85 results about "Inulinase" patented technology

The invention belongs to the biotechnology field, in particular to a method of manufacturing ethanol by utilizing Jerusalem artichoke material. The method comprises the following steps: in material process step, the fresh Jerusalem artichoke is cleaned and juiced, or sliced up, dried and pulverized; in a seed liquid preparation step, kluyveromyces are cultivated in seed culture fluid taking Jerusalem artichoke powders or Jerusalem artichoke juice as a complete composition; in a ferment step, seed liquid is inoculated in culture medium confected by the complete Jerusalem artichoke powders or the complete Jerusalem artichoke juice, the ethanol is produced through anaerobic fermentation, and the ethanol is obtained by distillation at last. Both compositions of a seed culture medium and a ferment culture medium provided by the method are Jerusalem artichoke powders or Jerusalem artichoke juice; any auxiliary composition is not needed; and beforehand saccharification processing of Jerusalem artichoke material in the ferment culture medium is not needed; inulase excreted by the kluyveromyces in the seed liquid directly hydrolyzes and saccharifys multi fructosan in the Jerusalem artichoke material; the ferment is processed at the same time; and concentration of ethanol product is high to 12 percent (v/v). The method has the advantages that the method is simple; the synchronization process of saccharification and ferment is implemented; and the production cost is reduced.

The invention belongs to the field of natural organic chemistry, and relates to a method for purifying fructo oligosaccharide in chicory by using an Aspergillus niger endo-inulinase-microwave extraction combined technology, macroporous resin decolorization and impurity removing, and combination of ultrafiltration and nanofiltration. The method has the following advantages that: 1, the Aspergillus niger endo-inulinase-microwave extraction combined technology is adopted so as to substantially improve the yield of the fructo oligosaccharide, greatly reduce the extraction time, and improve the extraction speed; the macroporous resin is selected so as to substantially improve the active ingredient content, and achieve the large-scale production, wherein the macroporous resin provides a large absorption amount for pigment, and the inulin loss is low during the decolorization process; and 3, the ultrafiltration and nanofiltration equipment is adopted, such that the extraction purity of the fructo oligosaccharide is high, the separation effect is significant, and the permeation flux of the membrane can be restored after the membrane is washed. By combining the Aspergillus niger endo-inulinase-microwave extraction combined technology, the macroporous resin decolorization and impurity removing, and the ultrafiltration and nanofiltration filtration process, the final product fructo oligosaccharide with the content more than 98% can be obtained. With the present invention, disadvantages of long time, existing of impurities, and high active ingredient loss of the conventional inulin extraction method are overcome.

The invention relates to the technical field of gene engineering and protein modification, in particular to a heat-resistant low temperature exo-inulinase mutant MutQ23 delta 3. Amino acid sequence ofthe mutant MutQ23 delta 3 is as shown in SEQ ID NO.1; the optimum pH of the MutQ23 delta 3 is 6.0, and the optimum temperature is 45 DEG C; the mutant MutQ23 delta 3 respectively contains 14%, 24%, 46% and 75% of enzyme activity at the temperature of 0 DEG C, 10 DEG C, 20 DEG C and 30 DEG C; the MutQ23 delta 3 still maintains 100% of enzyme activity after treatment for 60 minutes at the temperature of 50 DEG C; the enzyme can hydrolyze inulin to produce fructose. The heat-resistant low temperature exo-inulinase mutant MutQ23 delta 3 has the advantages of being applicable to industries such asfood, brewing and bioenergy.

The invention relates to a producing process of the inulin and the oligo fructose produced from the fruit of the snow lotus. The process is mainly that the fresh fruit of the snow lotus is cleaned, chipped, expressed juice, filtrated, dried, crushed characterized in that the slicing is dipped into the color fixative 1-3 hour and the inulin is purified by adding the 10% confected inulinase and the water and is fermented 48-72 hour in the aerobic atmosphere of the temperature of 20-30. The snow lotus inulin and the glucosidase fructose can be used for the health products, the food additive.The using of it as the health products has many merits of the low consumption of taking medicine, the good curative effect, taking conveniently, transporting and storing conveniently. So it can be added into the food such as the drink, the cake and the candy to form the functional food. The invention has some merits of the advance arts and crafts, the high quality.

The invention discloses a low-temperature exoinulinase mutant MutP126R stable at medium temperature, the mutant MutP126R has an amino acid sequence as shown in SEQ ID NO.1, the thermal activity and thermal stability of the mutant MutP126R are changed, the optimal temperature is increased, the thermal stability is better, and the mutant MutP126R is beneficial to production, storage, transportation and the like of enzymes. The optimum temperature of the purified wild enzyme InuAMN8 is 35 DEG C, and the optimum temperature of the mutant enzyme MutP126R is 40 DEG C; after the wild enzyme InuAMN8 is treated at 55 DEG C for 10-60 minutes, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the enzyme activity of the mutant enzyme MutP126R is reduced from 70% to 26%. The low-temperature excision inulase mutant MutP126R disclosed by the invention can be applied to the industries of food, wine brewing, biological energy and the like.

The invention belongs to the technical field of gene engineering, in particular to a technical proposal used to improve the expression level of recombinant protein. In the method, a promoter, alpha-signal peptide, target protein of an inulase gene are orderly connected with a terminal subcode sequence of the inulase gene first; then the sequence is inserted into a Kluyveromyces lactis expression vector; finally the Kluyveromyces lactis is transformed and fermented, and a fermented supernatant is taken and separated. The method can accelerate the remarkable growth of the yield of the recombinant protein expressed in the Kluyveromyces lactis. The technical proposal can be used to improve the expression amount of various proteins and provide a new method for reducing production cost and enlarging production scale.

The invention relates to a eukaryon expression method for exoinulinase, which mainly comprises the following steps: firstly, connecting a nucleotide sequence (as shown in SEQ ID No: 1) of the exoinulinase of Kluyveromyces marxianus with pichia expression plasmids pPICZ alpha A through a restricted enzyme site, and obtaining a recombinant expression vector; and secondly, introducing the recombinant expression vector into a thermococcus host strain X-33 or SMD1168, and expressing target proteins (the Kluyveromyces marxianus exoinulinase) through induction fermentation. The eukaryon expression method for the exoinulinase lays a foundation for developing the exoinulinase with industrial application value.

A method for fermenting carbohydrate-rich crops is provided. Sugar beet, sugar cane, sweet sorghum, tropical maize hybrids and fruits are rich in simple sugars; potato, sweet potato, cassava and yam are rich in starch; and Jerusalem artichoke is rich in inulin. This method uses vacuum infusion to infuse yeast into the intercellular space (apoplast) of the parenchyma tissue. The simple sugars diffuse into the apoplast, come into contact with the yeast and produce ethanol. Ethanol can be extracted from the crop by vacuum stripping or crushing or can be left inside the starchy crop to preserve it. In some variants, pectinase enzymes degrade the parenchyma cell walls to speed up diffusion of simple sugars to the yeast, speed up diffusion of amylase to starch granules or speed up diffusion of inulinase to insoluble inulin.

The invention discloses a double-promoter multi-copy recombinant pichia pastoris engineering strain for highly producing endo-inulinase INU2. The strain is named pichia pastoris GS115-A3-G2, and is preserved in the general microbiology center of the China Committee for Culture Collection of Microorganisms on April 16th, 2014, and the preservation number is CGMCC NO:9049. According to the strain disclosed by the invention, the endo-inulinase gene INU2 multi-copy is integrated into pichia pastoris GS115 genome by two plasmids pPIC9K and pGAPZaA, and the double-promoter multi-copy recombinant pichia pastoris engineering strain for efficiently expressing the endo-inulinase is obtained in a screening manner. The experiment proves that the inulinase enzyme activity can be up to 3006U/ml in 3L high-density fermentation, simply purified crude enzyme can be directly applied to hydrolysis of inulin, so as to generate fructo-oligose, the conversion efficiency is high, production of the fructo-oligose by using inulin enzymatic hydrolysis in industry is achieved, and the double-promoter multi-copy recombinant pichia pastoris engineering strain has great practical value.

The present invention provides a yeast gene engineering bacterium Pichia pastoris GS115/HY005 which is an endo inulase gene obtain by screening strain from Aspergillus by means of PCR method or DNA library hybridization method. It can be cloned and inserted into Pichia yeast integration expression vector, then the obtained expression vector containing endoinulase gene is introduced into Pichia yeast, then a yeast gene engineering bacterium capable of high-effectively expressing endoinulase can be screened. The enzyme activity of the endoinulase prepared with said yeast gene engineering bacterium can be up to above 300 u/ml, and the reaction time for hydrolyze inulin with it is short, and its conversion rate is high, so that it can high-effectively economically convert and prepare oligofructose.

The invention discloses an amino acid sequence and a nucleotide sequence of a heat-resisting alkali-resisting and salt stable inulase exonuclease, and construction and application of an inulase exonuclease recombinant expression vector. Results of enzymatic property research show that the optimum reaction conditions of the inulase exonuclease are as below: pH 7.0, 50 DEG C and 10% NaCl. The enzyme can catalyze the hydrolysis of inulase, levan, sucrose, raffinose, kestose, nystose and Kestopentaose. In a catalysis process using inulase as a substrate, only generation of fructose is detected rather than oligosaccharide or other carbohydrates. The inulase exonuclease provided by the invention can be applied to the field of food industry and biological energy source as a novel biocatalyst for fructose production.

The invention discloses a fructo-oligosaccharide producing method with fossilized endoinulinase in bacterial cellulose. The fructo-oligosaccharide producing method with the fossilized endoinulinase in bacterial cellulose is characterized in that the fructo-oligosaccharide producing method comprises the steps that 1 pretreatment is conducted on the surface of a bacterial cellulose film, the surface hole size of the bacterial cellulose film is enlarged, and modified bacterial cellulose film is acquired; 2 the modified bacterial cellulose film is added into the endoinulinase solution, and the endoinulinase is fixed to the surface of the bacterial cellulose film; 3 washing is conducted through 0.5 mol /L Tris-HCI buffer solution, and bacterial cellulose film which fossilizing the endoinulinase is obtained; 4 the bacterial cellulose film which fossilizing the endoinulinase is placed into inulin solution, the endoinulinase is used for resolving the inulin, and fructo-oligosaccharide solution is obtained; 5 purification is conducted on the obtained fructo-oligosaccharide solution, and fructo-oligosaccharide with the purity over 90% is obtained. According to the fructo-oligosaccharide producing method with fossilized endoinulinase in bacterial cellulose, the prepared fructo-oligosaccharide is high in purity and little in impurity, the content of the fructo-oligosaccharide is over 99%, and significant industrial application prospect is possessed.

The invention discloses lactobacillus plantarum 3-2 for generating acid inulase and an application of the lactobacillus plantarum 3-2 for generating acid inulase. The srtain is preserved in China Center for Type Culture Collection (CCTCC) on 25 November 2019, wherein the preservation number is CCTCC NO:M 2019972. The Lactobacillus plantarum 3-2 is screened from Guangxi Zhuang nationality traditional foods namely cold pressing rice powder, and in a fermentation medium that inulin is used as a carbon source and a beef extract is used as a nitrogen source, the lactobacillus plantarum 3-2 can growwell, and is high in acid resistance. After the acid inulase generated by the strain is placed under the condition that the pH is 4.5 for 1h, the survival rate is 96%, and the lactobacillus plantarum3-2 has the capacity for degrading inulin and levan substances and preparing high fructose syrup. For the first time, the lactobacillus plantarum for generating the acid inulase is reported, and a new strain and inulase thereof are provided for industrial degradation of the inulin and levan substances and preparation of the high fructose syrup.

The invention discloses a method for producing fructooligosaccharides by fermenting inulase mutants. An enzymolysis technology for producing the high-quality fructooligosaccharides includes the optimal adding quantity of jerusalem artichoke, the optimal pH, the optimal concentration of a buffer solution, the optimal adding quantity of enzymes, the optimal temperature, the optimal stirring speed and the optimal stirring time. The formed enzymolysis technology can achieve the efficient production process in which the fructooligosaccharides account for more than 95.78% of the total sugar content. Inulase adopted in the technology is the novel inulase mutants and has high enzyme activity, the enzyme activity of fermentation liquor in a 30-m<3> system can be 60,000 U/ml, the production efficiency of the inulase can be remarkably improved, and production cost can be reduced. The formed enzymolysis technology for the fructooligosaccharides is a new technology; when the fructooligosaccharides are prepared with the technology, operation difficulty can be greatly lowered, and economic cost can be saved.

The invention discloses a recombinant vector containing Jerusalem artichoke fructan 1-exohydrolase gene and application thereof in producing alcohol by fermentation. The recombinant plasmid is prepared by the following steps: inserting a Saccharomyces cerevisiae 3-phosphoglyceric kinase gene PGK1 promoter between EcoRV and SpeI enzyme digestion sites of an expression vector pUG6, inserting a CYC1-terminator-fused Jerusalem artichoke fructan 1-exohydrolase gene Ht 1-FEHs between SpeI and HpaI enzyme digestion sites of the expression vector pUG6, and inserting an 18SrDNA sequence segment of Saccharomyces cerevisiae genome between XbaI and BstEII enzyme digestion sites of the expression vector pUG6. The invention also discloses application of the recombinant plasmid in producing alcohol by fermentation. The Jerusalem artichoke inulase genes Ht-FEHI and Ht-FEHII can promote fermentation of the Saccharomyces cerevisiae to produce alcohol, thereby providing a new method for enhancing the alcohol yield of Saccharomyces cerevisiae fermentation.

A strategy to improve protein stability by domain insertion. TEM 1 beta-lactamase (BLA) and exo-inulinase, as model target enzymes, are inserted into a hyperthermophilic maltose binding protein from Pyrococcus furiosus (PfMBP). Unlike conventional protein stabilization methods that employ mutations and recombinations, the inventive approach does not require any modification on a target protein except for its connection with a hyperthermophilic protein scaffold. For that reason, target protein substrate specificity was largely maintained, which is often modified through conventional protein stabilization methods. The insertion was achieved through gene fusion by recombinant DNA techniques

The invention relates to a production method for preparing inulase. The method comprises the following four steps of: the preparation of a spore suspension, the preparation of liquid seeds, the preparation of an aspergillus niger fermentation liquor and the preparation of an inulase product. The method specifically comprises the following steps of: inoculating aspergillus niger stains on a slant culture medium to culture the spore suspension; inoculating the spore suspension into a liquid seed culture medium at a temperature of 28-30 DEG C to culture so as to obtain the liquid seeds; inoculating the liquid seeds into an aspergillus niger fermentation medium to ferment according to an inoculation quantity of 3-7 percent in a volume ratio under an aseptic condition; within a period of time of 0-60h under a certain fermentation condition, opening an ultrasonic transmitting device to carry out ultrasonic irradiation culture on the aspergillus niger fermentation medium and the liquid seeds; after the ultrasonic irradiation culture, continuously carrying out ultrasonic irradiation culture to obtain the aspergillus niger fermentation liquor; precipitating inulase from the aspergillus niger fermentation liquor by adopting a salting-out method; and filtering waste liquid through a filter press and drying a filter cake to obtain an inulase product. The production method is simple and stable, saves the production cost, and improves the output of the inulase.