WO2020093849A1 - 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 - Google Patents

一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 Download PDF

Info

Publication number
WO2020093849A1
WO2020093849A1 PCT/CN2019/111672 CN2019111672W WO2020093849A1 WO 2020093849 A1 WO2020093849 A1 WO 2020093849A1 CN 2019111672 W CN2019111672 W CN 2019111672W WO 2020093849 A1 WO2020093849 A1 WO 2020093849A1
Authority
WO
WIPO (PCT)
Prior art keywords
affinity
chitosanase
agarose gel
biomimetic
purification
Prior art date
Application number
PCT/CN2019/111672
Other languages
English (en)
French (fr)
Inventor
李尚勇
Original Assignee
青岛大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 青岛大学 filed Critical 青岛大学
Publication of WO2020093849A1 publication Critical patent/WO2020093849A1/zh
Priority to US16/878,591 priority Critical patent/US11293013B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0036—Galactans; Derivatives thereof
    • C08B37/0039—Agar; Agarose, i.e. D-galactose, 3,6-anhydro-D-galactose, methylated, sulfated, e.g. from the red algae Gelidium and Gracilaria; Agaropectin; Derivatives thereof, e.g. Sepharose, i.e. crosslinked agarose
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01132—Chitosanase (3.2.1.132)

Definitions

  • the invention relates to a novel biomimetic affinity purification material and its application in the purification of chitosanase, which belongs to the field of industrial biotechnology.
  • Chitosan is a derivative of chitin that is partially or completely deacetylated. It is mainly composed of D-glucosamine linked by ⁇ -1,4-glycosidic bonds. It is the only positively charged polysaccharide in nature and is known as Six elements of life. Chitooligosaccharides are functional oligosaccharides with antibacterial, anti-tumor, hypolipidemic, hypoglycemic, regulating body immunity, and promoting crop yield increase. It is one of the hotspots of functional oligosaccharide research and development in recent years.
  • Chitosanase is a type of glycoside hydrolase that catalyzes the cleavage of ⁇ -1,4 glycosidic bonds between glucosamine and specifically degrades chitosan.
  • Traditional chitosanase purification requires multiple steps: ultrafiltration, ammonium sulfate precipitation, desalting, Q column ion exchange chromatography, Sephacryl S-200 gel column chromatography, etc. The entire purification process takes a long time, has a high cost, and has a low recovery rate.
  • the biomimetic affinity purification method has the advantages of strong specificity, simple operation and easy scale-up.
  • chitosanases have histidine tags, which can be purified by metal chelation chromatography, which can obtain higher purification efficiency.
  • metal chelation chromatography due to the particularity of metal chelating ligands, a large amount of toxic imidazole needs to be used during the elution process of metal chelating chromatography, which will affect the biological activity of the chitosanase obtained by purification. Will add additional steps and costs. Therefore, it is necessary to develop more specific biomimetic affinity materials to establish a biomimetic affinity purification method suitable for chitosanase.
  • the present invention provides a novel biomimetic affinity material that uses chitosan as a biomimetic affinity ligand, the connecting arm is cyanuric chloride, and the basic medium is activated agarose 6B.
  • a bionic affinity purification method of chitosanase was established by using this material.
  • the chitosanase biomimetic affinity method of the present invention has a simple purification method, low cost and high purity, and is suitable for large-scale purification of chitosanase.
  • the present invention provides a synthesis method of a novel chitosanase biomimetic affinity material. Including the following 4 steps: 1), epichlorohydrin activated agarose gel; 2), epichlorohydrin activated agarose gel amination; 3), connecting cyanuric chloride connecting arm; 4), chelating shell II Sugar bionic affinity ligand.
  • the present invention also provides the application of the biomimetic affinity material in the biomimetic affinity purification of chitosanase.
  • the present invention also provides a method for measuring the dissociation constant and maximum binding capacity of the biomimetic affinity material for chitosanase.
  • the present invention also provides a biomimetic affinity purification method of chitosanase, and the selected biomimetic affinity material is the material described in the present invention.
  • the loading buffer is glycine-NaOH, the buffer pH is 8.0 to 10.0, and the optimal loading pH is 8.6;
  • the elution buffer is acetic acid-sodium acetate, the buffer pH is 4.0-6.0, and the optimal elution pH is 4.0;
  • the loading concentration is 25-150 mg / mL, and the optimal loading concentration is 50 mg / mL;
  • the NaCl concentration in the elution buffer is 0.2 to 1M, and the optimal NaCl concentration is 0.8M.
  • the present invention also provides an affinity column, and the packing is the biomimetic affinity material according to the present invention.
  • the chitosanase biomimetic affinity material of the present invention is a novel biomimetic affinity material, and the biomimetic affinity ligand used is chitobiose, which has the following advantages:
  • the preparation method of chitosan is simple and is chitosan
  • the minimal degradation product of the enzyme can be specifically combined with chitosanase and cannot be further degraded, which is a natural excellent ligand
  • chitosan is a basic oligosaccharide and naturally contains two amino groups, It can be firmly combined with the connecting arm, which provides convenient conditions for large-scale preparation of bionic affinity materials.
  • the chitosanase biomimetic affinity material of the present invention uses a circular connecting arm, which has strong mechanical strength, and the ligand is not easy to fall off; and the material has strong affinity for chitosanase, per gram of biomimetic affinity material
  • Q max maximum binding capacity for chitosanase CsnM is 24.1 mg / g. Easy to elute, the dissociation constant (K d ) is 24.2 ⁇ g / mL.
  • the chitosanase biomimetic affinity purification method established by using the biomimetic affinity material of the present invention is simple, low-cost, and easy to scale up.
  • the protein purity of chitosanase CsnM reached more than 95%. It is suitable for large-scale purification of chitosanase.
  • Figure 1 is the synthesis process of the bionic affinity material of the present invention
  • Figure 3 is the dissociation constant and maximum binding capacity of the biomimetic affinity material of the present invention.
  • FIG. 4 is a purification assay of chitosanase purified by the biomimetic affinity method of the present invention.
  • a is the purity of chitosanase CsnM by SDS-PAGE method;
  • b is the purity of chitosanase CsnM by HPLC method.
  • the synthesis process of the biomimetic affinity material includes 4 steps: 1), epichlorohydrin activates the agarose gel, making the agarose gel in an activated state to connect other groups; 2), amination of the agarose gel, Add amino groups to the activated agarose gel; 3), connect the cyanuric chloride linking arm, crosslink the cyanuric chloride linking arm with the linked amino group; 4), chelate the chitosan bionic affinity ligand, and then connect Chitobiose bionic affinity ligand is coupled to the arm. Specific steps are as follows:
  • the biomimetic affinity material synthesized in Example 1 is a macromolecular biomimetic affinity gel.
  • the biomimetic affinity material synthesized in step 1.2 of Example 1 was The ketone method detects the density of amino groups in the biomimetic affinity material, thereby characterizing the ligand density of the synthetic biomimetic affinity material. The detection by ninhydrin method revealed that the density of the synthetic biomimetic affinity ligand reached 20.9 ⁇ mol / ml.
  • the synthetic biomimetic affinity gel was added to 6M concentrated hydrochloric acid at the same ratio (w / v). After 6 hours of action, it was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. Dilute double distilled water 100 times, then add an equal volume (v / v) of acetonitrile, and perform mass spectrometry. Theoretically, the biomimetic affinity ligand cleaved with 6M HCl is C 18 H 31 ClN 6 O 12 , and its relative molecular weight is 558.9.
  • the dissociation constant (K d ) and maximum binding capacity (Q max ) of the biomimetic affinity material described in Example 1 were evaluated to determine the synthetic biomimetic affinity material for chitosan
  • the adsorption and dissociation ability of the enzyme CsnM was purchased from Qingdao Efit Biotechnology Co., Ltd.
  • the specific measurement method is as follows: 1 mL of chitosanase CsnM of different concentrations (0.1-0.9 mg / mL, 20 mM Gly-NaOH, pH 8.6) is mixed with 0.5 g of the biomimetic affinity material described in Example 1, at 4 ° C. Under conditions of shaking at 100rpm for 2h, the adsorption equilibrium was reached. The mixed solution was centrifuged at 1500g for 5 minutes to detect the remaining protease activity and protein content in the supernatant. The measured data is calculated according to the Scatchard equation method:
  • Q represents the amount of protein (mg / g) adsorbed in the bionic affinity material
  • Q max represents the theoretical maximum amount of protein (mg / g) adsorbed by the affinity material with chitosanase
  • [C *] solution The remaining amount of protein (mg / mL)
  • K d represents the adsorption constant.
  • the Scatchard equation method is used to draw a graph, and the dissociation constant K d of the chitosanase CsnM described in Example 1 is calculated to be 24.2 ⁇ g / ml; the maximum adsorption amount Q max is 24.1 ⁇ g / g.
  • sample buffer 0.1M Tris-HCl, pH8.0
  • filter through 0.22 ⁇ m filter to equilibrium and load the biomimetic protease described in Example 1.
  • Affinity column with material column length 25mm ⁇ inner diameter 7mm.
  • the column is then eluted with elution buffer to collect the active component.
  • the invention is optimized for various elution and loading conditions in the purification process.
  • Glycine-sodium hydroxide buffer solutions of different pH were prepared, and chitosanase was purified by FPLC.
  • the specific enzyme activity of the eluted peak samples was measured, and the effect of different pH loading buffers on the purification effect was analyzed.
  • the pH of the glycine-sodium hydroxide buffer was 8.6, and the elution effect was the best. Therefore, the pH of the buffer was 8.6.
  • the flow rate is 1mL / min when loading, rinse 5-10 column volumes with washing buffer A
  • the washing flow rate is 1.5 mL / min
  • the column pressure does not exceed 0.3 mPa
  • the washing buffer A is 100 mM glycine-sodium hydroxide buffer (pH 8.6) containing 100 mM NaCl.
  • the elution flow rate is 1.5ml / min, the column pressure does not exceed 0.3mPa, select the lowest Five common acidic buffers that can reach pH 4.0: acetic acid-sodium acetate, citric acid-sodium citrate, disodium hydrogen phosphate-citric acid, citric acid-sodium hydroxide-hydrochloric acid, acetic acid-potassium acetate, using Example 1
  • the biomimetic affinity material separates and purifies the chitosanase CsnM, collects the elution peak and measures its enzyme specific activity. Based on factors such as elution results and cost, the elution buffer was determined to be acetic acid-sodium acetate buffer.
  • Elution buffers of different pH were prepared, and the chitosanase was purified by protein purification chromatography (FPLC) under the premise of unchanged other conditions. Measure the specific enzyme activity of the elution peak samples, analyze the effect of different pH elution buffers on the purification effect, and determine the optimal pH of the elution buffer. Using acetic acid-sodium acetate buffer to prepare three different pH conditions (3.0, 4.0, 5.0, 6.0), chromatographic purification, the results show that the lower the pH, the higher the amount of protein obtained by purification, but considering the low pH will affect the enzyme The vitality is destroyed, and the elution pH is selected to be 4.0.
  • FPLC protein purification chromatography
  • the optimal NaCl concentration is determined to be 0.8M. It is determined that the wash buffer is 100 mM acetic acid-sodium acetate buffer (pH 4.0) containing 0.8 M NaCl, and the wash buffer B is 100 mM Tris-HCl buffer (pH 8.0) containing 0.8 M NaCl.
  • washing buffer C is 100 mM Tris-HCl buffer (pH 8.0) containing 2M NaCl.
  • Example 4 the chitosanase powder was filtered through a 0.22 m filter membrane to equilibrium and loaded with an affinity column loaded with the biomimetic affinity material described in Example 1. Wash 5-10 column volumes with adsorption equilibrium buffer (glycine-sodium hydroxide pH 8.6) until the system equilibrates, no protein peaks appear, the flow rate is 1 mL / min, and the column pressure is set to protect (below 0.3 mPa). The loading concentration was 50 mg / mL, and the loading volume was 1 mL. After loading the sample, first wash the column with washing buffer A to remove impurities, and then wash the column with washing buffer B to obtain the protein of interest. Then wash the column with Wash Buffer C to remove strongly bound contaminants. The column is stored in 20% ethanol.
  • adsorption equilibrium buffer glycine-sodium hydroxide pH 8.6
  • the protein denaturing electrophoresis (SDS-PAGE) method was used to detect the chitosanase CsnM purified in Example 5 for SDS-PAGE purity analysis: the concentration of the separating gel was 10%, and the concentration of the concentrated gel was 5%.
  • the purified sample was added to 2 ⁇ Loading Buffer in a medium volume, boiled in boiling water at 100 ° C for 5 min, and subjected to SDS-PAGE electrophoresis analysis in a BioRad vertical electrophoresis tank.
  • the voltage of the concentrated gel was 80V, and the voltage in the separation gel was 120V. , Use the color of bromophenol blue as an indicator to control the electrophoresis time.
  • the samples obtained by the purification method in Example 5 were subjected to purity analysis by high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the TSK3000SW gel filtration column was used for detection at a wavelength of 280 nm.
  • the mobile phase was 100 mM PBS, 100 mM Na 2 SO 4 , 0.05% NaN 3 , and the flow rate was 0.6 mL / min.
  • the protein purity of the purified chitosanase CsnM was 95.9%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Materials Engineering (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本公开涉及一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用，属于工业生物技术领域。所述仿生亲和材料的亲和配基为壳二糖，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B。所述仿生亲和材料的解离常数(Kd)及最大结合能力(Qmax)分别为24.2μg/mL和24.1mg/g。利用上述仿生亲和材料建立了一种壳聚糖酶仿生亲和纯化方法，可高效率、低成本的生产高纯度壳聚糖酶，具有良好的工业化应用潜质。

Description

一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 技术领域
本发明涉及一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用，属于工业生物技术领域。
背景技术
壳聚糖是几丁质部分或完全脱乙酰基后的衍生物，主要由D-氨基葡萄糖通过β-1,4-糖苷键连接而成，是自然界唯一带正电荷的多糖，被誉为第六生命要素。壳寡糖是一种功能性低聚糖，具有抗菌、抗肿瘤、降血脂、降血糖、调节机体免疫、促进农作物增产等功效，是近几年功能性寡糖研究和开发的热点之一。
壳聚糖酶是一类催化氨基葡萄糖间的β-1,4糖苷键断裂，专一性降解壳聚糖的糖苷水解酶。传统的壳聚糖酶纯化需要经过多个步骤：超滤，硫酸铵沉淀，脱盐，Q柱离子交换层析，Sephacryl S-200凝胶柱层析等。整个纯化过程耗时长、成本高、回收率低。与传统的柱层析方法相比，仿生亲和纯化方法具有特异性强、操作简单、易于放大等优势。目前部分重组表达的壳聚糖酶带有组氨酸标签，可以用金属螯合层析进行纯化，可得到较高的纯化效率。但是由于金属螯合配基的特殊性，金属螯合层析过程中，洗脱过程中需要利用大量的毒性的咪唑，会影响纯化所得壳聚糖酶的生物活性，利用脱盐柱对咪唑进行处理会增加额外的步骤和成本。因此，需要开发更为特异性的仿生亲和材料去建立适用于壳聚糖酶的仿生亲和纯化方法。
发明内容
本发明针对现有技术的不足，提供了一种利用壳二糖作为仿生亲和配基，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B的新型仿生亲和材料。利用该材料建立了一种壳聚糖酶的仿生亲和纯化方法。本发明所述壳聚糖酶仿生亲和方法纯化方法简单、成本低、纯度高，适用于壳聚糖酶的规模化纯化。
另一方面，本发明提供了一种新型壳聚糖酶仿生亲和材料的合成方法。包括以下4个步骤：1)、表氯醇活化琼脂糖凝胶；2)、表氯醇活化琼脂糖凝胶的氨基化；3)、连接氰尿酰氯连接臂；4)、螯合壳二糖仿生亲和配基。
另一方面，本发明还提供一种所述的仿生亲和材料在壳聚糖酶的仿生亲和纯化中的应用。
另一方面，本发明还提供一种仿生亲和材料针对壳聚糖酶的解离常数和最大结合能力的测定方法。
另一方面，本发明还提供了一种壳聚糖酶的仿生亲和纯化方法，所选用的仿生亲和材料为本发明所述材料。
优选：所述纯化条件中上样缓冲液为甘氨酸-NaOH，缓冲液pH为8.0～10.0，最适上样pH为8.6；
优选：所述纯化条件中洗脱缓冲液为醋酸-醋酸钠，缓冲液pH为4.0～6.0，最适洗脱pH为4.0；
优选：所述纯化条件中上样浓度为25～150mg/mL，最适上样浓度为50mg/mL；
优选：所述纯化条件中洗脱缓冲液中NaCl浓度为0.2～1M，最适NaCl浓度为0.8M。
另一方面，本发明还提供了一种亲和柱，填料为本发明所述仿生亲和材料。
有益效果：
1.本发明的壳聚糖酶仿生亲和材料为新型仿生亲和材料，所用仿生亲和配基为壳二糖，具有以下优势：一方面，壳二糖制备方法简单，且为壳聚糖酶的最小降解产物，可与壳聚糖酶专一性结合，不能被进一步降解，为天然的优良配基；另一方面，壳二糖为碱性寡糖，天然含有两个氨基基团，可与连接臂进行牢固结合，为仿生亲和材料的大规模制备提供了便利条件。
2.本发明所述壳聚糖酶仿生亲和材料采用环状连接臂，具有很强的机械强度，配基不易脱落；而且该材料对壳聚糖酶的亲和力强，每克仿生亲和材料对壳聚糖酶CsnM的最大结合能力(Q max)为24.1mg/g。易于洗脱，解离常数(K d)为24.2μg/mL。
3.利用本发明所述仿生亲和材料建立的壳聚糖酶仿生亲和纯化方法简单、成本低廉、易于放大。壳聚糖酶CsnM蛋白质纯度达到95％以上。适用于壳聚糖酶的规模化纯化。
附图说明
图1为本发明仿生亲和材料的合成过程；
图2为本发明仿生亲和配基的质谱结果；
图3为本发明仿生亲和材料的解离常数和最大结合能力；
图4为本发明仿生亲和方法纯化所得壳聚糖酶的纯化测定。a为SDS-PAGE法测定壳聚糖酶CsnM的纯度；b为HPLC法检测壳聚糖酶CsnM的纯度。
具体实施方式
实施例1仿生亲和材料的合成过程
仿生亲和材料的合成过程包括有4个步骤：1)、表氯醇活化琼脂糖凝胶，使得琼脂糖凝胶处于活化状态从而连接其他基团；2)、琼脂糖凝胶的氨基化，活化后的琼脂糖凝胶添加氨 基；3)、连接氰尿酰氯连接臂，利用连接的氨基基团交联氰尿酰氯连接臂；4)、螯合壳二糖仿生亲和配基，在连接臂上偶联壳二糖仿生亲和配基。具体步骤如下：
1.1表氯醇活化琼脂糖凝胶
用双蒸水按1:10(v/v)的比例对琼脂糖凝胶(Sepharose 6B)进行彻底洗涤，使得流出液的pH平衡到7.0，洗涤过的琼脂糖凝胶在室温下干燥，溶解在100mL活化溶液(1M氢氧化钠，2.5g二甲基亚砜以及10mL表氯醇)中，在40℃摇床中100rpm震荡2.5h进行活化，得到表氯醇活化琼脂糖凝胶6B(图1a)。
1.2琼脂糖凝胶的氨基化
将35％饱和氨添加到活化琼脂糖凝胶6B中，在30℃摇床中100rpm孵化过夜，使得活化琼脂糖凝胶6B加入氨基基团，得到氨基化活化琼脂糖凝胶6B(图1b)。
1.3连接氰尿酰氯连接臂
将氨基化活化琼脂糖凝胶6B按等体积加入50％(v/v)丙酮，在冰浴搅拌的条件下以0.5mL/min的流速缓慢加入氰尿酰氯溶液(溶入70mL丙酮)，用1M的氢氧化钠溶液调节该溶液的pH值至7.0，再用50％(v/v)丙酮洗涤去除未结合的氨基基团，氰尿酰氯(连接臂)连接至活化琼脂糖凝胶6B上，得到连接氰尿酰氯连接臂的仿生亲和配基(图1c)。
1.4螯合壳二糖仿生亲和配基
将上述步骤1.3的过饱和的连接氰尿酰氯连接臂的仿生亲和材料溶解在2M的碳酸钠溶液中，然后在碳酸钠溶液中缓慢添加两倍质量的壳二糖。室温搅拌24h，用双蒸水洗涤凝胶，保存在0.02％(W/V)的叠氮钠，得到仿生亲和材料(图1d)。
实施例2仿生亲合材料的性能表征
实施例1合成的仿生亲和材料为大分子的仿生亲和凝胶，为了确定合成的仿生亲和材料的配基密度，将实施例1中1.2步骤中合成的仿生亲和材料，利用茚三酮法检测仿生亲和材料中的氨基密度，从而表征合成的仿生亲和材料的配基密度。通过茚三酮法检测发现，合成的仿生亲和配基的密度达到20.9μmol/ml。
为了确定合成的仿生亲和材料中仿生亲和配基结构，将合成的仿生亲和凝胶等比例(w/v)加入6M浓盐酸，作用6h后，4000rpm离心10min，取上清液，用双蒸水稀释100倍，然后加入等体积(v/v)的乙腈，进行质谱分析。理论上，利用6M的HCl切割下的仿生亲和配基为C 18H 31ClN 6O 12，它的相对分子量为558.9。但是，由于氯离子在酸性环境中不稳定，在6M的HCl中会被羟基取代，因此，其分子式应变构为C 18H 32N 6O 12，它的相对分子量为524.5。质谱结果(图2)显示，质谱所得仿生亲和配基的相对分子量为525.01，与其正离子一级质谱的理论分子量保持一致。
实施例3仿生亲和材料的解离常数和最大结合能力
利用斯卡查德(Scatchard)方程法对实施例1中所述仿生亲和材料进行解离常数(K d)及最大结合能力(Q max)评价，确定合成的仿生亲和材料对壳聚糖酶CsnM的吸附及解离能力。所用壳聚糖酶CsnM购自青岛艾菲特生物科技有限公司。
具体测定方法为：取1mL不同浓度(0.1-0.9mg/mL，20mM Gly-NaOH，pH 8.6)的壳聚糖酶CsnM与0.5g的实施例1中所述仿生亲和材料混合，在4℃条件下100rpm震荡培养2h，达到吸附平衡。混合液1500g离心5min，检测上清中剩余的蛋白酶活性及蛋白含量。所检测数据按照Scatchard方程法计算：
Figure PCTCN2019111672-appb-000001
其中，Q代表吸附在仿生亲和材料中的蛋白量(mg/g)，Q max代表理论上与亲和材料吸附壳聚糖酶的最大蛋白量(mg/g)，[C*]溶液中剩余的蛋白量(mg/mL)，K d代表吸附常数。如图3所示，通过Scatchard方程法做图，计算得到实施例1中所述仿生亲和材料对壳聚糖酶CsnM的解离常数K d值为24.2μg/ml；最大吸附量Q max为24.1μg/g。
实施例4壳聚糖酶的仿生亲和纯化方法的条件筛选
取30mg的壳聚糖酶CsnM酶粉，稀释到1mL的样品缓冲液(0.1M Tris-HCl,pH 8.0)，经过0.22μm的滤膜过滤至平衡后的装载有实施例1中所述仿生亲和材料的亲和柱(柱长25mm×内径7mm)。用吸附平衡缓冲液直至体系平衡，直至无蛋白峰出现。然后利用洗脱缓冲液洗脱柱子，收集活性组分。本发明针对纯化过程中各种洗脱和上样条件进行了优化。
4.1仿生亲和柱上样条件筛选
上样前，用5-10个柱体积的用0.22μm过滤过的双蒸水洗涤实施例1中所述仿生亲和材料，然后用5-10个柱体积的上样缓冲液平衡柱子，流速为1mL/min，柱子压力设置保护(低于0.3mPa)。根据纯化结果筛选最适的上样缓冲液种类，缓冲液的pH，以及上样量。
4.1.1最适上样缓冲液
选取最高能达到pH 9.0的五种常见碱性缓冲液，巴比妥钠-盐酸、甘氨酸-氢氧化钠、硼酸-硼砂、磷酸氢二钠-磷酸氢二钾、碳酸钠-碳酸氢钠，利用FPLC对壳聚糖酶进行纯化。收集洗脱峰样品并测定纯度，对比五种洗脱缓冲液的纯化效果。通过分析纯化结果可知，甘氨酸-氢氧化钠缓冲液纯化所得蛋白量最大，且该缓冲液对亲和柱损伤较小。因此，选用甘氨酸-氢氧化钠为上样缓冲液。
4.1.2最适上样缓冲液pH
配制不同pH的甘氨酸-氢氧化钠缓冲液，利用FPLC对壳聚糖酶进行纯化。测洗脱峰样 品的酶比活力，分析不同pH的上样缓冲液对纯化效果的影响。通过纯化效率分析，发现甘氨酸-氢氧化钠缓冲液pH为8.6时，洗脱效果最好，因此选用缓冲液的pH为8.6。
4.1.3最适上样量
配制25mg/mL、50mg/mL、75mg/mL、100mg/mL、125mg/mL、150mg/mL六种不同浓度的酶溶液，考察不同的上样浓度对纯化效果的影响。结果发现，最适上样浓度为50mg/mL，上样体积为1mL。
4.2洗涤去除杂蛋白
按照4.1.3中所述壳聚糖酶的样本样，上样至按4.1中平衡过的仿生亲和柱，上样时流速为1mL/min，用洗涤缓冲液A冲洗5-10个柱体积，去除杂蛋白，洗涤流速为1.5mL/min，柱子压力不超过0.3mPa，所述洗涤缓冲液A为含有100mM NaCl的100mM甘氨酸-氢氧化钠缓冲液(pH 8.6)。
4.3洗脱获得壳聚糖酶纯品
按照4.2所述方法去除杂蛋白之后，用洗涤缓冲液B洗脱，收取活性峰，即为壳聚糖酶的纯酶，洗脱流速为1.5ml/min，柱子压力不超过0.3mPa，选取最低能达到pH 4.0的五种常见酸性缓冲液醋酸-醋酸钠、柠檬酸-柠檬酸钠、磷酸氢二钠-柠檬酸、柠檬酸-氢氧化钠-盐酸、醋酸-醋酸钾，利用实施例1中所述仿生亲和材料对壳聚糖酶CsnM进行分离纯化，收集洗脱峰并测其酶比活力。综合洗脱结果及成本等因素，洗脱缓冲液确定为醋酸-醋酸钠缓冲液。
配制不同pH的洗脱缓冲液，在其他条件不变的前提下利用蛋白纯化色谱仪(FPLC)对壳聚糖酶进行纯化。测洗脱峰样品的酶比活力，分析不同pH的洗脱缓冲液对纯化效果的影响，确定最适的洗脱缓冲液pH。利用醋酸-醋酸钠缓冲液配制三种不同的pH条件(3.0，4.0，5.0，6.0)，进行色谱纯化，结果显示pH越低纯化所得蛋白量越高，但是考虑到低的pH会对酶的活力产生破坏，选择洗脱pH为4.0。
在洗脱缓冲液中添加NaCl能显著提高洗脱壳聚糖酶的效率，通过优化NaCl浓度，确定最适的NaCl浓度为0.8M。确定洗涤缓冲液为含有0.8M的NaCl的100mM醋酸-醋酸钠缓冲液(pH 4.0)，所述洗涤缓冲液B为含有0.8M NaCl的100mMTris-HCl缓冲液(pH 8.0)。
4.4洗涤并保存柱子
用洗涤缓冲液C洗涤柱子，去除牢固结合在柱子上的蛋白，再用0.22μm过滤过的双蒸水冲洗5-10个柱体积，去除洗涤缓冲液中的各种盐离子，再用20％乙醇保存柱子。所述洗涤缓冲液C为含有2M NaCl的100mMTris-HCl缓冲液(pH 8.0)。
实施例5壳聚糖酶CsnM的仿生亲和纯化方法
依据实施例4中摸索的各影响因素的最适条件，将壳聚糖酶酶粉经过0.22μm的滤膜过 滤至平衡后的装载有实施例1中所述仿生亲和材料的亲和柱。用吸附平衡缓冲液(甘氨酸-氢氧化钠pH 8.6)洗涤5-10个柱体积直至体系平衡，无蛋白峰出现，流速为1mL/min，柱子压力设置保护(低于0.3mPa)。上样浓度为50mg/mL，上样体积为1mL。上样后，先用洗涤缓冲液A洗涤柱子，去除杂蛋白，再用洗涤缓冲液B冲洗柱子，获得目的蛋白。再用洗涤缓冲液C洗涤柱子，去除牢固结合的杂蛋白。柱子保存于20％乙醇。
实施例6壳聚糖酶的纯度测定
蛋白质变性电泳(SDS-PAGE)法检测实施例5中纯化所得壳聚糖酶CsnM进行SDS-PAGE纯度分析：分离胶的浓度为10％，浓缩胶的浓度为5％。将纯化所得的样品中等体积的加入2×Loading Buffer，在100℃沸水中煮沸5min，在BioRad垂直电泳槽中中进行SDS-PAGE电泳分析，浓缩胶电压为80V，在分离胶中的电压为120V，以溴酚蓝的颜色为指示控制电泳时间。电泳完成后利用考马斯亮蓝R-350染色，并用脱色液脱色后在全自动凝胶成像系统拍照并分析纯度。如图4a所示，利用SDS-PAGE分析所得壳聚糖酶CsnM的蛋白纯度为96.7％。
利用高效液相色谱(HPLC)法对按照实施例5中纯化方法所得的样品进行纯度分析。利用TSK3000SW凝胶过滤柱在波长280nm处检测。流动相为100mM PBS,100mM Na 2SO 4,0.05％NaN 3，流速为0.6mL/min。如图4b所示，通过计算HPLC峰面积，纯化所得壳聚糖酶CsnM的蛋白纯度为95.9％。

Claims (9)

  1. 一种新型仿生亲和材料，其特征是，仿生亲和配基为壳二糖，连接臂为氰尿酰氯，基础介质为活化的琼脂糖6B，其连接结构式为
    Figure PCTCN2019111672-appb-100001
  2. 如权利要求1所述的仿生亲和材料的制备方法，其特征是步骤如下：
    1)、表氯醇活化琼脂糖凝胶：
    用双蒸水按1:10的比例对琼脂糖凝胶Sepharose 6B进行彻底洗涤，使得流出液的pH平衡到7.0，洗涤过的琼脂糖凝胶在室温下干燥，溶解在100mL活化溶液中，在40℃摇床中100rpm震荡2.5h进行活化，得到表氯醇活化琼脂糖凝胶6B，所述活化溶液为1M氢氧化钠，2.5g二甲基亚砜以及10mL表氯醇；
    2)、琼脂糖凝胶的氨基化：
    将35％饱和氨添加到活化琼脂糖凝胶6B中，在30℃摇床中100rpm孵化过夜，使得活化琼脂糖凝胶6B加入氨基基团，得到氨基化活化琼脂糖凝胶6B；
    3)、连接氰尿酰氯连接臂：
    将氨基化活化琼脂糖凝胶6B按等体积加入50％丙酮，在冰浴搅拌的条件下以0.5mL/min的流速缓慢加入氰尿酰氯溶液，用1M的氢氧化钠溶液调节该溶液的pH值至7.0，再用50％丙酮洗涤去除未结合的氨基基团，连接臂氰尿酰氯连接至活化琼脂糖凝胶6B上，得到连接氰尿酰氯连接臂的仿生亲和配基；
    4)、螯合壳二糖仿生亲和配基：
    将上述步骤3)的过饱和的连接氰尿酰氯连接臂的仿生亲和材料溶解在2M的碳酸钠溶液中，然后在碳酸钠溶液中缓慢添加两倍质量的壳二糖，室温搅拌24h，用双蒸水洗涤凝胶，保存在0.02％的叠氮钠，得到仿生亲和材料。
  3. 如权利要求1所述的仿生亲和材料在壳聚糖酶的仿生亲和纯化中的应用。
  4. 一种壳聚糖酶的仿生亲和纯化方法，所选用的仿生亲和材料为权利要求1所述仿生亲和材料。
  5. 如权利要求4所述纯化方法，其特征是，纯化条件中上样缓冲液为甘氨酸-NaOH，缓冲液pH为8.0～10.0。
  6. 如权利要求4所述纯化方法，其特征是，纯化条件中洗脱缓冲液为醋酸-醋酸钠，缓冲液pH为4.0～6.0。
  7. 如权利要求4所述纯化方法，其特征是，纯化条件中上样浓度为25～150mg/mL。
  8. 如权利要求4所述纯化方法，其特征是，纯化条件中洗脱缓冲液中NaCl浓度为0.2～1M。
  9. 一种亲和柱，所述填料为权利要求1所述仿生亲和材料。
PCT/CN2019/111672 2018-11-09 2019-10-17 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用 WO2020093849A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/878,591 US11293013B2 (en) 2018-11-09 2020-05-19 Biomimetic affinity purification material and its application in chitosanases purification

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811329059.X 2018-11-09
CN201811329059.XA CN109320630B (zh) 2018-11-09 2018-11-09 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/878,591 Continuation US11293013B2 (en) 2018-11-09 2020-05-19 Biomimetic affinity purification material and its application in chitosanases purification

Publications (1)

Publication Number Publication Date
WO2020093849A1 true WO2020093849A1 (zh) 2020-05-14

Family

ID=65261006

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/111672 WO2020093849A1 (zh) 2018-11-09 2019-10-17 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用

Country Status (3)

Country Link
US (1) US11293013B2 (zh)
CN (1) CN109320630B (zh)
WO (1) WO2020093849A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109320630B (zh) * 2018-11-09 2019-12-20 青岛大学 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用
CN110479184B (zh) * 2019-08-05 2021-09-07 华侨大学 一种琼脂糖基乳化剂及其制备方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101078008A (zh) * 2007-01-29 2007-11-28 四川大学 壳聚糖酶的分离纯化工艺
CN108144586A (zh) * 2016-12-05 2018-06-12 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法
CN109320630A (zh) * 2018-11-09 2019-02-12 青岛大学 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用
CN110038524A (zh) * 2019-03-20 2019-07-23 江西师范大学 一种用于分离纯化壳聚糖酶的亲和层析介质的制备方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4319975A (en) * 1980-10-20 1982-03-16 Fmc Corporation Derivatized agarose and method of making and using same
US6262255B1 (en) * 1995-04-05 2001-07-17 Biomm, Inc. Non-immunogenic, biocompatible macromolecular membrane compositions, and methods for making them
SE0102171D0 (sv) * 2001-06-19 2001-06-19 Protista Internat Ab Method for the separation of bioproducts
SE0202067D0 (sv) * 2002-06-28 2002-06-28 Amersham Biosciences Ab Surface-modified base matrices
CN101250512B (zh) * 2008-04-17 2010-04-14 上海交通大学 内切木聚糖酶的仿生亲和纯化方法
US20090275130A1 (en) * 2008-05-02 2009-11-05 Biotex, Inc. Biomimetic nucleic acids

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101078008A (zh) * 2007-01-29 2007-11-28 四川大学 壳聚糖酶的分离纯化工艺
CN108144586A (zh) * 2016-12-05 2018-06-12 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法
CN109320630A (zh) * 2018-11-09 2019-02-12 青岛大学 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用
CN110038524A (zh) * 2019-03-20 2019-07-23 江西师范大学 一种用于分离纯化壳聚糖酶的亲和层析介质的制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, SHANGYONG ET AL: "Design and Synthesis of a Chitodisaccharide-Based Affinit} Resin for Chitosanases Purification", MARINE DRUGS, 21 January 2019 (2019-01-21), pages 1 - 13, XP055704875, ISSN: 1660-3397, DOI: 10.3390/md17010068 *

Also Published As

Publication number Publication date
CN109320630A (zh) 2019-02-12
US11293013B2 (en) 2022-04-05
CN109320630B (zh) 2019-12-20
US20200277589A1 (en) 2020-09-03

Similar Documents

Publication Publication Date Title
WO2020093849A1 (zh) 一种新型仿生亲和纯化材料及其在壳聚糖酶纯化中的应用
CA2784278C (en) Wash solution and method for affinity chromatography
JP3676817B2 (ja) 新規因子ｉｘ精製法
AU2006316039B2 (en) A method of chromatography using semi-synthetic heparin ligands
CN104645949B (zh) 一种以四肽为功能配基的亲和层析介质及其制备方法
CN107243336B (zh) 一种层析介质及其制备方法和应用
Chibata et al. Immobilized tannin—a novel adsorbent for protein and metal ion
CN110066314A (zh) 一种高效提高多聚体分离分辨率的亲和纯化工艺
WO2014034457A1 (ja) ミックスモード抗体アフィニティー分離マトリックスとそれを用いた精製方法および標的分子
Dalmia et al. Characterization of glucoamylase adsorption to raw starch
CN108355618B (zh) 牛来源透明质酸酶亲和介质及其吸附方法
CN108059673B (zh) 一种人血清中分离免疫球蛋白IgG的方法
CN108191956B (zh) 组合型配基、组合型仿生层析介质及其制备方法和应用
CN108144586B (zh) 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法
Boi et al. Adsorption of lectins on affinity membranes
Nagasaki et al. Enzymic and structural properties of crystalline yeast cell lytic enzyme from a species of fungi imperfecti
CN107033236A (zh) 一种从酵母发酵液中分离人血白蛋白的混合模式层析方法
CN106268556A (zh) 一种蛋白纯化用磁珠的制备方法
CN111065735B (zh) 用于纯化硫酸酯酶蛋白的方法
Sun et al. The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins
CN108905980A (zh) 一种以苯丙氨酸—酪氨酸—组氨酸—谷氨酸为功能配基的四肽层析介质及其应用
CN112876547B (zh) 一种制备型高效液相色谱纯化乳酸链球菌素的方法
CN110180505B (zh) 一种以四肽为功能配基的亲和仿生层析介质
JP2019182757A (ja) ９糖シアリルオリゴ糖ペプチド及びその製造方法
Sorci et al. Adsorption and Elution of Lectins by Affinity Membranes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19881059

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19881059

Country of ref document: EP

Kind code of ref document: A1