Method for purifying recombinant type I mussel mucin and removing endotoxin

Abstract

The invention discloses a method for purifying recombinant type I mussel mucin and removing endotoxin, and relates to the technical field of preparation of recombinant type I mussel mucin. According to the method, recombinant I-type mussel mucin fermentation liquor which is subjected to fermentation induced expression by escherichia coli engineering bacteria is adopted as a raw material, and the purification efficiency can be greatly improved, the content of endotoxin can be reduced, and the quality of the mussel mucin can be improved by combining Triton X-100 and disodium ethylene diamine tetraacetate, controlling the cooling temperature of feed liquid and optimizing the endotoxin removal process and the addition amount of Triton X-114. Therefore, the invention provides the method for purifying the recombinant I-type mussel mucin of which the purity is greater than 95% and the endotoxin content is less than 5EU / mg.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of recombinant type I mussel mucin, and in particular to a method for purifying and removing endotoxin from recombinant type I mussel mucin expressed by fermentation induction of Escherichia coli engineering bacteria. Background Art

[0002] Mussel adhesive protein (MAP) is a protein complex secreted by the secretory glands of the mussel foot. Different types of proteins are secreted at the proximal, distal and plaque regions of the byssus, producing adhesions of different strengths. Currently, three types of collagen, two types of silk matrix proteins and six types of foot proteins have been identified. Type I mussel adhesive protein is secreted by the cuticle glands and is the only foot protein found on the surface of the byssus. Type I mussel adhesive protein has attracted widespread attention due to its high strength, high toughness, waterproofness and degradability, and has been vigorously promoted and applied in biomedicine.

[0003] Among them, the strength of the adhesion effect of mussel mucin is positively correlated with the amount of L-DOPA. Through synthetic biology, the amino acid sequence of natural type I mussel mucin is optimized so that it contains more L-DOPA after modification, thus having the characteristics of high adhesion and strong biosafety. Therefore, the preparation of high-quality type I mussel mucin products has very broad application prospects in medicine.

[0004] At present, the fermentation production of recombinant type I mussel mucin using biotechnology has become an important direction of technological breakthroughs. The preparation of recombinant type I mussel mucin by Escherichia coli fermentation expression is currently a hot topic. However, the existing technology has problems such as the difficulty in developing the purification process of Escherichia coli fermentation expression products and the high content of bacterial endotoxin (hereinafter referred to as "endotoxin"). Therefore, it is urgent to develop a purification process for recombinant type I mussel mucin expressed by Escherichia coli fermentation with high yield, high purity and low endotoxin content. Summary of the invention

[0005] The invention provides a purification method for obtaining a recombinant type I mussel mucin (theoretical molecular weight 22.81KD) with high yield and high purity and having L-3,4-dihydroxyphenylalanine (L-DOPA) like natural mussel mucin and low endotoxin content by purifying a feed liquid expressed by fermentation of Escherichia coli engineered bacteria obtained by biotechnology. The method aims to solve the problems of low purification yield of recombinant type I mussel mucin expressed by fermentation of Escherichia coli engineered bacteria and high endotoxin content of the product.

[0006] In order to solve the above technical problems, the purpose of the present invention is achieved through the following technical solutions.

[0007] Provided is a method for purifying and removing endotoxin from recombinant type I mussel mucin, comprising the following steps:

[0008] (1) adding a combination of Triton X-100 and disodium ethylenediaminetetraacetic acid to a fermentation broth of recombinant type I mussel mucin expressed by fermentation with an engineered Escherichia coli bacterium and heating and mixing;

[0009] (2) dissolving the liquid treated in step (1) with acid and filtering it to collect the filtrate;

[0010] (3) ultrafiltering the filtrate after treatment in step (2), concentrating and collecting the concentrate;

[0011] (4) cooling the liquid treated in step (3) and maintaining it at 2-10° C., adding Triton X-114, dissolving and mixing, and then adjusting the pH of the liquid to 6.5-7.0, wherein the concentration of Triton X-114 in the liquid is 10-15 g / L;

[0012] (5) the feed solution obtained in step (4) is kept at a temperature of 2-10° C., a composite cation exchange filler is added for chromatography, and then added to a sodium chloride solution for leaching, and the leaching solution is collected by filtration, wherein the concentration of the sodium chloride solution is 25-35 g / L;

[0013] (6) The extract treated in step (5) is ultrafiltered and then freeze-dried to obtain recombinant type I mussel mucin freeze-dried powder.

[0014] The amino acid sequence of the recombinant type I mussel mucin is shown in SEQ.ID.NO.1.

[0015] Preferably, in step (1), in the composition, the mass ratio of Triton X-100 to disodium ethylenediaminetetraacetate is 1000:(4-6); the content of the composition in the fermentation broth is 10-15 g / L; and the heating is performed to 65-75°C.

[0016] Preferably, in step (2), the slurry treated in step (1) is first cooled to 35-40°C, and then acid-dissolved with an acid, wherein the acid is citric acid or acetic acid, and the amount of the acid added to the slurry is 50-55 g / L; the filtration is performed using a filter membrane with a pore size of 0.45 μm. In some specific embodiments, the specific operation of the acid-dissolving is to stir until completely dissolved and then continue stirring for not less than 1 hour.

[0017] Preferably, in step (3), the ultrafiltration is specifically ultrafiltration until the conductivity of the permeate is less than 0.5 mS / cm, and the concentration multiple is 10-11 times. More preferably, in step (3), the step further includes collecting the concentrated solution and performing an enzymatic reaction.

[0018] Preferably, in step (4), the dissolution and mixing time is not less than 1 hour.

[0019] Preferably, in step (5), the adding of the composite cation exchange filler for chromatography comprises, specifically, adding the composite cation exchange filler and mixing thoroughly for not less than 1 hour; filtering and intercepting the composite cation exchange filler, and eluting the composite cation exchange filler with purified water of not less than 5 times the volume of the composite cation exchange filler; and in the extraction step, the amount of the sodium chloride solution used is not less than 3 times the volume of the composite cation exchange filler.

[0020] Preferably, in step (5), the composite cation exchange filler is MMC filler.

[0021] Preferably, the MMC filler is selected from any one of Capto MMC, MMC Bestarose FF, Diamond MMC, Agarosix MC90-MMC, MMC Focurose HP, and MMC HR.

[0022] Preferably, the ultrafiltration described in the present invention is performed using a rolled membrane. More preferably, the rolled membrane is a 3KDa rolled membrane.

[0023] The present invention also provides a recombinant type I mussel mucin, which is prepared by the above method; the purity of the recombinant type I mussel mucin is greater than 95%, the L-3,4-dihydroxyphenylalanine content is greater than 1.0%, and the endotoxin content is less than 5EU / mg.

[0024] In the present invention, by controlling the ratio between Triton X-100 and disodium ethylenediaminetetraacetate, the temperature required for sufficient disruption of Escherichia coli cells can be greatly reduced from the conventional 80°C to 65°C, which greatly reduces the difficulty of implementing the purification process and the requirements of the process on equipment, making it easy to operate and industrialize.

[0025] The present invention is different from the conventional process of using Triton X-114 to remove endotoxins. In conventional operations, Triton X-114 is first added under low temperature conditions to dissolve it, and then heated, and Triton X-114 is subjected to sedimentation separation treatment. However, this sedimentation centrifugal treatment method is difficult to achieve a thorough separation of Triton X-114 and endotoxins, which will not only cause a large loss of the target product during the separation process, but also a large amount of Triton X-114 will remain in the target product, resulting in poor yield and purity. Therefore, in this scheme, we first cool the feed liquid, add Triton X-114 under 2-10°C conditions, and fully stir and dissolve it under low temperature conditions. This step can ensure that Triton X-114 is fully combined with the endotoxin substance; continue to adjust the system pH under low temperature conditions and then perform chromatography through the composite cation exchange filler, so that Triton X-114 fully encapsulates the endotoxin substance and the endotoxin substance does not bind to the filler during the chromatography process, thereby achieving flow-through removal, and the target product is combined with the chromatography filler and eluted. The present application adopts a Triton X-114 combined chromatography process at low temperature, which can effectively remove endotoxins and separate and purify at the same time in one step, thereby achieving the purpose of removing endotoxins and separating and purifying the target protein.

[0026] The invention provides a method for purifying recombinant type I mussel mucin and removing endotoxins. The method adopts a fermentation liquid of recombinant type I mussel mucin expressed by fermentation induction of Escherichia coli engineering bacteria as a raw material, and greatly improves the purification efficiency and reduces the endotoxin content by combining Triton X-100 and disodium ethylenediaminetetraacetic acid and controlling the cooling temperature of the feed liquid, and optimizing the endotoxin removal process and addition amount of Triton X-114, thereby providing a method for purifying recombinant type I mussel mucin with a purity greater than 95%, an L-3,4-dihydroxyphenylalanine (L-DOPA) content greater than 1.0% and an endotoxin content less than 5EU / mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0028] Figure 1 This is the purity result of the recombinant type I mussel mucin freeze-dried powder detected by high performance liquid chromatography in Example 2.

[0029] Figure 2SDS-PAGE electrophoresis detection diagram of 2 mg / mL samples prepared from the recombinant type I mussel mucin lyophilized powder obtained in the examples and some comparative examples: wherein, lane 1: 5 μl of recombinant type I mussel mucin obtained in Example 2; lane 2: 5 μl of recombinant type I mussel mucin obtained in comparative example 14; lane 3: 5 μl of recombinant type I mussel mucin obtained in comparative example 9; lane 4: protein marker. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The fermentation broth preparation method in the following examples / comparative examples is as follows:

[0032] The fermentation broth is prepared by fermentation expression of Escherichia coli genetic engineering bacteria capable of expressing recombinant type I mussel mucin. The genetic engineering bacteria is constructed using Escherichia coli BL21 (DE3) strain as a host bacterium. The amino acid sequence of the expressed recombinant type I mussel mucin is shown in SEQ.ID.NO.1 (reference type I mussel mucin GenBank: AAX23968.1, peptide segments are selected to be combined into sequence units with a length of 10aa and then repeated in series, i.e., recombinant type I mussel mucin, with a theoretical molecular weight of 22.68KDa and a theoretical isoelectric point of 9.98). According to the designed amino acid sequence, the codons are optimized in combination with the Escherichia coli expression system, and the optimized sequence is synthesized, the structure of which is to add a ccatgg NcoI restriction site at the 5' end of the gene sequence, and to add a stop codon and aagctt Hind III restriction site at the 3' end. The designed nucleotide sequence (i.e., SEQ.ID.NO.2) is subjected to full gene synthesis to obtain a synthetic sequence fragment containing the target gene. The synthesized sequence was digested with NcoI / Hind III restriction endonucleases, the target fragment was recovered, and ligated to the corresponding site of the pET28a expression vector that was also digested with NcoI / Hind III restriction endonucleases to obtain a recombinant plasmid. Positive transformants were picked, and the recombinant plasmid was introduced into the host bacteria BL21 (DE3) after enzyme digestion identification and sequencing verification. The transformed colonies were then screened using LB solid plates containing kanamycin (50 μg / mL) to obtain single colonies of Escherichia coli engineered bacteria.

[0033] Pick a single colony of Escherichia coli engineering bacteria and inoculate it into a 50ml LB medium test tube (with kanamycin), and culture it at 37°C overnight. The overnight cultured seed liquid is transferred to 100ml LB liquid medium at a 1% inoculation amount, and cultured at 37°C, 200rpm until the OD value is about 2-3, and then transferred to a fermenter (LB medium) at a 2-5% inoculation amount, the temperature is 37°C, the pH is controlled at 6.8, the pressure is 0.05MP, the dissolved oxygen is adjusted to ≥30% by the rotation speed and ventilation volume, and the nutrition is maintained by adding carbon source / nitrogen source / inorganic salt. When the wet weight is ≥50g / L, lactose with a final concentration of 10mM (mmo l / L) is added for induction, and the fermentation liquid is obtained after induction for 6h.

[0034] The recombinant type I mussel mucin freeze-dried powder obtained in the embodiments of the present invention and the comparative examples adopt the following purity detection method: refer to the high performance liquid chromatography method in General Chapter 0512 of the "Pharmacopoeia of the People's Republic of China" (Volume 4, 2020) for detection.

[0035] The recombinant type I mussel mucin freeze-dried powder obtained in the embodiments of the present invention and the comparative examples all adopt the following bacterial endotoxin detection method: refer to the general rule 1143 Bacterial Endotoxin Inspection Method 1 Gel Method of the "Pharmacopoeia of the People's Republic of China" (Volume 4 of the 2020 edition) for bacterial endotoxin detection.

[0036] The method for detecting the content of L-3,4-dihydroxyphenylalanine (L-DOPA) used in the recombinant type I mussel mucin freeze-dried powder obtained in the embodiments and comparative examples of the present invention is: referring to the method in Appendix B of YY / T 1293.6-2020 Contact Wound Dressings Part 6: Mussel Mucin Dressings.

[0037] Example 1

[0038] 1) Add 6 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 800 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, heated to 69° C., and stirred for 1 hour;

[0039] 2) The feed liquid was cooled to 37°C, 4000g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0040] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.46 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0041] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 6°C, 108 g of Triton X-114 was added, stirred for 60 min, the pH was adjusted to 6.6 with sodium hydroxide, Capto MMC filler was added and stirred for 60 min, the filler was filtered through a mesh and retained, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected through a mesh;

[0042] 5) The extract was transferred to a 3KDa spiral membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 95uS / cm. The feed liquid was continued to be concentrated, and 5.7L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.4L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 72.5g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0043] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.929g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.71%; after bacterial endotoxin detection, its endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0044] Example 2

[0045] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, heated to 65° C., and stirred for 1 hour;

[0046] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0047] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0048] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0049] 5) The extract was transferred to a 3KDa spiral membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 72.8g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0050] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.933g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.89%; after bacterial endotoxin detection, its endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0051] The purity of the recombinant type I mussel mucin freeze-dried powder obtained in Example 2 was determined by high performance liquid chromatography. Figure 1 As shown, the target protein showed a single peak after liquid phase detection, with a peak time of 14.183 and a purity of 97.89% (relative peak area). (This indicates that the liquid phase purity of the recombinant type I mussel mucin prepared by the method of the present invention can reach more than 97%).

[0052] Example 3

[0053] 1) Add 5 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 1100 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 75° C., and stirred for 1 hour;

[0054] 2) The feed liquid was cooled to 39°C, 4275g of acetic acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0055] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.48 mS / cm. The feed liquid was continued to be concentrated, and 5.2 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.5 L of the recovered liquid was collected, and a total of 9.7 L of ultrafiltration concentrate was collected; 14.5 g of disodium hydrogen phosphate dodecahydrate, 15.5 g of sodium dihydrogen phosphate dihydrate, 0.385 g of copper sulfate, and 0.19 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.13 with 2 molar sodium hydroxide, and stirred for 3.5 h for enzymatic reaction;

[0056] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 10°C, 142g of Triton X-114 was added, stirred for 62min, the pH was adjusted to 6.9 with sodium hydroxide, Diamond MMC filler was added and stirred for 60min, the filler was intercepted by sieve filtration, the filler was rinsed with 11L of purified water, the filler was added to 6L of purified water containing 35g / L sodium chloride and leached for 32min, and 5.8L of the leaching solution was collected by sieve filtration;

[0057] 5) The extract was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 94uS / cm. The feed liquid was continued to be concentrated, and 5.7L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.2L of ultrafiltration concentrate was collected, and 71.9g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0058] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.922g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.31%; the purity of the freeze-dried powder sample is 97.68%; after bacterial endotoxin detection, its endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0059] Comparative Example 1

[0060] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 650 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0061] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0062] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0063] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0064] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 66.2g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0065] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.849g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.31%; after bacterial endotoxin detection, the endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0066] It can be seen that the amount of the composition of Triton X-100 and disodium ethylenediaminetetraacetic acid added in step 1 of the purification method of Comparative Example 1 is too low, the Escherichia coli breakage rate is low, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0067] Comparative Example 2

[0068] 1) Add 2 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0069] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0070] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43mS / cm. The feed liquid was continued to be concentrated, and 6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.6L of the recovered liquid was collected, and a total of 10.6L of ultrafiltration concentrate was collected; 12.8g of disodium hydrogen phosphate dodecahydrate, 12g of sodium dihydrogen phosphate hydrate, 0.319g of copper sulfate, and 0.13g of tyrosinase with an indicated activity of 25EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3h for enzymatic reaction;

[0071] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0072] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 67.3g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0073] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.863g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.41%; after bacterial endotoxin detection, its endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0074] It can be seen that when preparing the composition of Triton X-100 and disodium ethylenediaminetetraacetate in step 1 of the purification method of Comparative Example 2, the amount of disodium ethylenediaminetetraacetate added is too low, the Escherichia coli breakage rate is low, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0075] Comparative Example 3

[0076] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 61° C., and stirred for 1 hour;

[0077] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0078] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0079] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0080] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 63.8g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0081] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.818g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.89%; after bacterial endotoxin detection, the endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0082] It can be seen that the heating temperature in step 1 of the purification method in comparative example 3 is too low, the cell disruption is insufficient, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0083] Comparative Example 4

[0084] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0085] 2) The feed liquid was cooled to 28°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0086] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0087] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0088] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 65.3g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0089] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.837g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.1%; the purity of the freeze-dried powder sample is 97.36%; after bacterial endotoxin detection, its endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0090] It can be seen that the cooling temperature in step 2 of the purification method in Comparative Example 4 is too low, the acid dissolution efficiency is low, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0091] Comparative Example 5

[0092] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 66° C., and stirred for 1 hour;

[0093] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0094] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 9 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 13.6 L of ultrafiltration concentrate was collected; 16.4 g of disodium hydrogen phosphate dodecahydrate, 15.4 g of sodium dihydrogen phosphate dihydrate, 0.410 g of copper sulfate, and 0.17 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0095] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0096] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 71.7g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0097] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.919g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 0.7%; the purity of the freeze-dried powder sample is 97.07%; after bacterial endotoxin detection, the endotoxin limit is less than 5EU / mg, and the test result is qualified.

[0098] It can be seen that the concentration multiple of the concentration process in step 3 in the purification method of comparative example 5 is insufficient, resulting in low efficiency of the subsequent enzymatic reaction, and the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced.

[0099] Comparative Example 6

[0100] 1) 4 g of disodium ethylenediaminetetraacetate was added to 1000 g of Triton X-100, and the mixture was weighed and stirred to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter was completed, the volume of the fermentation liquid was 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate was added, and the mixture was heated to 65° C. and stirred for 1 hour.

[0101] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0102] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0103] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 90 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0104] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 72.4g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0105] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.928g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.3%; the purity of the freeze-dried powder sample is 97.69%; after bacterial endotoxin detection, the endotoxin content limit is greater than 20EU / mg and less than 50EU / mg, and the test result is unqualified.

[0106] It can be seen that the amount of Triton X-114 added in step 4 of the purification method of Comparative Example 6 is too low, resulting in insufficient efficiency in removing endotoxin substances, and the endotoxin content of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly increased.

[0107] Comparative Example 7

[0108] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0109] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0110] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0111] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 15° C., 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0112] 5) The extract was transferred to a 3KDa spiral membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 70.9g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0113] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.909g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.1%; the purity of the freeze-dried powder sample is 97.32%; after bacterial endotoxin detection, the endotoxin content limit is greater than 10EU / mg and less than 20EU / mg, and the test result is unqualified.

[0114] It can be seen that the cooling temperature in step 4 of the purification method of Comparative Example 7 is insufficient, resulting in insufficient binding of Triton X-114 with endotoxin, thereby causing insufficient removal of endotoxin substances, and the endotoxin content of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly increased.

[0115] Comparative Example 8

[0116] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0117] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0118] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected; 5 L of purified water was used to top wash the residue in the recovery system, and 4.6 L of the recovered liquid was collected. A total of 10.6 L of ultrafiltration concentrate was collected, and 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0119] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 190 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0120] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 67.9g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0121] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.871g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.44%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0122] It can be seen that the amount of Triton X-114 added in step 4 of the purification method of Comparative Example 8 is too high, the target protein is not fully loaded on the column, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0123] Comparative Example 9

[0124] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0125] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0126] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0127] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 5.0 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0128] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 74.6g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0129] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.956g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 93.26%; after endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0130] It can be seen that the pH of the feed solution in step 4 of the purification method of Comparative Example 9 is too low, resulting in contamination by impurity proteins, and the purity of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced.

[0131] Comparative Example 10

[0132] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0133] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0134] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0135] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 7.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0136] 5) The extract was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 75.3g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0137] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.965g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 94.12%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0138] It can be seen that the pH of the feed solution in step 4 of the purification method of Comparative Example 10 is too high, resulting in contamination by impurity proteins, and the purity of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced.

[0139] Comparative Example 11

[0140] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0141] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0142] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0143] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was filtered through a mesh and retained, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 15 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected through a mesh;

[0144] 5) The extract was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 69.1g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0145] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.886g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 97.75%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0146] It can be seen that the sodium chloride content in the washing liquid in step 4 of the purification method of Comparative Example 11 is too low, the target protein is not fully extracted, the amount of recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth is significantly reduced.

[0147] Comparative Example 12

[0148] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0149] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0150] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0151] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 45 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0152] 5) The extract was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 75.2g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0153] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.964g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 94.12%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0154] It can be seen that the sodium chloride content in the washing liquid of step 4 in the purification method of Comparative Example 12 is too high, some impurities firmly bound to the chromatographic filler are eluted, and the purity of the recombinant type I mussel mucin freeze-dried powder finally collected is significantly reduced.

[0155] Comparative Example 13

[0156] 1) After the fermentation in the fermenter, the volume of the fermentation liquid was 78L, 780g Triton X-100 was added, heated to 66°C, and stirred for 1h;

[0157] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0158] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0159] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0160] 5) The extract was transferred to a 3KDa spiral membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 61.3g of recombinant type I mussel mucin freeze-dried powder was collected after freeze-drying.

[0161] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.786g / L; after measurement, the L-3,4-dihydroxyphenylalanine (L-DOPA) content of the freeze-dried powder sample is 1.1%; the purity of the freeze-dried powder sample is 97.23%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0162] It can be seen that in the purification method of Comparative Example 13, only Triton X-100 was added during the bacterial disruption in step 1, the Escherichia coli disruption rate was low, the amount of recombinant type I mussel mucin freeze-dried powder finally collected was significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid was significantly reduced.

[0163] Comparative Example 14

[0164] 1) Add 4 g of disodium ethylenediaminetetraacetate to 1000 g of Triton X-100 and stir thoroughly to prepare a composition of Triton X-100 and disodium ethylenediaminetetraacetate for later use. After the fermentation in the fermenter is completed, the volume of the fermentation liquid is 78 L, 780 g of the composition of Triton X-100 and disodium ethylenediaminetetraacetate is added, the mixture is heated to 65° C., and stirred for 1 hour;

[0165] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0166] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0167] 4) After the enzymatic reaction, the feed liquid was cooled to 3°C, 108g Triton X-114 was added, stirred for 62min, heated to 37°C and maintained for 30min, the supernatant was collected by centrifugation, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added, stirred at 4°C for 60min, the filler was retained by sieve filtration, the filler was rinsed with 11L purified water, the filler was added to 6L purified water containing 25g / L sodium chloride and leached for 32min, and 5.8L of the leaching solution was collected by sieve filtration;

[0168] 5) The extract was transferred to a 3KDa rolled membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 63.2g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0169] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.810g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.2%; the purity of the freeze-dried powder sample is 94.26%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0170] It can be seen that the Triton X-114 in step 4 of the purification method of Comparative Example 14 was removed by conventional heated centrifugation, and direct chromatography under low temperature conditions was not used, resulting in serious protein loss in the process, and the impurity separation effect was poor during the chromatography of the heated liquid. The purity of the recombinant type I mussel mucin freeze-dried powder finally collected was reduced, and the yield of the recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth was significantly reduced.

[0171] Comparative Example 15

[0172] 1) After the fermentation in the fermenter is completed, add 4 g of disodium ethylenediaminetetraacetate to the fermentation liquid volume of 78 L, heat to 65 ° C, and stir for 1 hour;

[0173] 2) The feed liquid was cooled to 36°C, 3900g of citric acid was added, and stirring was continued for 1h after dissolving. The feed liquid was discharged into the liquid storage tank of the 0.45μm hollow fiber microfiltration membrane system, and filtration was started to collect the filtrate. During the process, 20L of purified water was added twice to wash the residue, and a total of 107L of filtrate was collected;

[0174] 3) The filtrate was transferred to the liquid storage tank of the 3KDa spiral membrane ultrafiltration system, and the ultrafiltration was started. Purified water was used as the dialyzate, and the permeate conductivity was ultrafiltered to 0.43 mS / cm. The feed liquid was continued to be concentrated, and 6 L of the concentrate was collected. The residue in the recovery system was top washed with 5 L of purified water, and 4.6 L of the recovered liquid was collected, and a total of 10.6 L of ultrafiltration concentrate was collected; 12.8 g of disodium hydrogen phosphate dodecahydrate, 12 g of sodium dihydrogen phosphate dihydrate, 0.319 g of copper sulfate, and 0.13 g of tyrosinase with an indicated activity of 25 EU / mg were added, and the pH was adjusted to 7.01 with 2 molar sodium hydroxide, and stirred for 3 h for enzymatic reaction;

[0175] 4) After the enzymatic reaction, the feed solution was cooled and maintained at 3°C, 108 g of Triton X-114 was added, stirred for 62 min, the pH was adjusted to 6.6 with sodium hydroxide, MMC Bestarose FF filler was added and stirred for 60 min, the filler was intercepted by sieve filtration, the filler was rinsed with 11 L of purified water, the filler was added to 6 L of purified water containing 25 g / L sodium chloride and leached for 32 min, and 5.8 L of the leaching solution was collected by sieve filtration;

[0176] 5) The extract was transferred to a 3KDa spiral membrane ultrafiltration system storage tank, and ultrafiltration was started. Purified water was used as the dialyzate, and the ultrafiltration was performed until the conductivity of the permeate was 92uS / cm. The feed liquid was continued to be concentrated, and 5.6L of the concentrate was collected. The residue in the recovery system was top washed with 5L of purified water, and 4.5L of the recovered liquid was collected. A total of 10.1L of ultrafiltration concentrate was collected, and 27.5g of recombinant type I mussel mucin freeze-dried powder was collected after freeze drying.

[0177] According to the above purification method, the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation liquid is 0.353g / L; after measurement, the content of L-3,4-dihydroxyphenylalanine (L-DOPA) in the freeze-dried powder sample is 1.1%; the purity of the freeze-dried powder sample is 97.25%; after bacterial endotoxin detection, the endotoxin content limit is less than 5EU / mg, and the test result is qualified.

[0178] It can be seen that in the purification method of Comparative Example 15, only disodium ethylenediaminetetraacetic acid was added during the bacterial disruption in step 1, and the Escherichia coli could not be fully disrupted at 65°C. The amount of recombinant type I mussel mucin freeze-dried powder finally collected was significantly reduced, and the yield of recombinant type I mussel mucin freeze-dried powder per liter of fermentation broth was significantly reduced.

[0179] The SDS-PAGE electrophoresis test of the 2 mg / mL sample of the recombinant type I mussel mucin freeze-dried powder obtained in the examples and some comparative examples is as follows Figure 2 As shown; wherein, lane 1: 5 μl of recombinant type I mussel mucin obtained in Example 2; lane 2: 5 μl of recombinant type I mussel mucin obtained in Comparative Example 14; lane 3: 5 μl of recombinant type I mussel mucin obtained in Comparative Example 9; lane 4: protein marker. (The electrophoretic molecular weight of recombinant type I mussel mucin is 31 KDa. Compared with the theoretical molecular weight, the electrophoretic characterization error leads to a higher molecular weight in the electrophoretic diagram). Compared with the high-purity recombinant type I mussel mucin prepared in Example 2, the electrophoretic bands of the recombinant type I mussel mucin prepared in Comparative Examples 14 and 9 both show residues of impurity bands above and below the target protein band, indicating that the purified proteins in Comparative Examples 14 and 9 are of lower purity.

[0180] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for purifying recombinant type I mussel mucin and removing endotoxin, characterized in that: The following steps are involved: (1) adding a combination of Triton X-100 and disodium ethylenediaminetetraacetate to a fermentation broth of recombinant type I mussel mucin expressed by fermentation with engineered Escherichia coli bacteria and heating and mixing; (2) dissolving the feed liquid treated in step (1) with acid and filtering it to collect the filtrate; (3) ultrafiltering the filtrate after treatment in step (2), concentrating and collecting the concentrate; (4) cooling the solution treated in step (3) and maintaining it at 2-10° C., adding Triton X-114, dissolving and mixing, and then adjusting the pH of the solution to 6.5-7.0, wherein the concentration of Triton X-114 in the solution is 10-15 g / L; (5) the feed solution obtained in step (4) is kept at a temperature of 2-10° C., a composite cation exchange filler is added for chromatography, and then added to a sodium chloride solution for leaching, and the leaching solution is collected by filtration, wherein the concentration of the sodium chloride solution is 25-35 g / L; (6) The extract treated in step (5) is ultrafiltered and then freeze-dried to obtain recombinant type I mussel mucin freeze-dried powder.

2. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: The amino acid sequence of the recombinant type I mussel mucin is shown in SEQ.ID.NO.

1.

3. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: In step (1), in the composition, the mass ratio of Triton X-100 to disodium ethylenediaminetetraacetate is 1000:(4-6); the content of the composition in the fermentation broth is 10-15 g / L; and the heating is performed to 65-75°C.

4. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 3, characterized in that: In step (2), the liquid treated in step (1) is first cooled to 35-40°C.

5. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: In step (3), the ultrafiltration is specifically ultrafiltration until the conductivity of the permeate is less than 0.5 mS / cm, and the concentration multiple is 10-11 times.

6. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: In step (4), the dissolution and mixing time is not less than 1 hour.

7. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: In step (5), the adding of the composite cation exchange filler for chromatography is specifically, adding the composite cation exchange filler and mixing it thoroughly for not less than 1 hour; filtering and intercepting the composite cation exchange filler, and eluting the composite cation exchange filler with purified water of not less than 5 times the volume of the composite cation exchange filler; in the leaching step, the amount of the sodium chloride solution is not less than 3 times the volume of the composite cation exchange filler.

8. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: In step (5), the composite cation exchange filler is MMC filler.

9. The method for purifying and removing endotoxin from recombinant type I mussel mucin according to claim 1, characterized in that: The ultrafiltration is carried out by using a rolled membrane.

10. A recombinant type I mussel mucin, characterized in that: The recombinant type I mussel mucin is prepared by the method described in claims 1-9; the purity of the recombinant type I mussel mucin is greater than 95% and the endotoxin content is less than 5EU / mg.

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