294 results about "Specific enzyme" patented technology

The invention discloses an arginine decarboxylase diaA enzyme mutant and application thereof in preparation of butanediamine, and belongs to the field of bioengineering. According to the invention, rational charge overturning transformation is simultaneously carried out on a pentamer meridian oligomeric interface and a dimer latitudinal oligomeric interface, so that stable assembly and efficient catalysis of the decamer under the condition of neutral to alkaline pH (7.0-9.0) are realized. Wherein positive charges are introduced into a meridian interface to weaken electrostatic repulsion, and negative charges are introduced into a latitudinal interface to enhance dimer compactness and substrate transfer efficiency. The specific enzyme activity of the representative double mutant AdiAD110K / H736E at pH 8.0 is about 35 times that of a wild type, and the representative double mutant AdiAD110K / H736E keeps a complete decamer state in a pH range of 7.0-9.0. The yield of butanediamine is up to 145.9 g / L under the whole-cell catalysis of the mutant.

The invention discloses a rational copolymerization strategy for improving the stability of an oligomeric structure of acid-induced high-order oligomeric decarboxylase AdiA in a neutral to alkaline environment and application of the rational copolymerization strategy, and belongs to the field of bioengineering. According to the strategy, rational charge overturning transformation is carried out on a pentamer radial oligomeric interface and a dimer weft-wise oligomeric interface at the same time, and stable assembly and efficient catalysis of a decamer under the condition that the pH value is from 7.0 to 9.0 from neutral to alkaline are achieved. Wherein positive charges are introduced into a meridian interface to weaken electrostatic repulsion, and negative charges are introduced into a latitudinal interface to enhance dimer compactness and substrate transfer efficiency. The specific enzyme activity of the representative double mutant AdiAD471K / E467K / H736E is 45.5 times that of a wild type when the pH value is 8.0, and the representative double mutant AdiAD471K / E467K / H736E keeps a complete decamer state when the pH value is 7.0-9.0. The yield of butanediamine is up to 156.5 g / L by using the mutant to catalyze whole cells.

Provided are S-adenosylmethionine synthetase mutants and the use thereof, belonging to the technical field of biological enzyme engineering. Site-directed combinatorial mutations are performed on positions 189, 200, 234, 266 and 371 of S-adenosylmethionine synthetase MAT to obtain a series of mutants with improved enzyme activity, wherein the specific enzyme activity of I189V / V266H and V266H / N371R is increased by more than 10 times relative to the parent. The yields of S-adenosylmethionine synthesized by whole cells of recombinant bacteria expressing mutants I189V / V266H / N371R, V266H / Q234N / N371R and I189V / V266H / Q234N / N371R all reach 1550 mg / L or more. The provided enzyme mutants have broad application prospects in the production of S-adenosylmethionine and the construction of genetically engineered microorganisms for S-adenosylmethionine.

The present invention provides a manganese metal-organic framework material with oxidase properties, a preparation method, and an application thereof. The material is prepared by the following method: using 1,3,5-trimethic acid as an organic ligand, manganese acetate tetrahydrate as a manganese source, and a water-ethanol solution as a solvent, to obtain a manganese metal organic framework (MOF) precursor via a solvothermal synthesis technique; the manganese MOF precursor is post-synthetically modified using an oxidant, wherein the oxidant is a mixed solution of an aqueous alkali metal hydroxide solution and H2O2, and the ratio of the manganese MOF precursor to the alkali metal hydroxide is (400-600) mg:(0.5-2) mmol. By using specific types and amounts of manganese MOF precursors and oxidants, the resulting metal-organic framework nanomaterial has specific enzyme-like properties, can exert the function of uricase, and has optimal and high uricase activity under neutral pH conditions.

The invention discloses a mutant with improved catalytic activity of S-adenosylmethionine synthetase and application of the mutant, and belongs to the technical field of biological enzyme engineering. According to the present invention, the site 189, the site 234, the site 266 and the site 371 of the S-adenosylmethionine synthetase MAT are subjected to site-specific combinatorial mutation to obtain a series of mutants with improved enzyme activity, the I189V / V266H improvement is the most obvious, and the specific enzyme activity is 3.5833 + / -0.1373 U.mg <-1 >, which is improved by 1353.08% compared to the parent WT; and for a whole-cell catalysis system, the yield of S-ademetionine synthesized by the mutant I189V / V266H / Q234N / N371R reaches 1751.01 mg / L, and the increase is most obvious. The enzyme mutant provided by the invention has a wide application prospect in the production of S-adenosylmethionine and the construction of genetic engineering microorganisms of S-adenosylmethionine.

The application discloses a galactosyltransferase mutant and application thereof in preparation of flavonoid compounds, and belongs to the technical field of enzymology. The application provides a UDP-galactosyltransferase mutant, which is obtained by site-directed mutation of one amino acid in the amino acid sequence of wild-type galactosyltransferase VcUFGT into alanine (A). The effects of a series of mutants H82A, V139A, G141A, P186A, N245A, V282A and S307A are verified in the examples, the mutants can perform enzyme catalysis reaction with quercetin and UDP-galactoside as substrates, and generate flavonoid compound hyperoside, and the specific enzyme activity of the mutants is more than 1.5 times that of the wild type, thereby providing an effective application basis for biosynthesis of flavonoid compounds.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

The invention discloses a glutathione bifunctional synthetase mutant with improved catalytic activity and application, and belongs to the field of gene engineering and fermentation engineering. A series of mutants with improved enzyme activity are obtained by carrying out site-specific combinatorial mutation on a site 61, a site 136, a site 485, a site 498 and a site 722 of the glutathione bifunctional synthetase GshFst, the specific enzyme activity reaches 13.58 U.mg <-1 >-27.34 U.mg <-1 >, the half-life period at 37 DEG C reaches 195-320 min, and the enzyme activity and the thermal stability are remarkably improved compared with those of a wild type. The enzyme mutant provided by the invention has a wide application prospect in the production of glutathione and the construction of genetically engineered microorganisms for producing glutathione.

The invention discloses a method for preparing butanediamine and spermidine by an enzyme method, and belongs to the field of bioengineering. The novel arginine decarboxylase mutant is successfully provided, the mutant shows excellent catalytic performance under the alkaline condition, and the specific enzyme activity of the mutant reaches 24.9 U / g and is improved by 8.3 times compared with that of wild type enzyme. The invention develops a two-step synthesis process based on the constructed arginine decarboxylase mutant, and the two-step synthesis process is used for efficiently preparing spermidine. Experimental results show that the process successfully realizes the remarkable increase of the yield of spermidine, and the final yield reaches 200.4 mg / L.

The invention relates to the technical field of plant-based beverages, in particular to a chickpea beverage based on enzymolysis regulation and a preparation method thereof. The preparation method of the chickpea beverage comprises the step of performing enzymolysis on chickpeas by adopting alpha-amylase, acidic lipase and flavourzyme. According to the method, the sugar spectrum of the chickpea beverage is accurately regulated and controlled through a specific enzyme combination and enzymolysis process, the sugar composition of the chickpea beverage is improved, the beverage contains low sugar, medium sugar and oligosaccharide at the same time, the proportion of the low sugar, the medium sugar and the oligosaccharide is moderate, the flavor and taste are improved while the sweetness is well controlled, and the chickpea beverage is made to better conform to the taste preference of consumers and has good market prospects. Meanwhile, the nutritional value and the stability of the product are improved, and the market requirements can be better met.

The invention discloses a fluorescent probe device with high-sensitivity detection in the field of biological fluorescence detection.The fluorescent probe device comprises a micro-fluidic chip, a closed micro-channel allowing a sample to flow is arranged in the micro-fluidic chip, and the micro-channel comprises a reaction bin, a sample bin and an enzyme liquid bin; the sample bin and the enzyme liquid bin are symmetrically distributed on the two sides of the reaction bin, delay channels of zigzag structures are communicated between the sample bin and the reaction bin and between the enzyme liquid bin and the reaction bin, the sample bin and the enzyme liquid bin are provided with a first filling port and a second filling port respectively, the reaction bin is provided with vent holes communicated with the outside, and a plurality of aptamer fluorescent probes are fixed to the bottom wall of the reaction bin. A nucleic acid aptamer fluorescent probe is fixed in the micro-fluidic chip, a CRISPR-Cas12a enzyme digestion system is added, a plurality of signal fragments are released in the process that a single aptamer is combined with antibiotics, signal cascade amplification of CRISPR-Cas12a non-specific enzyme digestion activation is achieved, and the micro-fluidic chip has the function of detecting existence of drug-resistant genes. And the possibility of occurrence of false negative detection results is reduced as much as possible.

The invention provides a manganese metal organic framework material with oxidase characteristics as well as a preparation method and application thereof, and the material is prepared by the following method: taking 1, 3, 5-trimesic acid as an organic ligand, manganese acetate tetrahydrate as a manganese source and a water-ethanol solution as a solvent, and obtaining a manganese MOFs precursor through a solvothermal method synthesis technology; the manganese MOFs precursor is subjected to post-synthesis modification with an oxidizing agent, the oxidizing agent is a mixed solution of an alkali metal hydroxide aqueous solution and H2O2, and the dosage ratio of the manganese MOFs precursor to the alkali metal hydroxide is (400-600) mg: (0.5-2) mmol. The manganese MOFs precursor and the oxidizing agent with specific types and using amounts are adopted, so that the obtained metal organic framework nano material has specific enzyme-like characteristics, can play a role of urate oxidase, has optimal urate oxidase activity under a neutral pH condition, and is relatively high in activity.

The invention provides a glutaminase mutant and application of the glutaminase mutant. Wherein the glutaminase mutant comprises (a) a protein which is subjected to amino acid mutation and has a glutaminase activity function on at least one of the following sites of S467, Y334, K400, N521, R337, E402 or D447 of an amino acid sequence as shown in SEQ ID NO: 2, and (b) a protein which is subjected to amino acid mutation on at least one of the following sites of S334, Y334, K400, N521, R337, E402 or D447 of the amino acid sequence as shown in SEQ ID NO: 2; and (b) a protein which has 80% or more homology with the amino acid sequence limited by (a) and has a glutaminase function. The glutaminase mutant with the amino acid sequence disclosed by the invention has relatively high specific enzyme activity during enzyme catalytic reaction, and is low in cost and strong in market competitiveness when being used for commercial food processing.

The present invention relates to enzymatic detergents and cleaning compositions comprising specific enzyme stabilizers and having improved enzyme stability. More specifically, the present invention relates to detergent and cleaning compositions comprising an enzyme and an effective amount of a specific type of enzyme stabilizer, such as an alkanediol having a carbon chain of 5 to 14 carbon atoms, caprylin, 4-hydroxyacetophenone, ethylhexylglycerol, tropolone or a mixture of said enzyme stabilizers, and a surfactant, and to the use of said detergent and cleaning compositions for the preparation of detergent and cleaning products for household, public institution and industrial applications. In addition, the present invention relates to the use of specific enzyme stabilizing substances as defined herein for stabilizing enzymes in detergents and cleaning compositions. Furthermore, the present invention relates to a process for the preparation of a detergent and cleaning composition by adding to the detergent and cleaning composition an effective amount of a specific type of enzyme stabilizer such as an alkanediol having a carbon chain of 5 to 14 carbon atoms, caprylin, 4-hydroxyacetophenone, ethylhexylglycerin, or a mixture thereof. A method of stabilizing an enzyme in the detergent and cleaning composition by a mixture of a cycloheptenolone or the enzyme stabilizer. Finally, the invention relates to an enzyme composition comprising a particular type of enzyme stabilizer.

The invention discloses a microsphere entrapped with probiotics and ginsenoside as well as a preparation method and application of the microsphere, and aims to realize efficient treatment of flora imbalance inflammatory bowel disease (IBD). The microspheres adopt a bi-component synergistic entrapment technology, can stably bear probiotics and ginsenoside, effectively avoid inactivation or degradation of the probiotics and ginsenoside in gastric acid and cholate environments, and ensure directional release and efficient utilization of the probiotics and ginsenoside in intestinal tracts. The microspheres not only provide a protection effect as a delivery carrier, but also construct a'microreactor 'system, and provide a suitable microenvironment for survival of probiotics and conversion of ginsenoside. Probiotics secrete a specific enzyme to promote prototype ginsenoside to be converted into rare ginsenoside with higher activity and stronger solubility while regulating intestinal flora balance and relieving intestinal inflammation, so that the bioavailability and efficacy of the rare ginsenoside are remarkably improved. According to the microsphere system, the technical bottlenecks of the existing treatment means in the aspects of activity maintenance, directional release and conversion utilization are broken through by realizing the synergistic effect of the probiotics and the ginsenoside, and a brand-new strategy and a technical platform are provided for treatment of flora imbalance related diseases such as IBD.

The invention discloses a type III pullulan hydrolase mutant with improved enzyme activity and a construction method thereof, and relates to the technical field of gene engineering and enzyme engineering. The amino acid sequence of the III type pullulan hydrolase mutant is as shown in SEQ ID NO. 3. According to the present invention, on the basis of the type III pullulan hydrolase TK-PUL from Thermococcus kodakarensis, the type III pullulan hydrolase mutant with the improved enzyme activity is developed through the site-specific mutagenesis, such that the type III pullulan hydrolase mutant with the improved enzyme activity is provided; the specific enzyme activity of the III type pullulan hydrolase mutant to cassava starch is improved by 1.83 times. The cassava starch degradation capacity of the III type pullulan hydrolase mutant is obviously improved, the enzymatic property of the III type pullulan hydrolase mutant meets the requirements of the production and preparation process of the straight-chain maltooligosacharide, and the application potential of the III type pullulan hydrolase mutant in the production and preparation of the straight-chain maltooligosacharide is improved.

The invention discloses a serine hydroxymethyltransferase mutant and an application thereof in production of L-serin.The serine hydroxymethyltransferase mutant with the specific enzyme activity remarkably improved is obtained by conducting saturated mutation on serine hydroxymethyltransferase from escherichia coli and conducting screening, and the serine hydroxymethyltransferase mutant is applied to synthesis of L-serine. The serine hydroxymethyltransferase disclosed by the invention can be used for effectively improving the efficiency of catalyzing glycine and formaldehyde to synthesize L-serine by using the serine hydroxymethyltransferase, reducing the enzyme dosage, remarkably reducing the production cost and reducing the separation difficulty of downstream products.

The present invention discloses a genetically engineered bacterium expressing oligosaccharide debranching enzyme and its application, belonging to the technical field of microbial engineering. The present invention provides an oligosaccharide debranching enzyme with a specific base sequence, and successfully heterologously expresses the oligosaccharide debranching enzyme in Escherichia coli. By using the method of the present invention, the catalytic activity of the crude enzyme solution of the expressed oligosaccharide debranching enzyme can reach 258.83 U / mL. The oligosaccharide debranching enzyme provided by the present invention is a novel oligosaccharide debranching enzyme from a new source, which has not been reported before, has a high specific enzyme activity, and strong substrate specificity. The oligosaccharide debranching enzyme of the present invention can debranch short chain segments with DP<6 in maltodextrin, filling the blank in the substrate specificity of commonly used debranching enzymes at present, and has high potential application prospects in industrial fields such as starch sugar, resistant starch, beer production and ethanol fuel.

The invention relates to the field of nutritional health food, and discloses a casein modified active peptide for promoting bone repair and a preparation method thereof.The structure of the active peptide sequentially comprises a bone targeting sequence with high affinity to hydroxyapatite, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions, a casein phosphopeptide core sequence capable of chelating calcium ions and a casein phosphopeptide core sequence capable of chelating calcium ions from the C-terminal to the N-terminal. The digestion sequence can be specifically hydrolyzed by matrix metalloproteinase, and the alkynyl functional group is used for covalent cross-linking. The active peptide can be mixed with an azido-containing multi-arm cross-linking agent, and hydrogel can be rapidly formed under physiological conditions through cycloaddition reaction. The system can be gelated in situ and anchored to a bone defect part in a targeted manner, and is triggered by a specific enzyme in a pathological microenvironment to controllably release active peptide fragments capable of chelating calcium ions, so that the concentration of local mineral ions is adjusted, the cell living environment is improved, and an effective new way is provided for promoting bone repair.

The present invention discloses phi29 DNA polymerase mutants with improved performance, their preparation methods and applications, which relate to the field of biotechnology. Specifically, the present invention discloses phi29 DNA polymerase mutants that contain mutation sites Y163K, F181K, Y224K, W277K / W277P, Y298K, Y369K, Y439K, F526R relative to wild-type phi29 DNA polymerase; the amino acid sequence of the wild-type phi29 DNA polymerase is as shown in SEQ ID NO:1. Compared with the wild-type phi29 DNA polymerase, it has higher specific enzyme activity, stability and higher amplification efficiency, and has good application prospects in the fields of nucleic acid amplification and sequencing, etc.

The invention discloses a recombinant D-amino acid oxidase mutant, a coding gene and application thereof, and belongs to the technical field of enzyme engineering. A D-amino acid oxidase mutant is constructed, the catalytic activity of enzyme is greatly improved, and the specific enzyme activity can be improved to 54.32 U / g. The constructed mutant is used for catalyzing D / L-glufosinate-ammonium, under the participation of glutamate dehydrogenase and ammonium formate dehydrogenase, a catalytic reaction can be carried out under the 1M substrate concentration, the catalytic conversion rate is 99%, the yield is 97%, and the e.e. Value is gt; and the effect is 99.9%.

This invention discloses a mutant of raw starch hydrolase with improved stability and its applications. Using α-amylase AmyZ2 as the starting enzyme, and through computer-aided design to clarify the mutation site, a mutant was heterologously expressed in Bacillus subtilis, resulting in an α-amylase with improved stability. When using raw corn starch as a substrate, the mutant exhibits 1.57 times the specific enzyme activity and 2 times the stability of the starting enzyme. While maintaining the specific enzyme activity, the mutant enzyme shows a significant improvement in thermostability. This mutant has potential application value in the production of starch sugars using raw corn starch as a substrate.

The application discloses a D-allulose 3-epimerase mutant, a preparation method and application thereof, and relates to the fields of genetic engineering and enzyme engineering. The amino acid sequence of the D-allulose 3-epimerase mutant is shown as SEQ ID NO. 1. The specific enzyme activity of the D-allulose 3-epimerase mutant provided by the application is increased from 10.21 U / mg of a control (before mutation) to 15.29 U / mg at 80 DEG C and pH 6.0; and the equilibrium conversion rate is increased from 29.98% of the control (before mutation) to 36.26% when the substrate is 700 g / L of fructose. The D-allulose 3-epimerase mutant provided by the application has higher catalytic activity, and has a very broad application prospect in the efficient production and preparation of D-allulose.

The invention discloses an enzyme-containing anti-inflammatory composition for pet skin and hair follicle health. The enzyme-containing anti-inflammatory composition comprises an enzyme preparation, a hair follicle maintenance agent, a functional auxiliary agent and a carrier raw material. The core innovation lies in that a hair follicle targeted enzymolysis technology is adopted, and a specific enzyme modified by polyethylene glycol can efficiently dredge hair follicles; the anti-inflammatory and hair follicle repairing dual effects are achieved through anti-inflammatory-maintenance synergistic matching and adding sequence optimization; the damaged barrier is rapidly repaired by combining a composite barrier repairing agent and a gradient permeation process; enzyme activity and mildness are guaranteed by adopting a microcapsule embedding and low-temperature preparation technology. The composition can specifically solve the problems of hair follicle blockage, inflammation and unhairing, is suitable for pets of all ages and skin types, and is high in practicability.

The invention provides a method and a device for secondary filtration and re-removal of cholesterol ester after continuous blood purification and blood fat filtration, and belongs to the technical field of medical blood purification. According to the method and the device, the technology of combining enzymolysis, adsorption and membrane separation is adopted for residual CE in a CBP filtering product. The method comprises the following steps: firstly, decomposing CE into small molecules by using specific enzyme, and weakening the combination of CE and other components; residual impurities are further adsorbed through a high-performance adsorption material; and finally, efficient removal of CE is realized by virtue of a high-precision membrane separation technology, so that the residual quantity is reduced to be below a safety threshold value. Compared with a traditional technology, the problems that the CE-containing filtrate is high in recycling risk and low in resource utilization rate are effectively solved, the purity of a filtered product is remarkably improved, technical support is provided for industrial scenes such as plasma exchange, filtrate feedback, biopharmacy and extracorporeal circulation in the medical field, and the application prospect is wide. The safety and the resource utilization efficiency of the blood purification technology are promoted.

The application discloses a beta-1,4-xylanase mutant and a construction method thereof, and comprises the following steps: constructing a beta-1,4-xylanase recombinant expression vector; introducing the recombinant vector pNZ8048-P5-XynA into a competent lactococcus lactis by using an electrotransformation technology to construct a wild-type expression vector; on the basis, designing and constructing a multi-site mutant recombinant plasmid by using a site-directed mutation method, wherein the eighth asparagine (Asn) of the beta-1,4-xylanase is mutated into tyrosine (Tyr), the 22nd serine (Ser) is mutated into proline (Pro), and the 59th alanine (Ala) is mutated into lysine (Lys); introducing the multi-site mutant recombinant plasmid into the competent lactococcus lactis to construct a heterologous expression strain of the mutant beta-1,4-xylanase (Mut-1); and stably obtaining the mutant xylanase beta-1,4-xylanase (Mut-1) with high activity by using a metal affinity chromatography (Ni 2+ ) method. The specific enzyme activity of the mutant reaches 1929.30±5.36 U / mg, and the optimal temperature is increased by 15 DEG C compared with that of the wild type.

The invention discloses xylanase XynA00838, a truncated body of the xylanase XynA00838 for removing CBM1 and application of the xylanase XynA00838, and belongs to the technical field of microorganisms. The invention provides the XynA00838, the amino acid sequence of the truncation of the XynA00838 and the nucleotide sequence of the coding gene of the XynA00838, and provides characterization information, the XynA00838 and the truncation of the XynA00838 have obvious xylanase activity, and the specific enzyme activity of the truncation of the XynA00838 is obviously higher than that of wild type xylanase XynA00838 when six different plant polysaccharides are degraded, so that the XynA00838 and the truncation of the XynA00838 can be used for degrading various plant polysaccharides. When the truncated body degrades low-viscosity wheat arabinoxylan as a substrate, the specific enzyme activity reaches 4.46 + / -0.12 U / mg and is remarkably higher than that of wild type xylanase XynA00838. The CBM1 in the xylanase XynA00838 disclosed by the invention has a relatively good maintaining effect on the temperature stability of an enzyme catalysis module of the xylanase XynA00838. The truncated body XynA00838 delta CBM1 of the xylanase XynA00838 without the CBM1 has high enzyme activity and pH stability, so that the truncated body XynA00838 delta CBM1 has relatively great development and application potential, and a basic theoretical basis is provided for preparing a commercial feed enzyme preparation.

The present disclosure discloses a thermophilic recombinant type II pullulanase and the application thereof, and belongs to the technical field of genetic engineering. The present disclosure obtains a thermophilic recombinant type II pullulanase by heterologously expressing type II pullulanase in Escherichia coli. Its optimum pH is 6.6, it has better pH tolerance under the conditions of pH 5.8-8.0, and its optimum temperature is 95° C. After incubating at 95° C. for 10 h, the remaining enzyme activity is greater than 50%. It can exhibit higher specific enzyme activity under strong reducing conditions. For example, adding DTT to the culture environment can increase the specific enzyme activity of Sumo-PulPy by 237.2%. The present disclosure also provides the combined truncation mutant Δ28N+Δ791C of type II pullulanase Sumo-PulPy. The specific enzyme activity of the enzyme mutant is 32.18±0.92 U / mg, which is 5.99 times as high as that of the wild-type enzyme, thereby having important industrial application value and potential.

The invention discloses a glucose-6-phosphate dehydrogenase mutant as well as an expression strain and application thereof. According to the invention, on the basis of glucose-6-phosphate dehydrogenase from marine microorganisms, after a mutation site is selected by combining Evcouplings design with Fold X scoring, a recombinant plasmid is constructed, and a mutant gene is obtained. After the engineering bacteria containing the mutant plasmids are subjected to induced expression, the glucose-6-phosphate dehydrogenase G6PDH202-Des3 with improved specific enzyme activity is obtained. Under the condition of 37 DEG C, the specific enzyme activity of the mutant is improved to 1.6 times, and the stability is improved to 1.2 times. The mutant has potential application value in an in-vitro diagnostic kit for detecting creatine kinase activity by an enzyme method.

The application discloses a marine-derived cholesterol oxidase and a preparation method and application thereof, and relates to the technical field of biology. The amino acid sequence of the marine-derived cholesterol oxidase (SkChOx) is shown in SEQ ID NO. 1 or 3, the specific enzyme activity of the marine-derived cholesterol oxidase can reach 31.9 U / mg, the marine-derived cholesterol oxidase has excellent anti-reverse performance, maintains high stability in the pH range of 5.0-10.0, has good temperature tolerance, organic solvent tolerance and metal ion tolerance, and exhibits unique salt activation effect and salt tolerance. The enzyme is compounded with dithiothreitol (DTT) and nonylphenol polyoxyethylene ether (NP-40) to form a liquid enzyme preparation, and after incubation at 37 DEG C for 7 days under accelerated conditions, the catalytic activity of the liquid enzyme preparation still remains about 87%, the liquid enzyme preparation has excellent long-acting storage stability, and has wide application prospects in the fields of clinical diagnosis, food physical and chemical inspection, biosensor development and industrial preparation of steroid drugs.