fragment of artificially synthetic peptide is fused on C terminal. Obtained variant alpha-amylases respectively have temperature toleration increased by 2.7 DEG C, catalytic activity increased by 2.23 times compared with the parent alpha-amylase; and the variant alpha-amylases are more beneficial to industrialized production in the starch industry than the parent alpha-amylase.

The invention provides the construction of a Pichia pastoris engineering bacterial strain GS115 / appA NR for producing heat-resisting recombinant phytase and a method for producing the heat-resisting phytase by using the bacterial strain. A heat-resisting phytase gene is synthesized by adopting the two-step PCR method and a recombinant plasmid is constructed to be transferred into the Pichia pastoris bacterial strain GS115 through electric shock. The invention comprises the following steps of: (1) cloning the heat-resisting phytase gene synthesized by the two-step PCR method into a Pichia pastoris secretive expression vector to obtain a Pichia pastoris recombinant expression plasmid; (2) transferring the ombinant expression plasmid to the Pichia pastoris host GS115 through electric shock, screening by using an MD flat board and a substrate flat board containing calcium phytate, and then screening out a bacterial strain with largest hydrolysis circle; and (3) choosing positive clone and inducing to express by using 0.5% methanol. The heat stability of the recombinant heat-resisting phytase expressed by the Pichia pastoris engineering bacterial strain GS115 / appA NR is much higher than that of E.coli K12 wild type phytase through measurements, and specifically, 92% of enzymatic activity can still be maintained after a water bath at 95 DEG C for 10 min. The fermentation crude enzymatic activity of the recombinant phytase produced by fermenting the Pichia pastoris engineering bacterial strain can reach 978 U / mL.

The invention provides a high-efficiency directed evolution method of a lipase gene. The method comprises the following steps: (1) constructing a display expression vector of saccharomyces cerevisiae;(2) constructing display expression recombinant plasmids of the saccharomyces cerevisiae of a lipase; (3) obtaining vector-carried lipase gene fragments in a homologous sequence and constructing an expression mutation library; and (4) analyzing the display expression mutation library and mutants. The method synchronizes the construction of the mutation library and the expression of the mutants ina general directed evolution technology and overcomes the defects that directed evolution of lipase molecules has complicated operation steps, difficult mutant selection, and the like in the prior art.

A preparing method for a co-immobilized enzyme for accelerating aging and clarifying Shanxi aged vinegar comprises the steps that an esterifying enzyme solution and a saccharifying enzyme solution are prepared; then, sodium alga acid and xanthan gum are taken at the mass ratio of 5:1 to be prepared into a carrier material, the carrier material is dissolved with 55-60-fold water with the temperature of 50-60 DEG C by mass, a quantitative esterifying enzyme is added at the room temperature, and the mixture is evenly stirred and then subjected to standing; after bubbles completely disappear, the mixture is slowly dripped into a calcium chloride aqueous solution with the concentration of 2% at the condition of 4-5 DEG C, and particles are generated after curing is carried out for 1 h; the particles are filtered out with two-layer gauze, washed 3-5 times with an acetic acid-sodium acetate buffer solution with the pH being 3.6, calcium chloride on the surface is removed, moisture on the surface is absorbed away, and a particle immobilized enzyme with the diameter ranging from 2.0 mm to 2.3 mm is prepared; the immobilized enzyme is put into the saccharifying enzyme solution for adsorption for 6-7 h with the mass volume ratio of 1:1; then the particles are filtered out with gauze and washed 3-5 times with an acetic acid-sodium acetate buffer solution with the pH being 4.6, the enzyme solution on the surface is removed, moisture on the surfaced is absorbed away, and the co-immobilized enzyme is prepared.

The invention provides a novel feruloyl esterase gene from soil. The nucleotide sequence and the amino acid sequence of the novel feruloyl esterase gene are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. The esterase gene is heterologously expressed in escherichia coli BL21(DE3), and the molecular weight of purified recombinase (FAEXuan) is 29kDa. The catalytic activity of the FAEXuan for a substrate (ferulic acid methyl ester) is highest, the enzyme activity is 40U / mg, the optimum temperature is 30 DEG C, and the optimum pH is 5.0. After reaction for 4 hours under the pH of 3.0-10.0, theferuloyl esterase can keep the activity still by 75% or more, so that stronger pH stability is shown. The feruloyl esterase FAEXuan has better tolerance to metal ions and organic solvent. The substrate utilization preference and the phylogenetic analysis show that the FAEXuan belongs A-type feruloyl esterase. Due to good enzymatic properties, the FAEXuan has wide application prospect in industrialproduction of the fields of foods, pharmacy and feed and the like.

The invention belongs to the technical field of molecular biology and protein engineering, and discloses a recombinant polymerase, an encoding gene, a preparation method, a carrier, a kit and application; wherein the recombinant polymerase is derived from a hybrid DNA polymerase obtained by covalently connecting a protein Cl7 to the N end of a Taq DNA polymerase protein sequence in a Thermous aqueatus YT1 strain through a flexible connection sequence, and the amino acid sequence is shown as SEQ ID No.4; or the sequence is an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids and has the same function. The expression vector pET30a-Cl7-Taq disclosed by the invention is used for converting escherichia coli and inducing expression to obtain a target protein Cl7-Taq. The continuous synthesis capability of TaqDNA polymerase in PCR nucleic acid amplification is greatly improved, and the TaqDNA polymerase has a wide application prospect.

The invention discloses alpha-glucosidase QsGH97a derived from deep sea bacteria as well as a coding gene and application thereof. The alpha-glucosidase QsGH97a gene is screened from a bacterial genome library provided by the second ocean research institute of the State Ocean Bureau; it is found that the alpha-glucosidase QsGH97a has relatively good enzymatic properties through expression and purification of the alpha-glucosidase QsGH97a; and the alpha-glucosidase QsGH97a can be used for industrial glucose production, and can be applied to food processing and chemical industry related to saccharide hydrolysis. The obtained alpha-glucosidase gene can be cloned into a proper host to realize heterologous expression, the purification method is simple, the industrial production of alpha-glucosidase can be realized, the alpha-glucosidase gene is applied to the chemical industry of mass production of glucose, and in addition, the excellent alkali resistance and salt resistance of the alpha-glucosidase gene enable the alpha-glucosidase gene to have a prospect in special industrial production.

The invention discloses a phenylalanine amino mutase mutant derived from pantoea agglomerans, and belongs to the technical field of enzyme engineering. The sequence of the phenylalanine amino mutase mutant is shown in SEQ ID NO.2, and the mutant enzyme still has 60% residual enzyme activity after being processed at 50 DEG C for one hour and has higher temperature stability compared with wild enzyme. Meanwhile, the mutant also has better pH stability, and is good for future industrial production.

The invention discloses a PGL (Polygalacturonate Lyase) mutant capable of improving secretion performance and belongs to the technical field of bioengineering. A 58-th isoleucine I point of PGL deriving from Bacillus sp.WSHB04-02 is mutated into valine V. The extracellular enzyme activity of the mutated PGL is improved to 337.58 U / mL, and is 2.60 times that of the PGL which is not mutated, and the expression quantity of extracellular secretion is obviously increased. In addition, the optimum reaction temperature of the mutant is improved by 5 DEG C, and the optimum enzyme activity can be better shown under the condition of high temperature; Km is obviously improved, the capacity of the mutant in combination with a substrate is improved to be 4.5 times than that of the mutant which is not mutated, and kcat, t1 / 2,kcat / Km and the like are improved. The enzymatic property of the PGL provided by the invention is greatly improved, and requirements for industrial production and social production are met.

The invention discloses microbulbifer arenaceous beta-galactosidase as well as an encoding gene and application thereof. The invention provides the microbulbifer arenaceous beta-galactosidase which isa protein of which the sequence is shown in a sequence 2 in the description. The microbulbifer arenaceous beta-galactosidase provided by the invention is excellent in enzymatic property, and the specific enzyme activity to a natural substrate, namely lactose, is 38.69U / mg. Meanwhile, the lactose can be efficiently hydrolyzed, 80% or greater of the lactose in milk can be hydrolyzed with an enzymeaddition amount of 1U / mL at a refrigeration temperature of 4 DEG C after 36 hours of a reaction, after 48 hours of the reaction, 90% or greater of the lactose can be hydrolyzed, after 72 hours of thereaction, all lactose in the milk can be hydrolyzed. In addition, after 84 hours of the reaction, 90% or greater of the lactose in a 5% (w / v) whey solution can be hydrolyzed. The protein disclosed bythe invention has great application value for production of low-lactose or lactose-free milk products in the food industry.

The invention provides a heat-resistant N-acetylglucosamine deacetylase and an encoding gene and application thereof. The N-acetylglucosamine deacetylase is a) a protein encoded by the amino acid sequence shown in SEQ ID NO.2, or b) a fusion protein obtained by connecting an N-terminal and / or C-terminal of the protein shown in SEQ ID NO.2 and a tag, or c) a protein, with same functions, obtained by performing substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence shown in SEQ ID NO.2. The invention further relates to application of the protein in the preparation of glucosamine. The invention further relates to a biological material related to the heat-resistant N-acetylglucosamine deacetylase. The deacetylase can catalyze the deacetylation reaction of N-acetylglucosamine, and is applied to industrial preparation of glucosamine, the reaction conditions are mild, the energy consumption is less, no contaminant is generated, and the deacetylase has better industrial application prospects.

The invention provides a novel immobilized oxidordeuctase electrode and a preparation method thereof. The preparation method comprises the steps: firstly, a modifier is prepared from halogenated carboxylic acid and an imidazole compound; the modifier and carbonyldiiazole are added into anhydrous dimethylsulfoxide in a mixed mode, and a magnetic stirring reaction is conducted to obtain an active body; the active body is added into oxidordeuctase liquid to be dialysed to obtain an acylated oxidordeuctase solution; then immobilized oxidordeuctase powder is prepared from a zinc nitrate solution, 2-methylimidazole, the acylated oxidordeuctase solution and a polyvinylpyrrolidone mixed solution; and finally, the oxidordeuctase electrode is prepared. The characteristics of large specific surface area, more types, large loading capacity, satble structure and the like of materials of metal-organic frameworks (MOFs) are combined, by improving a preparation process, modified enzymes with monomersare embedded in the MOFs, thus it can be guaranteed that the activity of the immobilized enzymes is not obviously lowered, and the reuse stability of the enzymes can further be improved.

The invention discloses rhizomucor miehei-derived beta-1,3-glucanase and application thereof. The rhizomucor miehei-derived beta-1,3-glucanase is thermophilic fungus rhizomucor miehei-derived endo-type beta-1,3-glucanase (RmLam81A) that specifically acts on the beta-1,3 glycosidic bond in curdlan polysaccharide. Pichia pastoris is used for heterologous expression, and after high-density fermentation culture, the final enzyme activity is up to 694U / mL. Food grade beta-1,3-glucan (the curdlan polysaccharide, laminarin, and the like) are used as raw materials for enzymatic hydrolysis to obtain beta-1,3-glucosyl-oligosaccharides with degree of polymerization (DP) of 2-5. The yield of the soluble oligosaccharides is about 75-85%. The beta-1,3-glucosyl-oligosaccharides can be obtained by slag liquid separation, material liquid enrichment and spray-drying of hydrolysate. The beta-1,3-glucanase disclosed in the present invention has excellent enzymatic properties. Compared with that of the chemical or enzymatic methods in the prior art for preparing the beta-1,3-glucosyl-oligosaccharides, the method using the rhizomucor miehei-derived beta-1,3-glucanase mild in reaction conditions, environmentally-friendly, and higher in the yield of the oligosaccharides, and has important application value in enzymatic preparation of the beta-1,3-glucosyl-oligosaccharides.

The invention discloses L-aspartate beta-decarboxylase mutants and an application thereof, and belongs to the technical field of biological engineering. The catalytic enzyme activity of the L-aspartate beta-decarboxylase mutants N35D and A179E provided by the invention are improved by 238% and 244% respectively under the pH of 7.0, the remaining enzyme activity of the mutants N35D and A179E treated for 12 h under the pH of 4.5 is 83.9% and 85.5%, respectively, and compared with a control wild-type enzyme having residual enzyme activity of 24.8%, the mutants provided by the invention have significantly-improved acid stability, and therefore the L-aspartate beta-decarboxylase mutants N35D and A179E provided by the invention have better enzymatic properties.

The invention discloses a preparation method of proline endonuclease. The preparation method comprises the following steps of preparing aspergillus niger strain liquid, culturing and fermenting, and extracting and treating fermenting liquid, wherein a BMGY liquid culture medium is used as a culture medium for preparation of the strain liquid, and culture conditions are as follows: shake-flask culture is adopted, the temperature is 28 to 35 DEG C, the speed is 200 to 250r / min, and the culture time is 12 to 18h; the prepared strain liquid is subjected to shake-flask culture, an aspergillus niger strain high-density fermenting culture medium is used as a culture medium, and culture conditions are as follows: the temperature is 28 to 35 DEG C, and the speed is 200 to 250r / min; or the prepared strain liquid is cultured in a fermenting tank, the spergillus niger strain high-density fermenting culture medium is used as a culture medium, and culture conditions are as follows: the temperature is 28 to 35 DEG C, and the speed is 200 to 800r / min; by filtering, concentrating and blending the fermenting liquid, and finely filtering or drying, liquid proline endonuclease or solid proline endonuclease is obtained. The preparation method has the advantages that the natural aspergillus niger strain is used, and proline is used as an inducing substrate, so that the proline endonuclease with high enzyme activity, wide temperature and pH (potential of hydrogen) application range and good stability is prepared under the moderate fermenting condition.

The present invention discloses one new kind of artificially synthesized organophosphate degrading enzyme gene suitable for expressing in eukaryotic cell and possessing the nucleotide sequence of SEQ ID No. 1. The gene of the present invention is prepared through molecular reformation and modification on available organophosphate degrading enzyme gene and subsequent artificial synthesis. Compared with original gene, the reformed recombinant gene has 154 altered bases and 135 amino acids, and G+C content changed from original 62.89 % to 50.27 % and is suitable for expressing in eukaryotic cell in high efficiency. The present invention also provides recombinant expression vector containing the above gene, host cell containing the recombinant expression vector and the method of preparing recombinant organophosphate degrading enzyme.

The invention discloses a nitrile hydratase mutant and application thereof, and belongs to the field of genetic engineering and enzyme engineering. The half-life period of the nitrile hydratase mutantStr.t NHase-betaL48D at the temperature of 65 DEG C is about 43 minutes, and the thermal stability is remarkably improved compared with the thermal stability of other NHase enzymes. By adopting the technical scheme, the substrate tolerance of reaction taking nitrile compounds such as nicotinonitrile, acrylonitrile, benzonitrile, 2-cyanopyrazine nitrile, isobutyronitrile, n-valeronitrile and cinnamonitrile as substrates is obviously improved.

The invention relates to a lipase and an application thereof, and concretely provides a polypeptide. The polypeptide is selected from a polypeptide (1) having a substitution mutation in at least one of the 150th position, the 222th position, the 285th position and the 291th position of an amino acid sequence represented by SEQ ID NO:2, and a polypeptide (2) composed of the polypeptide (1) and a polypeptide for promoting the expression and purification of the polypeptide (1). The invention also provides a polynucleotide sequence for encoding the polypeptide, a nucleic acid construct containingthe polynucleotide sequence, a host cell, and relevant uses. The polypeptide is the lipase, and can be used in oil refining, oil processing, oil chemical engineering, feed improvers, the food industry, biodiesel preparation and the medicine industry.

The invention relates to a construction method of an in-vitro artificial scaffold protein mediated trehalose multienzyme complex. The construction method mainly comprises the following steps: constructing a self-assembled scaffold protein module recombinant bacterium WB800n-ScafCCR; constructing a recombinant bacterium WB800n-P43-phoD-treYC-cdoc of a self-assembled catalytic module; constructing arecombinant bacterium WB800n-P43-phoD-treZ-Ctdoc of a self-assembled catalytic module; constructing a recombinant bacterium WB800n-P43-phoD-cgt-Rfdoc of a self-assembled catalytic module; performingsecretory expression on the recombinant bacteria, performing self-assembly in vitro, and obtaining a recombinant trehalose multi-enzyme complex; the efficiency of preparing trehalose by catalyzing thetrehalose multi-enzyme complex is higher than the catalytic efficiency of mixed free enzymes; the cellulose microspheres can be used for producing high-quality trehalose after being immobilized.

The invention discloses a pretreatment method capable of improving enzymolysis efficiency of raw starch and belongs to the technical field of preparation of sugar from starch. According to the pretreatment method capable of improving enzymolysis reaction efficiency of raw starch, starch milk is prepared by mixing water and raw starch and pretreated at the constant temperature, and the reaction efficiency of hydrolysis of the raw starch with amylase is improved. The invention further provides a method for enzymatic hydrolysis of the raw starch. According to the method, the raw starch obtained after pretreatment at the constant temperature is adopted and contacted with enzyme. The enzymolysis efficiency of the raw starch can be increased by 1-3 times, the liquidation effect of high-concentration starch milk with the enzymatic method is improved, the dosage of the enzyme during porous starch production can be obviously reduced, the reaction time can be halved, the dosage of amylase is reduced, and the production cost of porous starch can be reduced significantly.