200 results about "Alginate lyase" patented technology

The invention features alginate and alginate lyase compositions and methods that are useful for the treatment of various conditions and diseases. The invention also provides kits and instructions for use.

The invention provides Bacillus sp Alg07 capable of producing an alginate lyase. The preservation number of the Bacillus sp Alg07 is CGMCC No.9391. The Bacillus sp Alg07 provided by the invention has the advantages that requirement on nutrition is low and fermentation time is short; a crude enzyme is easily prepared, and the crude enzyme can be obtained through centrifugation; the alginate lyase produced by the Bacillus sp Alg07 has high enzyme activity, the specific enzyme activity of an unpurified crude enzyme can reach 20000-40000 U/mg (563U/mL); the alginate lyase produced by the Bacillus sp Alg07 has good stability, is not obviously changed in enzyme activity after being preserved at 40 DEG C for 24 hours and is high and stable in enzyme activity at pH of 5.5-8.5; and the produced alginate lyase is capable of degrading sodium alginate to generate alginate-derived oligosaccharide with biological activity.

The invention improves the extraction yield of algae essence by using cellulase for decomposing cellulose in algae after dry algae which is ground and desanded is soaked in water and subjected to colloid mill treatment, and then leads alginic acid with large molecular weight to be degraded to alginic acid with small molecular weight by using alginate lyase, thus leading effective compositions in algae to be less damaged and having high extraction yield.

The invention discloses a gene sequence of Incision alginate lyase Alg2B from Flavobacterium strain (Flavobacterium sp. S20). The invention also provides a method for preparing novel alginate lyase, which is characterized in that by using a gene engineering technical method, the gene of the alginate lyase is cloned to an escherichia coli expression vector, the escherichia coli recombinant strain capable of realizing heterogenous expression of enzyme can be obtained, the recombinant strain is used for heterogenous expression of the alginate lyase Alg2B, and thereby monosaccharide and oligosaccharides produced by sodium alginate can be degraded. The provided alginate lyase Alg2B can be widely used in the fields of agriculture, food, feed addictives, medicine and alga genetic engineering.

The invention discloses a recombinant alginate lyase and a construction method and an application thereof, which belong to the technical field of biology. The alginate lyase Aly-Cob is derived from anenvironment sample (a mixture of sea-tangle stacking place and factory production waste material). By using a gene engineering technology, a gene of the alginate lyase is cloned to an expression vector, and a recombinant bacterial strain for heterogenous expression of the alginate lyase is obtained. The alginate lyase Aly-Cob produced by the bacterial strain through fermentation has the functionfor degrading sodium alginate to prepare alginate oligosaccharide. The provided alginate lyase Aly-Cob can be widely used for the fields of agriculture, food, feed addition, medicine and alga processing.

The invention discloses an alginate lyase (Alg509) derived from marine bacteria and a gene thereof, and also disclosed a method for recombinant expression and preparation of the alginate lyase. According to the method, the alg509 gene is cloned into an E. coli expression vector, and the vector is transformed into an E. coli host strain to obtain a recombinant engineering strain which can heterologously express the enzyme. The alginate lyase Alg509 disclosed in the invention has high enzyme activity, the specific enzyme activity can reach up to 48000 U/mg and above, the optimum reaction pH is 10, the optimum reaction temperature is 55 DEG C, and the enzyme activity has no dependence on various metal ions. The enzyme is active to sodium alginate, poly-guluronic acid (polyG) and ploymannuronic acid (polyM), and can completely degrade sodium alginate to produce alginate oligomers such as alginate disaccharide, alginate trisaccharide, alginate tetrasccharide, etc. The enzyme exhibits strongbasophilia, has certain tolerance to high pH, has certain potential of industrial applications, and can be widely applied in the fields of agriculture, food, feed additive, medicine and the like.

The invention provides a bacterial strain capable of generating alginate lyase and a fermentation method thereof. The bacterial strain capable of generating alginate lyase provided in the invention is classified as Tamlana sp., named as ZJU HZ22, assigned with the accession number of CGMCC No. 5324 and preserved on Oct. 14th, 2011. The bacterial strain is capable of background expression of alginate lyase under the condition of no inducing substrate and can improve the level of expression through stimulation of a substrate. The fermentation method provided in the invention is simple; the alginate lyase ALG22 generated by the bacterial strain CGMCC No. 5324 is lyoenzyme and has striking salt tolerance; fermentation broth of the alginate lyase can obtain substantial and stable enzyme activity after simple bacteria removal and can both be directly used in degradation of alginate and be easily further separated and purified; so the alginate lyase has application potential in degradation of alginate and preparation of alginate-derived oligosaccharide.

The invention relates to truncated recombinant alginate lyase rAly1-T185N, and an encoding gene and application thereof. The amino acid sequence of the truncated recombinant alginate lyase rT185N is as shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene of the truncated recombinant alginate lyase rT185N is as shown in SEQ ID NO.1. 185 amino acid residues are cut off from the N end of the full-length alginate lyase rAly-1 to obtain T185N truncated enzyme; the expression quantity of the rT185N can reach 1259+/-2.2 mg/L fermentation liquid, the specific enzyme activity can reach 2895+/-10.3 U/mg, the expression quantity is increased by about 2.6 times compared with that of full-length protein rAly-1, the aims of increasing yield, saving energy and improving economic benefit are fulfilled, and rT185N can realize one-step purification of fusion protein through nickel column separation.

The invention discloses a method for promoting in-vitro and in-vivo degradation and cell extension adherence of alginate-based 3D printed bioink. The method comprises the following steps: step 1, adopting alginate as an extracellular matrix main body skeleton and constructing celliferous 3D bio-printed bioink; step 2, mixing alginate lyase with alginate-based bioink; step 3, adding cell suspensioninto the obtained bioink, choosing print parameters and printing out a 3D printing block through a 3D bio-printer; step 4, regulating and controlling in-vitro and after-transplantation-in-vivo degradation time and cell extension adherence degree of the 3D printing block through adjusting the concentration of the alginate lyase according to actual demands. The method disclosed by the invention canpromote the degradation and cell extension adherence of the bioink with the alginate as the skeleton under in-vivo and in-vitro environments, and enables the alginate 3D printed bioink to be adaptedto more in-vivo and in-vitro experiments and be better applied to soft tissue repairing and transplantation.

The invention belongs to the field of biotechnologies, and particularly relates to a marine bacterium Cobetia sp. WG-007 screened from a waste material sample and an alginate lyase secreted from the marine bacterium and a preparation method thereof. By combining morphology and physiological and biochemical characters, the strain belongs to a Cobetia category, and is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No.7964. The strain secretes the alginate lyase, and the alginate lyase is purified by ultrafiltration and concentration, Q-Sepharose strong anion exchange chromatography, Phenyl Sepharose 6 Fast Flow hydrophobic chromatography and Superdex-G100 gel filtration technologies so as to obtain the alginate lyase with electrophoresis purity; SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) determines that the molecular weight of the alginate lyase generated by the strain is 35.0KDa.

The invention provides a method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis. An amino acid sequence of alginate lyase used in the method is SEQ ID NO:3. According to the method provided by the invention, the alginate oligosaccharides are directionally prepared by utilizing the genetically-modified recombinant alginate lyase; compared with an existing method for preparing the alginate oligosaccharides by acid hydrolysis, the method provided by the invention overcomes the defects of large destructiveness and low efficiency; compared with an existing method for preparing the alginate oligosaccharides by enzymatic hydrolysis, the method provided by the invention overcomes the defects that the enzymolysis time is long, the cost is high, and the alginate oligosaccharides with specific degree of polymerization cannot be directionally prepared by applying the improved recombinant alginate lyase. The method provided by the invention is simple in process, low in cost and suitable for industrialized production, and can reduce the alginate viscosity within a short time; the total yield of the prepared oligosaccharide is 90 percent or above. The prepared alginate oligosaccharides has the efficacies of resisting tumor, resisting inflammation, reducing blood fat, improving the immunity and the like, can be used in the field of food and health products, and has wide application prospect.

The invention discloses an anaerobic alginate decomposing bacterium Marinicatena alginatilytica SH-52. The strain is preserved in the China Center for Type Culture Collection and the preservation number of the strain is CCTCCNO: M2013073. The bacterial strain of the decomposing bacterium disclosed by the invention is an anaerobe capable of effectively degrading alginate and can be applied to the production of alginate lyase; and the decomposing bacterium is short in time period, high in enzyme yield, simple to collect collection; the operation is convenient. The strain can be put into industrial implementation easily.

The invention discloses an alginate lyase biosynthesis gene cluster which comprises five alginate lyase encoding genes: algV1, algV2, algV3, algV4 and algV5.The gene cluster is derived from Vibrio alginolyticus ATCC17749. The contained all gene and protein information related to the alginate lyase biosynthesis, provided by the invention, can help people to understand the biosynthesis mechanism of the alginate lyase so as to provide a great deal of substance and genetic information for the large-scale application of the alginate lyase.

The invention relates to the field of genetic engineering technology, especially to alginate lyase and a preparation method and application thereof. Maturation protein of the alginate lyase contains an amino acid sequence as shown in the SEQ ID NO. 1, and the precursor protein contains an amino acid sequence as shown in the SEQ ID NO.4. The alginate lyase has stable properties, and specific enzyme activity reaches up to 4600 U/mL and above. Alginate can be subjected to enzymolysis to obtain micromolecular alginate-derived oligosaccharides with special biological activity. The product has the potential for industrial application.

The invention discloses recombinant alginate lyase, an engineering bacterium thereof and a method for preparing alginate oligosaccharide through hydrolysis. The method is characterized by screening an alginate lyase gene AlgM according to a strain Vibrio sp.BZM-1 newly isolated from seaweeds; constructing an expression vector pHBM905BDM-AlgM containing the gene, and converting pichia sp GS115 competent cells to obtain a single copy gene engineering bacterium; and constructing, culturing and verifying the multi-copy gene engineering bacterium to prepare recombinant alginate lyase through high-efficiency expression, and producing alginate oligosaccharide by hydrolyzing sodium alginate with alginate lyase. The recombinant alginate lyase gene engineering bacterium GS115/AlgM4 (CCTCC No:M2015500) containing four copies has a higher expression level. The activity of recombinant alginate lyase is as high as 31000U/mL.

The invention provides a bacillus (sp.W003). The collection number of the bacillus is CGMCC NO.15010. The invention further provides a bacillus (sp.W024). The collection number of the bacillus (sp.W024) is CGMCC NO.15011. The invention provides a method adopting the bacilli to prepare alginate lyase. The method includes following steps: activating the bacilli, and coating activated bacteria liquidonto a slope culture medium; picking and inoculating the bacilli on the slope culture medium into a container filled with an enzyme-generating culture medium for culture, and culturing on a shaking table at temperature of 25-35 DEG C and rotating speed of 150-230r/min for 12-16h to logarithm middle and later period to obtain seed liquid; inoculating the seed liquid into a fermentation tank filledwith a fermentation enzyme-generating culture medium, and fermenting in conditions of 25-35 DEG C in temperature, 150-230r/min in rotating speed and 7-8 in PH for 2-4d to obtain alginate oligosaccharide fermention liquid. The invention further provides application of the bacilli in generating alginate lyase and fermenting a culture medium for producing alginate oligosaccharide. The alginate lyasegenerated by the bacilli is high in enzyme activity and fermentation efficiency.

The present invention discloses an alginate lyase Alga genetic sequence and a preparation method and application thereof. Alginate lyase Alga is derived from Vibrio alginolyticus 40B. The present invention provides a process for preparing the novel alginate lyase Alga, a genetic engineering method is used, an alginate lyase Alga coding gene is cloned into an E. coli expression vector to obtain a recombinant E. coli strain capable of heterologous expression of the alginate lyase Alga, and the alginate lyase Alga prepared by heterologous expression of the recombinant E. coli strain has the function of degrading sodium alginate to prepare sodium alginate oligosaccharide. The alginate lyase Alga can be widely used in chemical industry, agriculture, food, feed additives, pharmaceuticals and algae genetic engineering and other fields.

The invention discloses a preparation method of sargassum oligosaccharide. The preparation method comprises the steps that fine polysaccharide obtained through degreasing and deproteinizing of sargassum is taken as a raw material, ultrasonic treatment is utilized, alginate lyase, mannase, xylanase and pectinase are sequentially added to perform enzymolysis on the polysaccharide, the polysaccharide which is not degraded fully is removed through an ethyl alcohol sedimentation method, supernate is centrifuged and screened by a molecular sieve to obtain retained matter, freeze drying is performed, and the sargassum oligosaccharide is prepared. The prepared sargassum oligosaccharide has the high inhibitory activity on alpha-glucosidase, the IC50 value is 4.82 mg/mL, the dose dependency is presented, and meanwhile the obvious promoting effect on glucose consumption of insulin-resistant HepG2 cells is achieved. According to the preparation method, the non-specific commercial enzymes are adopted, the process route is simple and reasonable, the method is suitable for industrialization, the preparation amount of the hypoglycemic active oligosaccharide is increased, and meanwhile the enzyme consumption and production cost are reduced; the method is an effective method for preparing the sargassum oligosaccharide and can be applied to hypoglycemic drugs, health care products and food.

The invention discloses a strain for producing alginate lyase and a use thereof. The strain is preserved in the China general microbiological culture collection center on October 9, 2013, has an accession number of CGMCC No.8312 and is named as Halomonas elongata SH-19. A 16S rDNA polynucleotide sequence of the strain is shown in the formula of SEQ ID No.1. The strain SH-19 can efficiently express alginate lyase and can be used for high-efficiency production of alginate lyase. A strain SH-19 fermentation medium comprises simple ingredients commonly used in a laboratory, has substantial fermentation effects, greatly reduces an alginate lyase production cost and can be used for large-scale production.

The invention provides a vibiro splendidus OU02 strain highly yielding alginate lyase, the preservation number of which is CGMCC No.13478. The vibiro splendidus OU02 strain screened by the invention is used for producing and preparing alginate lyase. The alginate lyase produced by the vibiro splendidus OU02 strain screened can degrade alginate to generate alginate oligomers with potential biological activity. The microorganism is short in fermenting period and high in enzymatic activity, and almost activities intracellular activities, so that a sample is favorably collected and stored, thereby laying a foundation for follow-up industrial application.

The invention provides alginate lyase, host cells capable of secreting alginate lyase and application of the alginate lyase and the host cells. The alginate lyase has any one of amino acid sequences shown as (I) and (II): (I) an amino acid sequence shown as SEQ ID NO. 1; (II) an amino acid sequence obtained by carrying out substitution, deletion or addition of 1 to 20 amino acid residues on the amino acid sequence shown as SEQ ID NO. 1; the obtained amino acid sequence has the activity of the alginate lyase. According to the alginate lyase, whole genes of the alginate lyase are truncated to reduce the molecular weight, so that the expression amount of the host cells on the alginate lyase is remarkably improved; the secreted alginate lyase has high activity, good stability and strong degradation capability on sodium alginate.

The invention discloses a paenibacillus sp. strain HB172198; the systematic name of the strain HB172198 is Paenibacillus sp.; the preservation number is CGMCC No.15412; the preservation date is 5th March, 2018; the preservation institution is China General Microbiological Culture Collection Center, CGMCC; and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3building No.1 yard Beichengxi road, Chaoyang district, Beijing. The invention also discloses an alginate lyase and a preparation method thereof, and an application of the paenibacillus sp. strain andthe alginate lyase in degradation of brown algae.

The invention discloses a sphingomonas alginate lyase gene ZH0-III and an efficient prokaryotic expression vector pGEX-4T-1-ZH0-II thereof. A Ptac promoter is utilized to control the expression of the prokaryotic expression vector pGEX-4T-1-ZH0-II in colon bacillus. The expression of the alginate lyase gene is performed by the colon bacillus expression vector, so that the alginate lyase ZH0-III as an express product can be obtained in a short time. The obtained recombinant alginate lyase gene ZH0-III is wide in substrate specificity, wherein the PloyG or the PloyM can be served as a substrate; and the enzyme activity can reach 63.8U/mg. The prokaryotic expression vector and the overall expression system are easy to operate, and are convenient for industrialized production.

The invention relates to incision difunctional alginate lyase Aly2 generating various monosaccharide products, an encoding gene of the Aly2 and an application of the Aly2. The amino acid sequence of the Aly2 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the encoded Aly2 is as shown in SEQ ID NO. 1. The specific activity of prepared gene engineering recombinant protein rAly2 for alginate, polyguluronic acid and polymannuronic acid is 2025U/mg, 3672U/mg and 1324U/mg, so that the gene engineering recombinant protein rAly2 is a difunctional enzyme. The enzyme is an incision enzyme, and final main products are unsaturated disaccharide and unsaturated trisaccharide when an alginate polysaccharide substrate is thoroughly degraded. When different oligosaccharide substrates are degraded, various monosaccharide products such as saturated M and G or unsaturated delta can be generated. Besides, the rAly2 is stable in biochemical property, is a potential tool enzyme, can be applied tothe field of medicines, food and the like and has a wide application prospect.