417 results about "Pullulanase" patented technology

The invention provides a preparation method of slowly digestive starch by utilizing Pullulanase to modify and process raw starch by an enzyme method, changing the particle structure of the starch, enabling molecular chains of the starch to realign and recombine, controlling action conditions to damage amorphous structure and adjust crystal structure and further changing crystallization conditions so that the crystallinity of the starch is improved and can be controlled. The method comprises the following steps: preparing the raw starch, adding the Pullulanase to be enzymatically hydrolyzed, sterilized, cooled, refrigerated, baked, pulverized, screened, and the like. The invention provides an enzymatic preparation technology for a novel functional food raw material-slowly digestive starch and can be used in raw starch and modified starch enterprises, and the slowly digestive starch and slowly digestive starch products, which are produced by the technology, can be applied to the food industry and the medicine industry.

The invention discloses a bacillus subtilis strain capable of efficiently expressing an exogenous secretory proteinase. A bacillus subtilis host bacteria capable of expressing exogenous protein is disclosed. A gene apr of a bacillus subtilis alkaline protease, and a gene npr and a spore forming gene spoIIAC of a bacillus subtilis neutral metalloproteinase are knocked out, wherein the bacillus subtilis alkaline protease and the bacillus subtilis neutral metalloproteinase are two exocrine proteinases in the bacillus subtilis host bacteria. The obtained bacillus subtilis host bacteria can be used for high-yield exogenous proteinases by genetic engineering manners. Based on this, a genetically engineered bacterium secreting pullulanase is constructed and can produce the pullulanase with a high yield. The genetically engineered bacterium secreting has a wide development prospect in industrial production by fermentation industry research.

A production method and the application of a high temperature stable slowly digestible starch belong to the non-chemical modified starch field. The material starch of the present invention, such as cornstarch, wheat starch, rice starch, potato starch, tapioca starch, sweet potato starch, mung bean starch, chick pea starch, sorghum starch, sago starch, and canna starch, is gelatinized, and the complex debranching treatment is conducted after adding the commodity diastases, pullulanase and alpha amylase, and finally, the slowly digestible starch is gained after preservation, recrystallization, and hydrothermal treatment. The content of the slowly available glucose is greater than or equal to fifty percent, and the residue of the slowly digestible starch is greater than or equal to 80 percent after high temperature processing treatment. The present invention relates to a plurality of fields, such as baking food, short order, candy, flavoring, dairy products, athlete special food, food for diabetes, slimming foods, oral intestinal target controlled release film coating, and animal feed.

The invention relates to novel variants of the enzymatic peptide pullulanase, the gene sequences encoding said novel peptides, expression vectors comprising those gene sequences as well as organisms expressing the novel pullulanase variants. The novel pullulanase variants of the present invention were made empirically by the use of codon-optimization procedures, selective truncation of “wild-type” molecules and through the replacement of selected amino acid residues. Furthermore, the invention relates to the use of these novel pullulanase peptides in the textile, fermentation, food and other industries.

The invention belongs to the technical field of natural polymer modification and discloses a preparation method of a starch and fatty acid compound. The method comprises the following steps: adding de-ionized water into starch and balancing moisture in a closed container; carrying out heat treatment to obtain the starch subjected to wet and hot treatment; then adding a buffering solution to prepare a starch solution; after pre-heating, adding 2 to 5u / g pullulanase liquid to react for 4 to 5 hours; after carrying out enzyme deactivation, centrifuging and separating to obtain enzyme treatment modified starch; then stirring and gelatinizing the starch in a boiled water bath for 25 to 45 minutes and cooling to 60 to 90 DEG C to obtain gelatinized starch; then transferring the gelatinized starch into a homogenizing machine and adding fatty acid to carry out homogenization and mixing; and keeping the heat of the water bath to synthesize for 30 to 40 minutes to obtain the starch and fatty acid compound. According to the preparation method, a greener and more environment-friendly new way is provided for synthesizing the starch and fatty acid compound through wet and hot treatment and controlled enzymolysis treatment by adopting a gelatinizing method.

The inventors have modified the amino acid sequence of a pullulanase to obtain variants with improved properties, based on the three-dimensional structure of the pullulanase Promozyme(R). The variants have altered physicochemical properties, e.g. an altered pH optimum, improved thermostability, altered substrate specificity, increased specific activity or an altered cleavage pattern.

The invention discloses a method for processing and preparing resistant starch by Pullulanase cooperated with acid alcohol, comprising the following steps: heating starch milk of which the weight concentration is 10-45% and the pH value is 3.5-6.5 to 40-70 DEG C, adding Pullulanase of which the dosage is 1-40ASPU per gram of starch, and keeping for 8-48hrs; inactivating enzymes, assisting by acid alcohol processing, cooling, filtering, drying, crushing and sieving to obtain the product. The invention takes starch as a raw material, adopts the biological technology and acid alcohol processing means, thus greatly improving the content of the resistant starch, simplifying the process for preparing the resistant starch, effectively enhancing the yield, and lowering energy consumption. The obtained product not only has the function of dietary fiber, but also has the characteristics of fat substitute, and can be widely used as functional food materials and biochemical drug carriers.

The invention relates to preparation process of amphipathic octenyl succinic short-straight chain starch nano-particles. The preparation process comprises the following steps: (1) performing enzymolysis on gelatinized starch by using pullulanase, and obtaining short straight chain starch; (2) preparing a short straight chain starch solution, performing gelatinization, adding an octenyl succinic anhydride solution which accounts for 25-100% of the weight of dry powder of short straight chain starch, and continuously stirring for 6-10 hours, so as to obtain a modified octenyl succinic short straight chain starch solution; preparing a 1-10mg / mL solution of the octenyl succinic short-straight chain starch, stirring for 6-10 hours at 37-40 DEG C, and cooling to the room temperature, thereby obtaining a amphipathic octenyl succinic short-straight chain starch nano-particle solution. The particle size of the nano-particles is within 5-100nm, the nano-particles are good in tissue adhesion property, are capable of wrapping hydrophobic active substances, are high in loading rate and low in cost, the conveying efficiency of lyophobic active components by gastrointestinal tracts can be improved, and the bioavailability of the nano-particles can be improved. The lyophobic active components can be protected, the stability of the lyophobic active components can be improved, and the release of adverse flavor can be shielded; in addition, the addition amount and the toxic and side effects of bioactive components can be effectively reduced.

The invention alters the physical characteristics of storage polyglucans including starch. Methods are provided to modify the polyglucan biosynthesis pathway by simultaneously altering the activity of a pullulanase debranching enzyme and the activity of another polypeptide in the polyglucan biosynthesis pathway. Compositions of the invention include transgenic plants and seeds having a modified polyglucan structure and / or content and elevated phytoglycogen levels. Additional compositions include a grain with increased energy availability for improved feed quality and industrial uses. Further compositions include a polyglucan with improved functional properties useful in a wide range of food and industrial applications.

The invention discloses a pullulanase mutant with improved secretion efficiency and heat stability and a preparation method of the pullulanase mutant, and belongs to the field of gene engineering and enzyme engineering. The secretion efficiency and the heat stability of pullulanase are improved through structural domain deletion mutation; deleted structural domains are CBM41, X45 and X25 structural domains or combinations thereof; and the mutation technical scheme capable of improving the secretion efficiency and the heat stability of pullulanase is provided. The obtained pullulanase structural domain deletion mutant has at least one of changed properties as follows: 1), the extracellular secretion efficiency is improved after recombinant bacteria are fermented; and 2), and the heat stability is improved under the conditions of pH 4.0-5.0 and 60 DEG C. Compared with natural pullulanase, structural domain deletion mutants are more suitable for production, preparation and applications of the pullulanase.

The invention discloses a preparation method of ultra-high maltose syrup, which liquefies and saccharifies rice starch taken as raw material sequentially. The liquefying comprises the steps of adding 0.3-0.5g / kg of calcium chloride and 0.2-0.3g / kg of alpha-amylase into starch slurry with the specific gravity of 14-17OBe and the pH value of 5.0-6.0, and then utilizing a spraying liquefier to carry out liquefying twice so as to obtain rice starch liquefying feed, wherein the first-time liquefying temperature is 110-115 DEG C, the temperature of 90-100 DEG C of the feed liquid is kept for 40-60min and the second-time liquefying temperature is 130-140 DEG C. The saccharification comprises the steps of adding 0.4-0.6g / kg of beta-amylase and 1.0-1.2g / kg Pullulanase into rice starch liquefying feed, and then saccharifying the Pullulanase at the temperature of 55-65 DEG C for 45-60h to obtain the saccharifying solution, separating and purifying the saccharifying solution by ion exchange resin and column chromatography, and then concentrating to obtain the ultra-high maltose syrup of which the maltose content is larger than 80%.

The invention relates to a preparation method of isomaltooligosacharide. The preparation method comprises the following steps of 1, adding water into a starch raw material, adjusting a concentration and a pH value of the slurry, and adding high temperature-resistant alpha-amylase into the slurry to obtain starch slurry, 2, carrying out liquidation on the starch slurry to obtain a liquefied liquid, 3, adding maltotriose generation enzyme (AMANO AMT1.2L) and pullulanase (GENENCOR OPTIMAX L-1000) into the liquefied liquid, carrying out thermal-insulation saccharification, and carrying out enzyme denaturalixation to obtain a saccharified liquid, 4, adding alpha-glucosidetransferase into the saccharified liquid for a reaction to produce a primary trans-glucoside saccharified liquid, adding alpha-glucosidetransferase into the primary trans-glucoside saccharified liquid for a reaction, and carrying out enzyme denaturalixation to obtain a trans-glucoside saccharified liquid, and 5, carrying out decoloration on the trans-glucoside saccharified liquid, carrying out filtration, carrying out ion exchange, carrying out chromatographic separation, carrying out concentration and carrying out drying to obtain isomaltooligosacharide. The preparation method greatly improves contents of sugars such as panose, isomaltotriose and tetrasaccharide having polymerization degrees greater than or equal to 3 in the reaction product liquid.

The invention provides a preparation method of resistant starch RS3. The preparation method of the resistant starch RS3 comprises the following steps: (1) pulping: preparing starch milk from starch (corn, wheat, rice, sweet potato and potato) as raw materials and pulping uniformly; (2) pressing and heating: adjusting the pH value of the starch milk by using hydrochloric acid, adding a certain amount of acidic alpha-amylase, placing in a temperature-controllable heating high-pressure container, and pressing and heating; (3) debranching: adding pullulanase into starch paste which is cooled after pressing and heating and debranching; (4) centrifuging: centrifuging the debranching liquid at once to obtain a heavy phase part and a light phase part; (5) crystallizing: crystallizing the heavy phase part and the light phase part respectively at low temperature; (6) washing sugar: performing sugar washing on the crystallized heavy phase and the crystallized light phase respectively by using distilled water with a certain temperature for many times, centrifuging and removing supernatant to obtain heavy phase precipitate and light phase precipitate respectively; (7) performing fluidized drying by using a fluidized bed: drying and crushing the heavy phase precipitate and the light phase precipitate respectively to obtain two different specifications of resistant starch RS3.

The invention discloses a pullulanase mutant having a high specific activity and a high thermal stability, and a preparation method thereof. The mutant comprises one, two or three substituents relative to debranching Bacillus spp Pullulanase active amino acid residues and related with the thermal stability of pullulanase; and the active amino acid residues comprise 503th aspartic acid, and can be mutated to form arginine, phenylalanine and tryptophan or tyrosine, the above mutation improves the high specific activity and the high thermal stability of the pullulanase, and at least one of the cases comprising the rise of a best reaction temperature, the improvement of the thermal stability at a pH value of 4.0-5.0 and the improvement of the specific activity at a pH value of 4.0-5.0 is realized. The mutant is more suitable for the starch saccharification production process than wild pullulanase.

The invention relates to a coding gene of heat-resistant type pullulanase from geobacillus stearothermophilus as well as recombinant expression thereof and application thereof. The coding gene successfully constructs a recombinant plasmid containing PluGS, so that various highly-expressed bacterial stains which comprise escherichia coli, bacillus, saccharomycete and mycete are obtained. A recombinant host cell is suitable for expressing a gene of the heat-resistant type pullulanase. The optimal pH value of pullulanase is 6.0, and relatively high enzyme activity is kept when the pH value is 5.0-7.0. The optimal temperature is 65 DEG C, thermal stability is good at a temperature of 60-70 DEG C, the heat is preserved for 3 hours at the temperature of 60 DEG C, and the pullulanase still keep 90% or more of enzyme activity, and is suitable for industrial application needs. The pullulanase which is in recombinant expression is identified as I type pullulanase through thin-layer chromatography and efficient liquid-phase chromatographic analysis, can be used for hydrolyzing pulullan, amylopectin and alphla-1,6-glucosidic bond of soluble starch, and can not hydrolyze amylopectin and cyclodextrin.

The invention relates to a method for producing isomalto-oligosaccharide by using high-concentration starch and belongs to the field of production of isomalto-oligosaccharide. By improving the initial concentration of starch milk, through performing insulation liquification and spraying liquification to prepare a high-concentration maltodextrin solution, by virtue of a synergistic effect of beta-amylase and pullulanase (incisal enzyme) in a process of converting glucoside by saccharifying, the content and the concentration of maltose in liquid glucose are increased; meanwhile, due to the fact that the pullulanase is added, the viscosity of the high-concentration liquid glucose can be reduced, and an optimal substrate environment is provided for alpha-glucosyltransferase, thus the yield of the isomalto-oligosaccharide is improved, and the time of converting glucoside by saccharifying is shortened. The starch resource is wasted due to the fact that a separation and purification technology of the isomalto-oligosaccharide is not enough advanced. According to the method disclosed by the invention, by adopting a nanofiltration membrane technology, the purity of the isomalto-oligosaccharide is increased, crystalline dextrose can be co-produced when high-purity liquid glucose can be produced as a byproduct, and thus the problem of low resource utilization rate is effectively solved.

The invention discloses a new raw material of novel resistant starch, and simultaneously discloses a preparation method and foods of high-content resistant starch. The preparation method comprises the following steps: preparing coarse starch of canna edulis ker; adding diluted hydrochloric acid to regulate the PH value to be between 3.5 and 5.5; adding Pullulanase at the temperature of between 40 and 70 DEG C; and preserving the temperature, heating, cooling and the like to remove residual acid or impurities to prepare the resistant starch which can be deposited in cold water for a long time for storage. In the method of content measurement, an alpha-starch amylase enzymolysis method is mainly adopted, and the content of the resistant starch is calculated by measuring the content of glucose in a standard curve method. The result of resistant starch content measurement indicates that: the content of resistant starch in the untreated canna edulis ker is 26.5 percent, and is far higher than the content of resistant starch in conventional staple foods, such as rice, wheat and the like, while the content of resistant starch in the process-treated canna edulis ker is 70.5 percent; therefore, the resistant starch is an ideal raw material of functional foods. The invention also discloses a preparation method of a series of foods which take the resistant starch as a raw material.

The invention relates to a technology for preparing thermally stable slowly digestible starch, belonging to the technical field of preparation of slowly digestible starch in food processing. Ordinary starch is quickly digestible starch, and a thermally stable slowly digestible starch product can be obtained through the following processes (enzymolysis, tea polyphenol adding and spray drying): adding 20-50g of water to 1kg of starch raw material to blend into a 2-5 percent starch suspension, adding pullulanase (the addition is 1-5U / g) at 50 DEG C with a pH value of 5.0, and carrying out enzymolysis for 4-10h; adding 20-80g of tea polyphenol, mixing, heating to 100 DEG C for enzyme inactivating for 125n and cooling to room temperature; homogenizing by a high-voltage homogenizer under 20MPa, and drying into powder by using a spray dryer (the air inlet temperature is 160-200 DEG C, and the air outlet temperature is 80-100 DEG C); and packaging to obtain the slowly digestible starch product.

The invention discloses a pullulanase mutant and application thereof and belongs to the technical field of genetic engineering and enzyme engineering. According to the constructed pullulanase mutant,the enzyme activity of an obtained single mutant K419R (the 419th lysine is mutated to arginine) is 1.3 times higher than that of a wild mutant, and the remaining enzyme activity reaches 67.3 U / mg after the single mutant K419R is maintained at 67 DEG C for 30 minutes; the enzyme activity of an iterative mutant K419R+Y102C+K383C (the 419th lysine is mutated to arginine, and the 102th tyrosine and the 383th lysine are mutated to cysteine separately) is two times higher than that of the wild , the remaining enzyme activity of the iterative mutant reaches 88.3 U / mg after the iterative mutant is maintained at 67 DEG C for 30 minutes and is 43.1% of previous enzyme activity before heat preservation, and the thermal stability is high. The pullulanase mutant can be applied to a starch saccharification process.

The invention relates to a method for preparing corn starch whole powered sugar, belonging to the technical field of bioengineering. The method adopts Alpha-amylase and diastatic enzyme (glucoamylase) to hydrolyze corn starch at different times; during the saccharification process, pullulanase is adopted for synergistic reaction so that the DE value of the final saccharification of the product can be enhanced to 98.80 percent. The method provides a new technical basis for using the corn starch to prepare high-quality whole powdered sugar. By the simultaneous use of a plurality of diastatic enzymes, the conversion rate of the product is enhanced and the production cycle is shortened, thus opening up wide application prospect for realizing the production of the high-quality whole powdered sugar in China.

The invention relates to pullulanase producing strain and manufacturing method. It is named alieycloeacillus. And its preservation register number is CGMCC No.1504. The formed strain D-1 is one new species of alieycloeacillus. Its pullulanase pH is 3.5-4.5. If pH is higher then 5.0 or lower than 3.0, enzyme is inactive; if it is 4.0, residue enzyme energy is 88%; the optimum temperature is 60 centigrade degree; enzyme reaction is not need metal ion while temperature is 55-65 centigrade degree. Thus the enzyme can be used in starch processing saccharification stage. Compared with acidity pullulanase, the enzyme has better heat resistance and pH stability.

The invention discloses a preparation method and a modification method of taro starch nanoparticles as well as application of the taro starch nanoparticles in a starch composite film. The preparation method of the taro starch nanoparticles comprises the following steps: preparing the taro starch into a starch solution having the mass concentration of 10-25% by use of a phosphate buffer solution of which the pH is 5.0, stirring evenly and then putting the starch solution into boiling water for gelatinizing for 20-40 minutes; next, cooling the gelatinized starch solution, adding a pullulanase in the dosage of 25-35ASPU / g of starch to perform enzymolysis for 6-10 hours, centrifuging, heating the liquid supernatant obtained by centrifuging in boiling water bath for 10-15 minutes and then terminating the reaction, storing a sample at 4 DEG C for 6-10 hours and then washing until the sample to be neutral, freezing at -18 DEG C for 20-28 hours, and finally, dying by use of a freeze dryer to obtain the taro starch nanoparticles. The prepared starch nanoparticles are modified by use of octenyl succinic anhydride. The preparation method is short in preparation time, high in nanoparticle yield, free of pollution on the environment, and green and efficient; after the prepared starch nanoparticles are added to the starch composite film, the properties of the composite film can be improved remarkably.

The invention provides a preparation method for a resistant starch. The means of complexing of auxiliaries and temperature-varying crystallization are utilized to prepare a heat-resisting type resistant starch with excellent palatability. The principle of the method is to form an amylase-lipid compound with high heat stability and difficulty in reacting with amylase through complexing of auxiliaries and to generate more crystals through temperature-varying crystallization, so as to guarantee better growth of crystals and increase the content of resistant starch. The preparation steps are as follows: preparing rice starch into starch milk in certain concentration; gelatinizing and then cooling to a certain temperature; adding pullulanase for degreasing and then concentrating, wherein the solid content reaches up to 35%-40%; adding crystallization auxiliaries, such as stearic acid and oleic acid, and performing low-temperature complex reaction at 90-100 DEG C; adopting a temperature-varying crystallization method for quickly cooling for 1-5min under low temperature environment at -80 to 4 DEG C at a cooling rate of 9-18 DEG C / min; preserving heat in 40-50 DEG C water bath, drying andsmashing, thereby acquiring an end product.

The invention discloses pullulanase XWPu2 and the gene thereof. The amino acid sequence of the pullulanase XWPu2 is shown in SEQID No. 1, and the nucleotide sequence of the pullulanase XWPu2 is shown in SEQID No. 2; and the recombinant vector and the recombinant strain of the pullulanase XWPu2 are provided. The pullulanase XWPu2 taking pullulanase as a substrate has the following properties that the optimum temperature is 50 DEG C; the optimum pH is 5.8-6.8; beta-mercaptoethanol having a final concentration of 1 mM, and metal ions Ca<2+>, Mn<2+>, Fe<2+> and K<+1> having a final concentration of 1 mM have a strong activation effect on enzyme; the residual enzyme activities after heat preservation for 140 minutes at 37 DEG C and at 50 DEG C are greater than 84% and greater than 52% in sequence; the residual enzyme activities after treatment for 90 minutes in a buffering solution having a pH of 5.0 and in a buffering solution having a pH of 8.6 are greater than 74% and greater than 57% in sequence; and the specific activity is 383.5 U / mg. Additionally, a qualitative analysis adopting thin-layer chromatography indicates that the pullulanase XWPu2 can be used for thoroughly hydrolyzing pullulanase to generate single maltotriose, hydrolyzing corn amylopectin to generate dextrin, and hydrolyzing cyclodextrin to generate glucose. The researches indicate that the pullulanase XWPu2 has a potential application prospect in the industries such as food, chemical industry, pharmacy and energy.

The invention relates to starch-polyphenol composite nano-granules and a preparation process thereof. The preparation process comprises the steps that 1, a biological enzyme method is adopted to prepare debranched starch nano-granules: enzymolysis is conducted on gelatinized starch by using pullulanase to prepare a short amylase solution, ethyl alcohol titration is performed to prepare the starch nano-granules, and freeze-drying is performed to obtain starch nano-granule powder; 2, polyphenol adsorption and loading are performed to prepare the starch-polyphenol composite nano-granules: starch nano-granule turbid liquid is prepared, different amounts of polyphenol are added, the liquid is put in an oscillating water bath kettle, adsorption is performed at room temperature for different time, and ultra-filtration centrifugation, precipitate washing and vacuum freeze-drying are performed. The starch-polyphenol composite nano-granules are small in size, attachment forces of the nano-granules to tissues can be increased, the polyphenol delivery efficiency of the gastrointestinal tract is improved, the retention time of the polyphenol in the gastrointestinal tract is prolonged, and the bioavailability is improved. The starch-polyphenol composite nano-granules protect the polyphenol having bioactivity, prevent light in the outside environment, a pH value, oxygen and the like from affecting the polyphenol and improve the stability of the polyphenol.

The invention discloses codon optimized pullulanase, an expression vector thereof and an construction method of the expression vector. On the basis of codon preference of bacillus subtilis 168 and onreference to pullulanase genes of bacillus acidpullulyticus, a base sequence is designed and expressed in bacillus subtilis with a method of autonomously replicating plasmids and integrative plasmids.A promoter, an mRNA stable sequence, signal peptide and a combination of the promoter, the mRNA stable sequence and the signal peptide of an expression cassette is selected, and a combination capableof performing efficient secretory expression in the bacillus subtilis is obtained. Industrial production is realized and meanwhile, the enzyme activity stability is improved.

The present invention relates to pullulanase variants, wherein the variants have improved properties, for example, altered pH optimum, improved thermostability, altered substrate specificity, increased specific activity or altered cleavage pattern. The present invention also relates to methods of making pullulanase variants having at least one altered property based on the three-dimensional structure of a parent pullulanase.

A method for fixing pullulanase with chitosan magnetic nanoparticles comprises the steps as follows: (1) the chitosan magnetic nanoparticles are prepared; (2) amination treatment is performed on surfaces of the chitosan magnetic nanoparticles; (3) the chitosan magnetic nanoparticles after amination treatment and the pullulanase form covalent bonds in the presence of a cross-linking agent to fix the pullulanase. Compared with a traditional pullulanase fixing method, the method has the advantages that a carrier prepared with a chitosan hydrogel induction method is firstly used for fixing the pullulanase, and the method is simple in step, mild in condition and environment-friendly.

The invention relates to pullulanase and a production method thereof, and belongs to an amylolytic enzyme acted on an alpha-1,6-glucosidic bond. The pullulanase and the production method are characterized in that: a, zymologic characteristic is that the optimal pH is 3.8 to 4.4, the stable pH is 3.5 to 4.5, and the optimal action temperature is 50 to 65 DEG C; and b, Bacillus licheniformis is selected as an enzyme producing strain and derived from China Center of Industrial Culture Collection (CICC No.10185), and the pullulanase is prepared by a liquid state fermentation process in a mode of compositely feeding a backing material and a feed supplement and the method meets the following technological indexes that: (1) the enzymatic activity of the product is more than or equal to 1,100u / ml; (2) the fermentation period is 72 to 80 hours; and (3) the liquid enzyme recovery rate is more than or equal to 86.5 percent. The pullulanase product has the advantages of high heat resistance and acid resistance, high enzymatic activity, stable performance and low cost; the liquid biological fermentation production method has the advantages of high fermentation enzymatic activity, high extracting yield and low manufacturing cost; and the pullulanase and the production method are suitable for the fermentation industry using starch as a raw material and manufacturing of modified starch products, amylose starch and high-amylose starch.

The invention discloses pullulanase with high secretion capacity and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. The pullulanase has high extracellular secretion capacity, and escherichia coli carrying the pullulanase is fermented in a shake flask for 48 hours, the total enzyme activity in the fermentation liquor can reach 212.0 U mL<-1>, wherein the extracellular enzyme activity reaches 200.5 U mL<-1>, which accounts for 94.5% of the total enzyme activity; therefore, the pullulanase is easy to prepare through fermentation, and the control difficulty and cost of the fermentation process can be obviously reduced; the pullulanase has mild action condition, and can hydrolyze alpha-1, 6-glucosidic linkage at a condition with a temperature of 40-60 DEG C and a pH value of 6.0-7.5, therefore, the pullulanase can reduce the cost in industrial transformation, and has potential utilization value in industries such as food, starch sugar and the like.