118 results about "Agarase" patented technology

The invention discloses an agarase and an acquisition method of an encoding gene thereof. The acquisition method utilizes various PCR methods to acquire the agarase gene aga41 from marine bacteria vibrio natriegens (the preservation number is CGMCC No.2428) for the first time and breaks through the conventional method which screens the agarase gene by constructing a library. The agarase gene aga41 belongs to a GH50 family in a beta-agarase. The whole length of the agarase gene aga41 is 2,973bp. 990 amino acids are encoded on the agarase gene aga41. The highest sequence similarity of the agarase gene aga41 with the existing agarase is 49 percent. The agarase gene aga41 is a novel beta-agarase. An activated recombinant agarase which is obtained by cloning the agarase gene of the invention on an expression vector pET28b, constructing a recombinant escherichia coli Rosetta bacterial strain and carrying out heterologous expression can be applied to the industrial production.

The present invention is directed to methods and systems of producing neoagarobiose, useful in whitening melanoma cells and in cosmetics, using polypeptides having neoagarobiosebiohydralase activity, including Aga86E from Saccharophagus degradans. The reaction can be enhanced by including other agarases, including Aga16B, also from S. degradans.

The invention aims to provide a beta-agarase and application thereof. The beta-agarase is one capable of degrading agarose to produce single Neoagarobiose, and the amino acid sequence is shown in SEQ ID NO:1. According to the invention, the beta-agarase can degrade agarose to produce single Neoagarobiose, thereby ensuring that the beta-agarase can be used for preparation of pure Neoagarobiose. The recombinant agarase expressed by an Escherichia coli recombinant strain (pETC/BL21) according to the invention accounts for 30% of the total protein, and is high in expression level and easy to purify; and the expression quantity is not reduced after continuous passage. Thus, the recombinant beta-agarase and the Neoagarobiose can be prepared in a large scale according to the invention.

The invention discloses a novel flavobacterium strain, a cultivation method for the strain and application of the strain in the aspect of producing agarase. The provided strain which can be used for producing the agarase is flavobacterium sp. CGMCC NO.2342, and is specially characterized in that the strain can generate the agarase without agar, and 0.05 to 0.25 percent of agar powder or a certain carbon source can be added in a liquid culture medium. The provided method for producing the agarase cultivates the flavobacterium sp. in a fermentation culture medium; and the crude enzyme liquid of the obtained agarase can be continuously concentrated and purified, and also can be directly used for degrading the agar to produce agar oligosaccharide. The agarase produced by the strain has high activity, produces various agarase, and can be expected to form various series of agar oligosaccharide.

The invention provides a novel agarase generating strain (roseobactersp.zjut) and application thereof in the preparation of agaro-oligosaccharide. The roseobactersp.zjut is preserved in the China Center for Type Culture Collection, the collection number is CCTCC No. M 209292, and the preservation date is December 3, 2009. The roseobactersp.zjut and the application thereof in the preparation of the agaro-oligosaccharide have the advantages that: (1) the roseobactersp.zjut of the invention has low requirements on nutrition and can be easily cultured; (2) agarase generated by the roseobactersp.zjut is an extracellular enzyme, separation and purification are convenient, and cell breakage is not required; (3) the roseobactersp.zjut has high agarase generating activity, and the activity of crude agarase liquid which is not separated and purified can reach 215.8U/ml under optimized conditions; (4) the agarase generated by the roseobactersp.zjut degrades agar under mild reaction conditions, and the reaction time is short; and (5) the agarase in the invention has high stability and basically has no enzyme activity loss (inactivation efficiency is less than or equal to 5 percent) after being stored in a refrigerator for 7 days at the temperature of less than 4 DEG C.

The invention provides an enzymolysis preparation method of new agaro oligosaccharides and relates to the field of biological engineering. Asparagus serves as raw materials, agar is prepared through high-temperature and high-pressure water extraction, agarase fermentation liquid produced through spontaneous fermentation in a laboratory is used for preparing the new agaro oligosaccharides, the oligosaccharides is purified through a gel chromatography, and a set of complete new agaro oligosaccharides preparation technology is established. The agar prepared through water extraction has the advantages that the yield is high, no chemical reagents need to be consumed, and the agar is environmentally friendly and low in cost, the agar can serve as raw materials for preparing agaro oligosaccharides, and high-value utilization of asparagus is achieved; an enzymatic method is used for replacing a traditional chemical method to prepare agaro oligosaccharides, environmental pollution is reduced, and high-bioactivity agaro oligosaccharides can be obtained.

The invention provides novel beta-agarase, namely, beta-agarase capable of specifically degrading agarose to produce neoagarotetraose, application of the novel beta-agarase, and a gene for encoding beta-agarase. The novel beta-agarase is characterized in that the amino acid sequence is as shown in SEQ ID No.: 1; and the nucleotide sequence of the gene is shown as SEQ ID No.: 2. The beta-agarase is used for degrading agarose to produce neoagarotetraose and can specifically degrade agarose to produce the unique product, namely, neoagarotetraose; and therefore, the beta-agarase can be used for preparing high-purity neoagarotetraose; the conversion rate of the beta-agarase to agarose within 48 hours is more than 90%, and the purity of neoagarotetraose is higher than 95%; and therefore, the beta-agarase has a good industrial application prospect.

The invention provides a preparation method of asparagus agar oligosaccharide, and relates to the technical field of oligosaccharide production. The preparation method comprises the following steps: (1) soaking the asparagus in salt brine for 3 to 5 hours, and then drying the asparagus; (2) pulverizing the asparagus in a pulverizer and filtering the smashed asparagus to obtain asparagus puree; (3)adding the asparagus puree into water at the mass ratio of the asparagus puree to the water tobe 1 to (21-26), and heating the mixture at the temperature of 105-115 DEG C for 90-105 min to obtain a water-extracted mixture; (4) carrying out microwave heating and agar extraction on the water-extracted mixture to obtain an agar solution; (5) after cooling, adding a buffer agent and beta-agarase, heating the mixture to 38-45 DEG C, and carrying out enzymolysis on the mixture for 3-10 h to obtain an asparagus oligosaccharide solution; and (6) drying the asparagus oligosaccharide solution to obtainthe asparagus oligosaccharide. According to the invention, the agar is extracted by the process of crushing, heating and microwave treatment of the asparagus, the extraction rate of the agar is increased and the prepared agar oligosaccharide has high antioxidant activity and high probiotics activity.

The invention provides a neoagarobiose hydrolase. The amino acid sequence of the neoagarobiose hydrolase is SEQ IDNO:1. The neoagarobiose hydrolase can degrade neoagarobiose to generate 3,6-anhydro-L-galactose (L-AHG) and D-galactose, and therefore the neoagarobiose hydrolase can be used for preparing pure L-3,6-inner ether galactose. Serving as a biocatalyst used for producing the L-AHG, the neoagarobiose hydrolase has a user-friendly reaction environment, high catalytic activity, a certain advantage over a chemical method and good application potential and prospects. The L-AHG is a kind of levoglucosan, has good skin whitening performance and anti-inflammatory activity and has very good potential application value in the aspects of medicine and cosmetics. According to recombined escherichia coli strains, recombined agarase expressed by pETNA/BL21 accounts for 28% of total protein. The expression level is high, the recombined agarase is easily purified and expression quantity is not reduced after continuous passage is carried out. The neoagarobiose hydrolase is adopted for preparing recombined neoagarobiose hydrolase and the L-AHG in a mass mode.

The invention relates to a beta-agarase, the amino acid sequence of the beta-agarase is SEQ ID NO:1, and the nucleotide sequence of encoding gene is SEQ ID NO:2. The beta-agarase has the high temperature resistance characteristic, and can specifically degrade agar to produce neoagarool igosaccharode with the polymerization of 4-10, and the final product is neoagarotetraose and neoagarohexaose, and the beta-agarase has good industrial application prospects.

The invention discloses a method for preparing an agar oligosaccharide from immobilized agarase. The method comprises the following steps: firstly, preparing magnetic Fe3O4 nanoparticles embedded in oleic acid by virtue of a coprecipitation method, and adding a KMnO4 solution to oxidize the nanoparticles so as to carboxyl-functionalized magnetic Fe3O4 nanoparticles; then, reacting the carboxyl-functionalized magnetic Fe3O4 nanoparticles with a coupling agent, namely glutaraldehyde, for 2h, wherein the concentration of the glutaraldehyde is 4%; after removing a cleaning fluid through magnetic separation, adding an enzyme fluid for immobilizing at 4 DEG C for 1h so as to obtain immobilized agarase, wherein enzyme addition is 1mL (850U); and under an oscillating condition, carrying out an enzymatic hydrolysis reaction on agar dissolved in a reaction medium and the immobilized enzyme, and after the reaction, magnetically separating to obtain a new agar oligosaccharide and recovering the immobilized enzyme. The immobilizing process disclosed by the invention is simple, low in operation cost, good in enzymatic activity recovery rate, good in the stability of the immobilized agarase, repeatable in use, easy in product separation and applicable to large-scale production.

The invention relates to a Thalassospira sp. fjfst-201307 strain and a method for preparing agarase by using the strain. The strain has been registered and collected at China Center for Type Culture Collection on December 25th, 2013, and the collection number is CCTCC M2013708. In a fermentation medium composed of a carbon source, a nitrogen source and inorganic salts by using the strain as the seed, the activity of the agarase is up to 11.2 U/ml under optimized culture conditions. The method for preparing the agarase comprises the following steps: salting out the fermentation liquor by ammonium sulfate, centrifugating, collecting the precipitate, dialyzing, passing the dialysate through an ion-exchange column and a gel column, and carrying out freeze-drying to obtain the agarase dry powder.

The invention provides agarase and a preparation method thereof and a method for cloning and expression of agarase in escherichia coli and yeast cells. The nucleotide sequence of agarase is as shown in SEQ NO.1, and the amino acid sequence of agarase is as shown in SEQ NO.2. Vectors of nucleotide molecules are escherichia coli plasmids or yeast plasmids. Cells of the nucleotide molecules are obtained by transforming the vector. Cells of the nucleotide molecules of agarase are escherichia coli containing the nucleotide molecules or transformed by the vectors or Pichia pastoris containing nucleic acid molecules or transformed by the vectors. A recombination production strain which can be expressed efficiently and secrete agarase is prepared, the production industrialization of agarase is achieved, and the agarase has good capability of hydrolysis of agar and gracilaria lemaneiformis.

The invention relates to a low temperature beta-agarase and a coding gene and an application thereof. The nucleotide sequence of the low temperature beta-agarase gene NJ21 is shown as SEQ ID NO. 1. The amino acid sequence of the low temperature beta-agarase NJ21 is shown as SEQ ID NO. 2. The invention further relates to the application of the low temperature beta-agarase NJ21 in industrial degradation of agarose. According to the invention, gene of agarase is cloned and obtained from Antarctic bacterial strains which generate agarase and can be cloned to appropriate hosts to realize heterologous expression, and finally industrialized production of agarase is realized. The low temperature beta-agarase NJ21 has stronger low temperature tolerance and provides an initial material of agarase which is low in cost for subsequent industrial application.

The invention relates to vibrio alginolyticus A-001 and a method for preparing agaro-oligosaccharide with agarase generated by the vibrio alginolyticus A-001. The vibrio alginolyticus A-001 is preserved in CCTCC (China Center for Type Culture Collection) in September 18, 2015, and a preservation number of the vibrio alginolyticus A-001 is CCTCC M2015555. The method includes: taking strains for culture, using a fermentation medium composed of a carbon source, a nitrogen source and inorganic salt to generate enzymes, salting out fermentation liquor through ammonium sulfate, centrifuging, dialyzing, passing through iron exchange columns and gel columns to obtain dry enzyme powder, allowing reaction between agarase and 0.5% agar substrate, filtering through an ultrafiltration membrane, collecting filtrate and performing freeze drying to obtain the agaro-oligosaccharide.

The invention belongs to the field of enzyme engineering research and development technology. The invention relates to an agarase immobilization method research. The invention comprises the following steps: preparing a 1:20 (W/V) of chitosan solution with 1.5-2% (V/V) of acetate for fully dissolving; dropping the dissolved chitosan solution drop by drop into a 1 mol/L sodium hydroxide solution, and filtering and collecting a chitosan bead; washing to neutral with distilled water, and then immersing with a 0.2 mol/L phosphate buffer solution of pH7.6 overnight; fetching the immersed chitosan bead carrier 2.5-5 g, and adding 2.5% of glutaraldehyde 20-25 ml, carrying out water-bath oscillation for 0.5 hours and room temperature crosslinking for 5-6 hours, and washing the chitosan bead cross-linked carrier with a phosphoric acid buffer; adding diluted agarase liquid 10-15 ml into the chitosan bead cross-linked carrier, stirring uniformly, fixing at 4 DEG C for 2-3 hours, washing with the phosphoric acid buffer; obtaining the immobilized agarase by vacuum-pumping. The agarase immobilization method provided by the invention has the advantages of cheap and easily available carrier, simple technology, mild condition and small loss of enzyme activity, and the enzyme activity recovery rate reaches to 77.6%.

The invention relates to a vampirovibrio sp. fifst-2013001 which has been registered and collected in the China Center for Type Culture Collection on December 8, 2013, with a collection number of CCTCC M 2013638, and a method for preparing an agarase from the vampirovibrio sp. fifst-2013001. The vampirovibrio sp. fifst-2013001 is taken as a strain and a fermentation culture medium composed of a carbon source, a nitrogen source and an inorganic salt is adopted, and under optimized culture conditions, the activity of the agarase reaches up to 4.17U/ml. A fermentation liquor is salted out by virtue of ammonium sulfate, a precipitate is collected after centrifuging, next, dialysis is carried out, a dialysate is put through an ion exchange column and a gel column, and then freeze-drying is carried out to obtain the dry powder of the agarase.

The invention relates to a beta-agarase and a recombinant expression strain. The recombinant expression strain of the beta-agarase can efficiently express the beta-agarase, the amino acid sequence of the beta-agarase is SEQ ID NO:1, and the expressed beta-agarase has the high temperature resistance characteristic. The recombinant expression strain of the beta-agarase can efficiently express the beta-agarase, the expressed beta-agarase has the high temperature resistance characteristic, and can specifically degrade agar to produce neoagarool igosaccharode with the polymerization of 4-10, and the final product is neoagarotetraose and neoagarohexaose, and the beta-agarase has good industrial application prospects.

The present invention provides a β-agarase, a composition containing the same and applications thereof. The present β-agarase provides the field a novel alternative and is favorable for the industrial utilities of the hydrolysis products of agarose. Furthermore, the hydrolysis product of agarose by the present β-agarase has high purity of neoagarotetraose therefore the present β-agarase is especially useful for the neoagarotetraose's utilities in the field.

The invention relates to an endo-cleaved Beta-agarase with homogeneous degradation products and an application thereof. Beta-Agarase is a new Beta-Agarase AgaB1, amino acid sequence shown in SEQ ID NO: 1. Beta. Provided by the present invention-Agarase is a new Beta-amylase designed artificially. Beta-agarase AgaB1 has a catalytic region (R1-V392) at the N terminal and a carbohydrate binding domain (G393-Y540) at the C terminal. Its amino acid sequence is identical to that of Beta-The sequence similarity of agarase is only 71%. Beta-Agarase AgaB1 is endo-cleaved and has homogeneous characteristics. The proportion of neo-agarose in the degradation product is up to 90.5%. Beta-Agarase AgaB1 has a stable property and a large yield, and has a certain potential for industrial application.

The invention discloses vibrio natriegens HJPH YXJ-1 for producing agarase and application of the vibrio natriegens; the strain is preserved in China Center for Type Culture Collection with preservation number of CCTCC M 2015244 on April 23, 2015, wherein the China Center for Type Culture Collection is located in Wuhan University, and post code is 430072. The agarase generated from the strain is an extracellular enzyme, which is simple in separation and purification and is free from cell breakage; enzyme-producing activity is relatively high, and under optimized conditions, the activity of unpurified crude enzyme reaches 103U/mL; to degrade agar, the agarase produced from the strain is mild in reaction condition, good in specificity and free from environmental pollution, and the is suitable for the large-scale preparation of neoagaro-oligosaccharides.

A process for preparing neoagarotetraose or neoagarohexaose includes such steps as hydrolyzing the agarose to obtain different types of neoagaroligose, and chromatographic separating to obtain the products with different degrees of polymerization. It features that the transgenic agarase used for said hydrolysis is exprssed by recombinant colibacillus DH5alpha-pET24-agaA. Its advantages are short period and low cost.

The invention provides a site-directed mutagenesis-modified agarase mutant with improved thermal stability and belongs to the field of genetic engineering and enzyme engineering. A mutant according tothe invention is obtained in beta;-Agarase rAgaZC-1 to glycine (D622G). The nucleotide sequence of mutant D622G and the corresponding amino acid sequence are shown in SEQ ID NO. 3 and SEQ ID NO. 4. The catalytic efficiency of D622G was higher than that of rAgaZC-1 There was a 17.6% increase. Thermal inactivation kinetics and hydrolysis kinetics analysis showed that the thermal stability of D622Gwas obviously improved, T5010 was increased by 1.5 DEG C, half-life t1/2 at 41 DEG C was prolonged by three times, and more agarose oligosaccharides could be accumulated at 43 DEG C, which enhanced the application potential of agarase in agarose oligosaccharides degradation.

The invention relates to a truncated modified agarase, and the truncated agarase is obtained by deleting a homologous fragment of a Por secretion system C-terminal sorting domain at the C-terminal ofwild agarase. The truncated agarase can catalyze an agar substrate to generate neoagaro-oligosaccharide 4, neoagaro-oligosaccharide 6, neoagaro-oligosaccharide 8, neoagaro-oligosaccharide 10 and neoagaro-oligosaccharide 12; however, the wild agarase only generates the neoagaro-oligosaccharide 4 and the neoagaro-oligosaccharide 6. According to the invention, the neoagaro-oligosaccharides, which aregenerated by using the modified agarase and have multiple degrees of polymerization, are final products of a reaction, and the moisture retention and resistance to oxidation of the neoagaro-oligosaccharides are obviously improved compared with existing products.

The invention relates to beta-agarase and a coding gene and application thereof. The amino acid sequence of the beta-agarase is shown in the sequence table SEQ ID No:1. The nucleotide sequence of the coding gene of the beta-agarase is shown in the sequence table SEQ ID No:2. The beta-agarase has the good heat stability and enzymatic activity and can specifically degrade agarose, generate a single end-product of neoagarotetraose and provide a new enzyme source for preparation of agaro-oligosaccharide.