A recombinant chondroitin sulfate exonuclease mutant, recombinant bacteria and application thereof

Abstract

The present invention relates to a recombinant chondroitin sulfate exonuclease mutant, a recombinant bacterium and an application thereof, and belongs to the field of enzyme engineering. The amino acid sequence of the mutant is shown in SEQ ID NO.1. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 is shown in SEQ ID NO.2. The SEQ ID NO.2 is translated into a nucleotide sequence encoding the original amino acid sequence of SEQ ID NO.3, and a gene fragment of the nucleotide sequence is obtained by PCR amplification and sequencing, and a mutant is obtained by performing an error-prone PCR reaction on the gene fragment. The present invention also provides a recombinant expression vector and a recombinant bacterium containing the mutant gene. The mutant, the recombinant expression vector and the recombinant bacterium can prepare oligoosteogenin using bone as a raw material. The oligoosteogenin can significantly increase the proliferation rate of chondrocytes, promote the proliferation and regeneration of chondrocytes, repair cartilage damage in rats with osteoarthritis, and has obvious effects of promoting chondrocyte proliferation and improving osteoarthritis.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and specifically relates to a recombinant chondroitin sulfate exonuclease mutant, a recombinant bacterium and applications thereof. Background Art

[0002] Chondroitin sulfate is a natural bioactive macromolecule belonging to the glycosaminoglycan class. Chondroitin sulfate has many biological functions, such as anti-arthritis, anti-oxidation, wound healing, anti-coagulation and thrombosis, cancer cell inhibition, intestinal flora regulation, etc. Due to the structural characteristics of chondroitin sulfate such as large molecular weight, many hydrophilic groups, and poor fat solubility, its absorption in the body is incomplete and its bioavailability is low (less than 13%). At present, the difficulty in the application of chondroitin sulfate resources is no longer in extraction, but in reducing the molecular weight to solve the problems of poor absorption and low bioavailability.

[0003] The degradation methods of high molecular weight chondroitin sulfate mainly include enzymatic degradation, oxidative degradation and acid degradation. The principle of acid degradation is that polysaccharides are broken in organic or inorganic acids to obtain relatively low molecular weight polysaccharides by destroying glycosidic bonds. This method usually leads to the shedding of sulfate groups, destroying the main structure and active ingredients of the product after chondroitin sulfate degradation, thereby limiting the application of this method. The principle of oxidative degradation is a method of obtaining low molecular weight chondroitin sulfate by attacking glycosidic bonds with oxygen free radicals produced by H2O2 or catalyzing H2O2. This method has low yield, complex process and is difficult to control, which limits the application of this method. The principle of enzymatic degradation is to act on the glycosidic bonds between disaccharide repeating units through the elimination mechanism and cut them off, and the enzymatic hydrolysis product is oligosaccharide, mainly unsaturated disaccharides. Compared with other degradation methods, enzymatic degradation reaction conditions are mild, the reaction process is controllable and highly specific. Degrading chondroitin sulfate by the recombinant chondroitin sulfate exoenzyme is an effective method for obtaining low molecular weight chondroitin sulfate.

[0004] The chondroitin sulfate enzymes reported so far generally have the problem of low enzyme activity. During industrial production, a large amount of enzyme is added, and the production is complicated. Therefore, chondroitin sulfate exoenase with high enzyme activity is a prerequisite for the industrial preparation of low molecular weight chondroitin sulfate. In addition, the use of microbial host fermentation to prepare recombinant chondroitin sulfate exoenase is an effective method to reduce production costs and obtain high-efficiency recombinant chondroitin sulfate exoenase. Summary of the invention

[0005] The present invention aims to solve the above technical problems and provides a recombinant chondroitin sulfate exonuclease mutant, a recombinant bacterium and applications thereof. The recombinant chondroitin sulfate exonuclease mutant has improved enzyme activity and can meet industrial application requirements.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A recombinant chondroitin sulfate exohydrolase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.1; specifically:

[0008] MTHEVSRRHILQGTAALTFAGLLTAGFAPLAQAAEAATPANLDALAERWVDQITGRLLIDPADPDFGAAIASQDRAVDKSVSLLAPRPLKMGVFTDAPFSSEAQMVTSYGRLAQMAAAWATPGSKHQGSPVLLDQILGALEDGNTYIYNDGQAEYGNNWSWEIGTSKAATDTLAILGSNVSPDLIAKYCAAIDHFIPDPTKQFPDSRGKILSEGANRVDICQAIIVRSIVGGDTARLAAAISALSPVHQYVTSGNGFYADGSFVQHTTIPYTGTYGVVLLGGLAKLFSLLGGSDHAVSDPSRTILFNTVEDSFAPFLHDGLMMDSVRGRAISRTQERGFDDGTITIEAILWLARAVDATTGNRMRALCKGWVARNQYSNPLAGASIPRTALLKELAASATDAAAETAGHSFFPGHDRSVYRGKGWAAALGLSSRRTTWYECGNGENNLGAQTGSGMTYLYAGNQGHFDDDFWPTANLSRLPGITVDTTPLPPKVEGENGAATPHNEWTGGVTLNGTGAVGMHLVGPGGTGLQARKAWFYTQDMVVALGADIHTASGAAVESIMEHRNLGADGGQAMTVDGRPHTAAAGTPVSYGHPRWAHLEGTGGYLLLGEGDLTVLREQRTGAKKDINTGGTPAVVSRQYATLLFEHGTDPASYAYAILPGASAMDTAKAAGKSAPRVLRNDKTGQGLELDKKTTAALFWAPGTVGNLTADGPACVLFNGNPGHGVLAVVDPTQTATSVTVTIRDARYIRISVTVGATLLVDRDGNVTITIPTAGLLGATVEVTLHR。

[0009] The nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.1 is shown in SEQ ID NO.2, and SEQ ID NO.2 is translated into the encoding nucleotide sequence based on the chondroitin sulfate exonuclease of the original amino acid sequence SEQ ID NO.3, and the gene fragment of the nucleotide sequence is obtained by PCR amplification and sequencing, and the mutant is obtained by performing an error-prone PCR reaction on the gene fragment.

[0010]

[0011] The amino acid sequence of the original chondroitin sulfate exonuclease (SEQ ID NO.3):

[0012] .

[0013] The present invention also provides a recombinant expression vector of chondroitin sulfate exonuclease, wherein the recombinant expression vector comprises the recombinant chondroitin sulfate exonuclease gene, and the vector is Pichia pastoris.

[0014] The present invention also provides a recombinant bacterium of chondroitin sulfate exonuclease, wherein the recombinant bacterium comprises the recombinant chondroitin sulfate exonuclease gene, and the strain is Pichia pastoris X33.

[0015] The present invention also provides an enzyme preparation, which comprises the mutant of the chondroitin sulfate exoenase and / or the recombinant bacteria of the chondroitin sulfate exoenase.

[0016] The present invention also provides the application of the mutant, recombinant expression vector and recombinant bacteria of the chondroitin sulfate exonuclease, wherein the application is to prepare oligoosteogenin by using the mutant, recombinant expression vector and / or recombinant bacteria.

[0017] Preferably, the application uses bone as raw material.

[0018] The present invention also provides the use of the oligoosteogen prepared by the method in preparing drugs for repairing osteoarthritis or bone damage.

[0019] The beneficial effects of the present invention compared with the prior art are as follows:

[0020] 1. The present invention obtains a recombinant chondroitin sulfate exonuclease by an error-prone PCR method, and constructs a recombinant expression vector mutation library thereof in Pichia pastoris.

[0021] 2. The present invention heterologously expresses the recombinant chondroitin sulfate exonuclease in Pichia pastoris X33 to obtain the expression product of the recombinant chondroitin sulfate exonuclease, and has a method for preparing the recombinant chondroitin sulfate exonuclease in Pichia pastoris X33.

[0022] 3. The present invention characterizes the enzyme activity level of the expression product of recombinant chondroitin sulfate exonuclease. The enzyme activity of the recombinant chondroitin sulfate exonuclease is significantly improved compared with the original sequence chondroitin sulfate exonuclease.

[0023] 4. The recombinant chondroitin sulfate exoenzyme of the present invention can be used to prepare oligomeric chondroitin products. Compared with the original chondroitin sulfate exoenzyme, the recombinant chondroitin sulfate exoenzyme of the present invention has a comparable effect of preparing low molecular weight chondroitin sulfate with an enzyme dosage of 8-15 U / g for substrate and about one-thirtieth of the original chondroitin sulfate exoenzyme under the same enzymolysis conditions. At the same time, the molecular weight distribution of low molecular weight chondroitin sulfate in the oligomeric chondroitin obtained by this enzymolysis technology is more concentrated.

[0024] 5. The oligomeric chondroitin product prepared by using recombinant chondroitin sulfate exoenzyme described in the present invention can significantly increase the proliferation rate of chondrocytes, promote the proliferation and regeneration of chondrocytes; can repair cartilage damage in osteoarthritis rats, and has obvious effects of promoting chondrocyte proliferation and improving osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of nucleic acid electrophoresis for gene amplification of recombinant chondroitin sulfate exonuclease;

[0026] Figure 2 Schematic diagram of the recombinant chondroitin sulfate exoenzyme and the enzymatic activity of chondroitin sulfate exoenzyme;

[0027] Figure 3A is the molecular weight distribution of chondroitin sulfate in the enzymatic hydrolysis product of complex enzyme A;

[0028] Figure 3B is the molecular weight distribution of chondroitin sulfate in the enzymatic hydrolysis product of complex enzyme B;

[0029] Figure 3C is the molecular weight distribution of chondroitin sulfate in the enzymatic hydrolysis product of compound enzyme C;

[0030] Figure 4A The molecular weight distribution of chondroitin sulfate in the oligomeric chondroitin product prepared by using recombinant chondroitin sulfate exoenzyme;

[0031] Figure 4B This is a Fourier infrared scan of the oligomeric chondroitin product prepared using recombinant chondroitin sulfate exoenzyme;

[0032] Figure 4C The circular dichroism spectrum of the oligomeric chondroitin product prepared by using recombinant chondroitin sulfate exoenzyme;

[0033] Figure 5A Schematic diagram of the effect of chicken low molecular weight chondroitin sulfate prepared by recombinant chondroitin sulfate exoenzyme on chondrocyte proliferation;

[0034] Figure 5B Schematic diagram of the effect of chicken chondroitin sulfate prepared by recombinant chondroitin sulfate exoenzyme on chondrocyte proliferation;

[0035] Figure 5C Schematic diagram of the effect of chicken oligosaccharide product prepared by recombinant chondroitin sulfate exoenzyme on chondrocyte proliferation;

[0036] Fig. 6A Schematic diagram of the effect of oligochondroitin products prepared by recombinant chondroitin sulfate exoenzyme on the inflammatory factor IL-1β in the serum of osteoarthritis rats;

[0037] Figure 6B Schematic diagram of the effect of the oligochondroitin product prepared by recombinant chondroitin sulfate exoenzyme on the inflammatory factor IL-6 in the serum of rats with osteoarthritis;

[0038] Figure 6C Schematic diagram of the effect of oligochondroitin products prepared by recombinant chondroitin sulfate exoenzyme on inflammatory factor TNF-α in the serum of rats with osteoarthritis;

[0039] Fig. 7A Schematic diagram of the effect of oligochondroitin products prepared by recombinant chondroitin sulfate exoenzyme on HE staining of cartilage structure in arthritic rats;

[0040] Figure 7B Schematic diagram of the effect of oligochondroitin products prepared by recombinant chondroitin sulfate exoenzyme on Alcian blue staining of chondrocytes in arthritic rats. DETAILED DESCRIPTION

[0041] 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 only 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.

[0042] Example 1 Error-prone PCR of recombinant chondroitin sulfate exonuclease.

[0043] The genome sequence of Arthrobacter sp. CS01 was obtained from GeneBank (accession no. MK459466.1); the coding gene of chondroitin sulfate exonuclease, i.e., the open reading frame: ORF, orf00115_1, was searched from the genome; the gene sequence of chondroitin sulfate exonuclease published in orf00115_1 was used as the original sequence, and the amino acid sequence formed by its translation was used as the original amino acid sequence. The original chondroitin sulfate exonuclease target gene fragment was obtained by PCR amplification from the genome of Arthrobacter sp. CS01. The gene fragment was used as a template for an error-prone PCR reaction. The DNA amplification enzyme used in the reaction was Super-Fidelity DNA Polymerase, the specific reaction system is: Buffer 25μL; dNTP 1μL; Phanta 0.5μL; front primer 2μL; rear primer 2μL; amplification template 1μL; ddH2O replenishes the system to 50μL. The annealing temperature in the reaction conditions is set to 58℃, and the extension time is 72℃ for 4min. The error-prone PCR reaction product is tested by agarose nucleic acid gel electrophoresis. The test results are as follows Figure 1 As shown, the DNA amplification band with the correct molecular weight is about 2370bp. The tested PCR product is further digested with DpnI enzyme and purified by Cycle Pure Kit PCR Purification Kit. The DNA fragment recovered after purification is the error-prone PCR product of chondroitin sulfate exonuclease.

[0044] Example 2 The error-prone PCR fragment was inserted into the Pichia pastoris X33 expression vector to construct a recombinant expression vector.

[0045] The inverse PCR method (F: 5'-GATGACTATCAAAGTAGGTATCAAC-3'; R: 5'-TTTGGAGATGTGAGCGATCA-3') was used to linearize the Pichia pastoris X33 expression vector pPICZαA. The inverse PCR amplification enzyme used was Super-Fidelity DNA Polymerase, the specific reaction system is shown in Example 1. The error-prone PCR product of chondroitin sulfate exonuclease and the original chondroitin sulfate exonuclease were respectively connected to the linearized expression vector by in vitro homologous recombination, and the connection system was: Exnase 1μL; Buffer 2μL; target gene 1μL; linearized vector 2μL; ddH2O supplemented the system to 10μL, and reacted at 37°C for 30min. The homologous recombination connection product was transferred into Escherichia coli DH5α by heat shock method, and single colonies were selected after culturing at 37°C for 12-16h. After the positive clone transformant was verified, the construction of the chondroitin sulfate exonuclease recombinant expression vector was completed.

[0046] Example 3 Expression of recombinant chondroitin sulfate exonuclease in Pichia pastoris.

[0047] The constructed recombinant expression vector was linearized by enzyme digestion using Sac I enzyme, and the linearized recombinant expression vector was transformed into Pichia pastoris X33 competent cells by electroporation. The specific transformation method was as follows: the linearized recombinant expression vector was transformed into competent cells, ice-bathed for 30 min, electroporated using an electroporator, 1 mL of pre-cooled 1 M sorbitol solution was added, and the cells were incubated in a 30°C incubator for 1 h, and the cells were spread on a YPD plate containing Zeocin antibiotics and cultured at 30°C for 2-3 days.

[0048] The grown yeast transformants were screened for positive transformants and inoculated into BMGY liquid medium for shake flask fermentation. The fermentation conditions were 30°C and 200rpm. 1% methanol was added every 24h for induction, and added 3 times in total. After the expression was completed, the bacteria were centrifuged and the supernatant was collected, which was the prepared chondroitin sulfate exonuclease. The enzyme activity of chondroitin sulfate exonuclease was detected in the fermentation supernatant, indicating that the recombinant chondroitin sulfate exonuclease was successfully expressed in Pichia pastoris X33.

[0049] Example 4 Determination of the enzymatic activity of recombinant chondroitin sulfate exoenzyme.

[0050] The activity of recombinant chondroitin sulfate exonuclease and original chondroitin sulfate exonuclease was determined by ultraviolet absorption method. The method for determining the activity of chondroitin sulfate exonuclease is as follows: after chondroitin sulfate is hydrolyzed, unsaturated disaccharides are produced, which have maximum light absorption at 232nm. 3.9mL 0.2% chondroitin sulfate solution and 4mL Tris-HCl buffer were added with 100μL enzyme solution, and the reaction was carried out at 37℃ for 5min. After the reaction, the OD was measured after boiling in water for 5min. 232 , add inactivated enzyme solution under the same conditions as reference. Parallel determination was performed three times, and enzyme activity was calculated according to the formula. 1 enzyme activity unit (U) is defined as the amount of enzyme required to degrade chondroitin sulfate to produce 1 μmol of unsaturated disaccharide per minute at 37°C.

[0051] Enzyme activity calculation formula: Enzyme activity

[0052] Where: ε refers to the millimolar extinction coefficient, 5.1, d refers to the thickness of the adaptive cuvette, 1 cm, t refers to the reaction time, min, A refers to the absorbance of the unsaturated disaccharide product at 232 nm, Vt refers to the total volume of the reaction, mL, and Vs refers to the volume of the sample, mL.

[0053] The results are as follows Figure 2 As shown in the figure, the enzymatic activity of the recombinant chondroitin sulfate exonuclease is significantly improved compared with the original chondroitin sulfate exonuclease, and the enzymatic activity is increased from 15.7U / ml to 536.8U / ml. This means that a recombinant chondroitin sulfate exonuclease with improved enzymatic activity has been obtained through the error-prone PCR technology, which has great application potential.

[0054] Example 5 Preparation of oligomeric chondroitin products using recombinant chondroitin sulfate exoenzyme.

[0055] The oligoosteoin products were prepared by using composite enzymatic hydrolysis technology, composite enzyme A: recombinant chondroitin sulfate exoenzyme, alkaline protease, papain; composite enzyme B: original chondroitin sulfate exoenzyme, alkaline protease, papain; composite enzyme C: alkaline protease, papain; the effects of different composite enzyme combinations on preparing oligoosteoin products were compared to verify the advantages of the recombinant chondroitin sulfate exoenzyme described in the present invention in preparing oligoosteoin products.

[0056] Weigh 8-10 kg of chicken cartilage, wash with water, and subject the cartilage to high pressure treatment at 120°C for 90 minutes. Grind the cartilage into a homogenate using a colloid mill, add an appropriate amount of water, adjust the solid concentration of the homogenate to be enzymatically hydrolyzed to about 10%, and further perform enzymatic hydrolysis.

[0057] Enzymatic hydrolysis was performed using three enzyme systems: compound enzyme A, compound enzyme B, and compound enzyme C. Compound enzyme A enzymatic hydrolysis: adjust the pH to 7.0, the temperature to 53°C, add protease for 5h, adjust the pH to 6.5 after enzymatic hydrolysis, the temperature to 36°C, add 8mL of recombinant chondroitin sulfate exonuclease, and enzymatic hydrolysis for 5h; compound enzyme B enzymatic hydrolysis: adjust the pH to 7.0, the temperature to 53°C, add protease for 5h, adjust the pH to 6.5 after enzymatic hydrolysis, the temperature to 36°C, add 8mL of chondroitin sulfate exonuclease, and enzymatic hydrolysis for 5h; compound enzyme C enzymatic hydrolysis: adjust the pH to 7.0, the temperature to 53°C, and add protease for 5h.

[0058] After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is heated to 85°C, kept warm for 20 minutes to inactivate the enzyme, centrifuged for 15 minutes, the supernatant is collected, and low-temperature vacuum concentration is performed, and further spray drying is performed to obtain the oligosaccharide product.

[0059] The molecular weight distribution of chondroitin sulfate in the products obtained by enzymatic hydrolysis of the three composite enzymes was determined to judge the enzymatic hydrolysis effect of chondroitin sulfate exoenzyme. The relative molecular mass was determined by high performance gel permeation chromatography (HPGPC). A TSK-GELG4000 PWXL (7.5mm×30.0cm) gel chromatography column was used, the column temperature was 35℃, and it was equipped with a differential refractive index detector. The mobile phase was 0.2mol / L sodium nitrate solution and 0.01mol / L sodium dihydrogen phosphate solution, and the flow rate was 0.5mL / min. Dextran (relative molecular masses were 1000Da, 3650Da, 5000Da, 12000Da, 21000Da and 80000Da, respectively) was used as the standard, and the standard curve was drawn with the retention time of the standard as the horizontal axis and the logarithm of the molecular weight of the standard as the vertical axis.

[0060] Prepare a sample solution with a mass concentration of 10 mg / mL, filter it through a 0.22 μm filter membrane, and use HPGPC analysis after filtration. The injection volume is 20 μL, and the retention time of the chromatographic peak is determined. The molecular weight distribution of each sample is calculated based on the standard curve. The molecular weight distribution liquid chromatogram of the product is shown in the figure below. Figure 3A-3C shown.

[0061] According to the molecular weight distribution of chondroitin sulfate in the product prepared by using three kinds of complex enzymes for enzymolysis, the sample prepared by using recombinant chondroitin sulfate exoenzyme, the content of low molecular weight chondroitin sulfate is higher than the sample prepared by original chondroitin sulfate exoenzyme, and is higher than the product prepared by not adding chondroitin sulfate exoenzyme. Under the condition of the same enzyme addition amount, the recombinant chondroitin sulfate exoenzyme of less amount can be used to obtain the low molecular weight chondroitin sulfate of the same degree. Therefore, the recombinant chondroitin sulfate exoenzyme of the present invention can be used as an effective tool enzyme for preparing oligomeric chondroitin products, can significantly reduce the usage amount of enzyme, and adapt to industrial production.

[0062] Example 6 Moisture, ash, protein, fat, chondroitin sulfate, and sulfate content of oligoosteogenin products.

[0063] Moisture content: Direct drying method, refer to GB 5009.3-2016, after measurement, the moisture content of the sample is 6.12%.

[0064] Ash content determination: Determination of total ash in food, refer to GB 5009.4-2016. After measurement, it was found that the ash content in the sample was 6.75%.

[0065] Protein content determination: Kjeldahl nitrogen determination method, GB 5009.5-2016. After determination, it was found that the protein content in the sample was 63.20%.

[0066] Fat content determination: Soxhlet extraction method, refer to GB 5009.6-2016. After determination, it was found that the fat content in the sample was 1.41%.

[0067] Chondroitin sulfate content: The content of glucuronic acid was determined by the m-hydroxybiphenyl method, and the content of chondroitin sulfate was converted by the content of glucuronic acid. After determination, it was found that the content of chondroitin sulfate in the sample was 22.52%.

[0068] Sulfate content determination: The sulfate content was determined by barium sulfate turbidimetry. The sulfate content in the sample was 3.36%. Sulfate plays a certain role in the functional activity of chondroitin sulfate. The determination showed that chondroitin sulfate exoenzyme did not cause the sulfate to fall off during the enzymatic hydrolysis process, which better maintained the functional activity of chondroitin sulfate to a certain extent.

[0069] Example 7 Chondroitin sulfate molecular weight distribution, Fourier transform infrared scanning, total amino acid composition, and circular dichroism scanning of the oligomeric chondroitin product.

[0070] Molecular weight distribution: The relative molecular mass was determined by high performance gel permeation chromatography, using a TSK-GELG4000PWXL (7.5mm×30.0cm) gel chromatography column, a column temperature of 35°C, and a differential refractive index detector. The results showed that more than 90% of the molecular weight distribution of chondroitin sulfate in the sample was less than 3000Da. Figure 4A shown.

[0071] Fourier infrared scanning: Take an appropriate amount of sample and KBr, place them in an agate mortar, grind them thoroughly and press them into tablets. Use an infrared spectrometer to scan the sample with a resolution of 4 cm -1 , record the infrared spectrum. Through the infrared spectrum, it is found that at 3400cm -1 A broad absorption peak appeared around 1800-1500cm, which is considered to be the stretching vibration of the OH bond, indicating the presence of a hydroxyl structure of polysaccharide in the component; after chondroitin sulfate is degraded, conjugated double bonds are generated, -1 The characteristic absorption peaks appearing on the left and right are usually called the "double bond stretching region". This region can be considered as the characteristic absorption peak produced by the double bonds of stretching vibration, indicating that the chondroitin sulfate in the product has been successfully degraded. Figure 4B shown.

[0072] Total amino acid composition: Weigh an appropriate amount of sample (protein content in the range of 25±5mg) into an ampoule, add 12mL6mol / L HCl (containing 5‰ mercaptoethanol), and seal the bottle with nitrogen. The sample was hydrolyzed at 110℃ for 22h, and after cooling, it was fixed to 50mL with 0.02mol / L HCl. 1mL of the fixed volume solution was taken, evaporated to dryness by rotary evaporation, and 5 drops of ultrapure water were added to evaporate to dryness to remove acid (repeat 3 times). After rotary evaporation, 2mL0.02mol / L HCl was added to re-dissolve, and filtered through a 0.22μm water filter membrane to obtain the test solution. The amino acid type and content were determined using an amino acid automatic analyzer. The total amino acid composition of oligosclerotin is shown in Table 1. It can be seen from Table 1 that glycine is the most important amino acid in the amino acid composition of oligosclerotin, because collagen is composed of repeating units of (Gly-XY)n. The content of hydroxyproline and proline is relatively high, and this composition is conducive to maintaining the planar peptide bond, thereby improving the stability of the structure. It can be proved that the protein in the extracted osteotin is type II collagen peptide;

[0073] Table 1 Total amino acid composition of oligosaccharides

[0074]

[0075] Circular dichroism scanning: Prepare a 0.3 mg / mL sample solution, pass the solution through a 0.22 μm polyethersulfone water filter, and use circular dichroism scanning to analyze its secondary structure. The sample cell thickness is 1 cm, the scanning wavelength range is 190-240 nm, the scanning speed is 100 nm / min, and the scanning temperature is 25 ° C. The results are as follows Figure 4C As shown in Figure 1, the CD spectrum of oligomerized osteotin solution can reflect the protein-related secondary structure. The sample shows the maximum rotation (positive band) at 223nm, the minimum rotation (negative band) at 196nm, and a consistent crossover point (zero rotation) at about 210nm, which are signs of the integrity of the triple helical conformation. Figure 4C shown.

[0076] Example 8 Evaluation of the efficacy of oligoosteogenin products in promoting chondrocyte proliferation.

[0077] The pathogenesis of osteoarthritis is closely related to chondrocyte activity. Changes in articular cartilage are considered to be the most important pathological process in the development of osteoarthritis. Chondrocytes are the only cellular component of articular cartilage and play a decisive role in maintaining cartilage function. In order to verify the chondrocyte proliferation-promoting effect of the prepared oligoosteogenin, SW1353 cells (human chondrosarcoma cell line) were used as a chondrocyte model, and the proliferation rate of SW1353 cells was used as the main evaluation index to evaluate the chondrocyte-promoting effect of the prepared oligoosteogenin.

[0078] SW1353 cell culture conditions: cultured in DMEM medium containing 10% FBS and 1% Penicillin-Streptomycin, 5% CO2, 95% air, 37°C constant temperature incubator. When the plated cells proliferated to 80-90%, they were digested with 0.25% trypsin.

[0079] SW1353 cells in the logarithmic growth phase were digested with 0.25% trypsin. After digestion, the cells were mixed to prepare a cell suspension. 3The cells were inoculated in a 96-well plate at a density of 100 μL / well, and each well was inoculated with 100 μL of cell suspension. The inoculated 96-well plate was placed in a 5% CO2 incubator (37°C) for 24 hours to ensure that the cells were completely attached to the wall; the 96-well cell culture plate was taken out, the cell supernatant was discarded, and 100 μL per well was added to intervene in the SW1353 cells with different concentrations of chicken oligosaccharide, chicken low molecular weight osteosulfate, and chicken chondroitin sulfate, and the cells were continued to be cultured for 24 hours; after 24 hours of culture, the 96-well cell culture plate was taken out, the original drugs in the well cell culture plate were removed, and the culture medium was added at 100 μL / well. 10 μL CCK-8 reagent was added to each well under light-proof conditions, and the plate was returned to the incubator for another incubation for 1.5 hours; after incubation, the cell culture plate was taken out, and the absorbance was detected at 450 nm after shaking with an ELISA reader, and the chondrocyte proliferation rate was calculated. The cell proliferation rate calculation formula is as follows:

[0080] Chondrocyte proliferation rate (%) = [(As-Ab) / (Ac-Ab)] x 100%; where As: absorbance of the experimental group, Ac: absorbance of the control group, Ab: absorbance of the blank group;

[0081] The results of cell proliferation showed that the chicken oligochondroitin and chicken low molecular weight chondroitin sulfate prepared by recombinant chondroitin sulfate exoenzyme had a good proliferation effect on SW1353 chondrocytes. Compared with the undegraded chicken chondroitin sulfate, the cell proliferation rate was significantly improved. This proves that the product has a good promoting effect on the proliferation of chondrocytes. Figure 5A-5C shown.

[0082] Example 9 Evaluation of the efficacy of oligoosteogenin products in improving osteoarthritis.

[0083] The osteoarthritis model was established by injecting papain into the joint cavity. The early osteoarthritis rats were induced by injecting papain, which caused the release of chondroitin sulfate in the cartilage, induced the loss of sulfated glycosaminoglycans in the medial and lateral cartilages, affected the proteoglycans in the extracellular matrix, and damaged the integration of bone collagen. The inflammatory factors IL-1β, IL-6, TNF-α in the rat serum, and the knee joint HE and Alcian blue pathological sections were used as evaluation indicators to evaluate the effect of the product in improving osteoarthritis.

[0084] A total of 48 SD rats were purchased from Jinan Pengyue Animal Experiment Breeding Co., Ltd., license number: SCXK (Lu) 20220006. They were divided into six groups, each with 8 rats, and were adaptively fed for 6 days before the experiment, quarantined and adapted to the barrier environment. The breeding environment was: 20-25℃, relative humidity: 50%-70%, suitable lighting, good ventilation, and clean environment. The daily food intake and weight changes of the rats were monitored, and the daily mental state of the rats was observed. Feed and water were given once a day at a fixed time.

[0085] The day before the experiment, the experimental animals were randomly divided into 6 groups, namely: model control group (MOD), blank control group (NOR), positive control group (DRU), low-dose group (L-CHE), medium-dose group (M-CHE), and high-dose group (H-CHE), 4 animals / cage, and two cages in each group.

[0086] After the adaptive feeding, the model was established for 1 week. The model control group (MOD), positive control group (DRU), low-dose group (L-CHE), medium-dose group (M-CHE), and high-dose group (H-CHE) were regularly established with 10% papain on the 1st, 3rd, 5th, and 7th days.

[0087] Pay attention to aseptic handling during the modeling process. The modeling reagent is 10% papain solution, and the anesthetic reagent is 3% sodium pentobarbital solution. The solutions are prepared immediately before use.

[0088] Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution at a volume of 0.3 mL / 100 g. The left hind knee joint was selected as the modeling site. When the rat showed slightly closed eyes, weak limbs, and no violent reaction when grasped, the anesthesia was successful. The hair in the 0.5 cm area around the left knee joint cavity was removed and disinfected with 75% alcohol cotton balls. The needle insertion site was the lateral socket of the rat knee joint. There was an obvious sense of emptiness after the needle was inserted, proving that the needle was inserted into the joint cavity. The modeling solution was injected into the left knee joint cavity to induce an osteoarthritis model. The blank control group followed the same operation method, and the injection solution was normal saline.

[0089] After modeling, rats were treated to improve osteoarthritis. L-CHE, M-CHE, and H-CHE were treated by gavage at 50 mg / kg, 100 mg / kg, and 200 mg / kg of product solution, respectively; DRU was treated by gavage at 5 mg / kg of meloxicam; MOD and NOR were treated by gavage with the same dose of saline. The treatment cycle was 35 days, with gavage treatment every day.

[0090] By measuring the relevant inflammatory factor indicators in rat serum, it was found that compared with MOD, the levels of IL-1β, IL-6, and TNF-α in the serum of DRU, L-CHE, M-CHE, and H-CHE were significantly decreased (P<0.05), proving that oral administration of the product to rats can effectively reduce the relevant inflammatory indicators in the serum of osteoarthritis model rats. Inflammatory factors are involved in the pathogenesis of osteoarthritis. IL-1β, TNF-α, and IL-6 are the main inflammatory cytokines in the pathophysiology of osteoarthritis. As an important cytokine, IL-6 can trigger osteoclast differentiation and bone resorption in cartilage lesions and reduce the expression of type II collagen. IL-1β and TNF-α inhibit the synthesis of proteoglycans, connexins, and type II collagen in chondrocytes by increasing in osteoarthritis synovial fluid, synovium, subchondral bone, and cartilage, stimulate MMP-1, MMP-3, and MMP-13, induce the production of cytokines and chemokines, and lead to extracellular matrix damage. Type II collagen can improve the structure of cartilage extracellular matrix, and chondroitin sulfate can reduce the expression of matrix metalloproteinases and inflammatory factors. Figure 6A-6C shown.

[0091] HE staining was performed on the knee joints of rats. The cartilage tissue structure of rats can be observed through HE staining. The edges of the normal group were smooth and without obvious damage. The edges of the model group were obviously damaged. In the high, medium and low dose groups, as the dose increased, it was obvious that the damage to the edges gradually slowed down, proving that cartilage enzymatic extracts have a relieving effect on osteoarthritis in rats. Fig. 7A shown.

[0092] The knee joint was stained with Alcian blue. Alcian blue staining is mainly used to observe the state of chondrocytes. In NOR, the chondrocyte structure is neat, the arrangement is tight, the number is large, and the tide line is clearly visible, indicating that this is the state of normal rat knee chondrocytes. In MOD, it can be clearly found that the blue color becomes lighter, the number of chondrocytes decreases, the arrangement is loose and disordered, the hierarchy is disordered, and the tide line is blurred, indicating that the model is successfully built. In DRU, the number of chondrocytes increases, the arrangement is more orderly, and the tide line is relatively clear, indicating that the positive drug meloxicam has a certain improvement effect on osteoarthritis. In L-CHE, the number and number of chondrocytes are improved compared with the model group, but the stratification is not obvious, and the arrangement of chondrocytes is still relatively loose; in M-CHE, the number and morphology of chondrocytes are further improved compared with the low-dose group, the arrangement is gradually neat, and the tide line is gradually clear; in the H-CHE group, the number and morphology of chondrocytes are significantly improved, the cells are arranged more neatly, the tide line is obvious, and the chondrocytes are mostly round and evenly distributed, indicating that the prepared oligosaccharide has an improvement effect on osteoarthritis. Figure 7B shown.

[0093] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0094] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant chondroitin sulfate exonuclease mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. A recombinant chondroitin sulfate exonuclease mutant according to claim 1, characterized in that: The nucleotide sequence encoding the amino acid sequence described in SEQ ID NO.1 is shown in SEQ ID NO.2, and SEQ ID NO.2 is translated into the encoding nucleotide sequence based on the chondroitin sulfate exonuclease of the original amino acid sequence SEQ ID NO.3, and the gene fragment of the nucleotide sequence is obtained by PCR amplification and sequencing, and the mutant is obtained by performing an error-prone PCR reaction on the gene fragment.

3. A recombinant expression vector of chondroitin sulfate exonuclease, characterized in that: The recombinant expression vector comprises the recombinant chondroitin sulfate exonuclease gene as shown in SEQ ID NO.2, and the vector is Pichia pastoris.

4. A recombinant bacterium of chondroitin sulfate exonuclease, characterized in that: The recombinant bacteria comprises the recombinant chondroitin sulfate exonuclease gene as shown in SEQ ID NO.2, and the strain is Pichia pastoris X33.

5. An enzyme preparation, characterized in that The enzyme preparation comprises the mutant of the chondroitin sulfate exoenase according to claim 1 and / or the recombinant bacteria of the chondroitin sulfate exoenase according to claim 4.

6. Use of the mutant of chondroitin sulfate exonuclease according to claim 1, the recombinant expression vector according to claim 3 and the recombinant bacteria according to claim 4, characterized in that: The application is to prepare oligoosteogenin by using the mutant, recombinant expression vector or / and recombinant bacteria.

7. The use according to claim 6, characterized in that: The application uses cartilage as raw material.

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