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 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 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 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 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 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 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 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 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.