Bacillus subtilis for secretory expression of collagenase and application thereof

Abstract

The invention discloses bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis. The invention provides bacillus subtilis for secretory expression of collagenase and application of the bacillus subtilis in fat cell extraction, and after optimization of an expression element promoter and a signal peptide and large-scale fermentation of a fermentation tank, the highest enzyme activity can reach 669U. ML <-1 >. After purification, adipose-derived cells are successfully extracted and separated from mouse adipose cells, and after passage, the adipose-derived cells present the form of adipose cells. The method shows that the method has a good application prospect in the aspect of fat cell extraction.

Description

Technical Field

[0001] The present invention relates to a Bacillus subtilis for secreting and expressing collagenase and its application, belonging to the technical field of genetic engineering. Background Art

[0002] Collagen mainly plays a role in binding tissues in vivo and is the most important component in the extracellular matrix. It is widely distributed in connective tissues such as tendons, bones, ligaments, skin, and blood vessels of mammals, playing a role in support, repair, and protection. However, due to its stable triple helix structure, collagen has become an insurmountable obstacle in tissue dissociation. Currently, relevant research shows that collagenase from microorganisms can completely separate tissues while ensuring the integrity of cells.

[0003] Collagenase is a class of enzymes that can specifically hydrolyze the triple-helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. Classified by source, collagenase can be strictly divided into two categories: animal collagenase and bacterial collagenase according to the strict definition. Animal collagenase refers to the collagenase extracted and isolated from animals. Animal collagenase is mainly divided into three categories: matrix metalloproteinases of the M10 family, cysteine proteases of the C1 family, and serine proteases of the S1 family. This type of collagenase has high activity and strong substrate specificity, but the extraction process is complex and costly, and it is not suitable for large-scale industrial production. Bacterial collagenase was the first to be discovered and studied. They have strong collagen hydrolysis activity, and their fermentation cycle is short and the yield is high. Among them, the collagenases ColG and ColH from the M9B subfamily of Clostridium histolyticum are the most widely studied collagenases at present. They have specific recognition and cleavage ability for the special sequence Pro-X-Gly-Pro in collagen, so they can efficiently hydrolyze natural collagen without the assistance of other proteases. In addition, certain Bacillus spp., Staphylococcus aureus, Lactobacillus spp., Treponema spp., Streptomyces spp., and proteases of the S1 and S8 families from certain bacterial sources also have the ability to degrade collagen. However, compared with the collagenase from Clostridium histolyticum, these collagenases have obvious defects, such as weak substrate specificity and the ability to damage the stemness of cells during cell isolation and extraction. Classified according to the broad definition, collagenases from different sources can also be divided into four categories: animal collagenase, plant collagenase, bacterial collagenase, and fungal collagenase. Among them, plant collagenase and fungal collagenase are not enzymes that only hydrolyze collagen in the traditional sense. They can hydrolyze some collagen more because of their broad substrate spectrum, such as the protease produced by Trichoderma spp. Therefore, the definition methods of collagenase enzyme activity are relatively diverse, and there is no unified standard for the substrates used in enzyme activity determination. The substrates include PZ peptide, FALGP peptide, fish scale collagen, bovine hoof collagen, porcine skin collagen, etc. The results obtained by different substrates or methods vary greatly, and the enzyme activity data reported in the literature are not comparable.

[0004] Collagenase ColH belongs to type II collagenase and is mainly used for the isolation of cells such as stem cells, muscle cells, and adipocytes. The gene of collagenase ColH is derived from Clostridium histolyticum. The application of this strain is limited due to certain pathogenic risks, especially in tissue isolation. Most of the current heterologous expressions use classical expression systems such as Escherichia coli, etc. These expression systems also have limitations in application. For example, the cell wall of Escherichia coli contains endotoxins (lipopolysaccharides), and these endotoxins may be released into the extract during the extraction process, which can cause an immune response in the human body. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a Bacillus subtilis secreting and expressing collagenase and its application in the extraction of adipocytes. In the starting strain, the pMA5 vector is used to heterologously express collagenase derived from Clostridium histolyticum, and the nucleotide sequence is as shown in SEQ ID NO.3. Through the optimization of promoter and signal peptide elements, the recombinant Bacillus subtilis efficiently expresses collagenase extracellularly. The highest hydrolysis activity of the prepared collagenase reaches 669 U / mL. After ammonium sulfate precipitation and Q column purification, it is successfully applied to the extraction of adipose-derived cells.

[0006] The first object of the present invention is to provide a Bacillus subtilis secreting and expressing collagenase, and the Bacillus subtilis heterologously expresses collagenase derived from Clostridium histolyticum using the pMA5 vector in the host bacterium.

[0007] Furthermore, the nucleotide sequence of the collagenase is as shown in SEQ ID NO.3.

[0008] Furthermore, the host bacterium is Bacillus subtilis B.subtilis WB600.

[0009] Furthermore, the collagenase is expressed through the signal peptide mdh and the promoter npre. The nucleotide sequence of the signal peptide mdh is as shown in SEQ ID NO.2, and the nucleotide sequence of the promoter npre is as shown in SEQ ID NO.1.

[0010] Furthermore, the coding gene of the collagenase is cloned between the Nde I and BamH I restriction sites of the pMA5 vector.

[0011] The second object of the present invention is to provide a microbial inoculum containing the above-mentioned Bacillus subtilis.

[0012] The third object of the present invention is to provide the application of the above-mentioned Bacillus subtilis or the above-mentioned microbial inoculum in the preparation of collagenase.

[0013] Furthermore, the above-mentioned Bacillus subtilis is inoculated into a fermenter at an inoculation amount of 3-10%, and fermented under the conditions of a fermentation temperature of 30-40°C, a rotation speed of 100-500 rpm, an aeration rate of 1-3 vvm, and a pH of 6.5-7.5 to obtain a fermentation broth containing collagenase.

[0014] Furthermore, the application also includes precipitating the fermentation broth containing collagenase with 20%-30% ammonium sulfate and purifying it with an anion chromatography column to obtain purified collagenase.

[0015] The fifth object of the present invention is to provide the application of the Bacillus subtilis in the extraction of adipose-derived cells.

[0016] Further, the application is to produce collagenase using the Bacillus subtilis, and to process fat blocks with the collagenase to obtain the fat-derived cells.

[0017] Further, the fat at the groin of a mouse is torn off with forceps, washed three times with a washing solution, and the fat blocks are cut into fine shredded fat blocks of 1 mm 3 . Subsequently, the shredded small fat blocks are transferred to a sterile 15 mL centrifuge tube, incubated with the above-purified collagenase at 37 °C for 1 h and then centrifuged. The extracted cells are cultured in a DEME low-glucose medium at 37 °C under saturated humidity conditions of 5% CO2. After the cells cover the plate, they are passaged and observed to grow into the morphology of adipose stem cells.

[0018] Advantages of the present invention

[0019] The present invention provides a Bacillus subtilis secreting and expressing collagenase and its application in the extraction of adipose cells. After optimizing the expression element promoter and signal peptide and fermenting in a fermentor, the enzyme activity can reach up to 669 U·mL -1 . After purification, adipose-derived cells are successfully extracted and isolated from mouse adipose cells, and they present the morphology of adipose cells after passage. It shows that it has good application prospects in the extraction of adipose cells. Description of the drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 SDS-PAGE detection of the extracellular protein expression of the recombinant Bacillus subtilis B. subtilis WB600 / PMA5-ColH in Example 1 of the present invention; wherein, M: protein maker, 1: fermentation supernatant of the control group without the ColH gene, 2-9: fermentation supernatants of the experimental group at 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h;

[0022] Figure 2 Relative enzyme activity of the signal peptide modified strain;

[0023] Figure 3 Relative enzyme activity of the promoter modified strain;

[0024] Figure 4 Determination of the growth enzyme activity in the fermentor in Example 2 of the present invention;

[0025] Figure 5 It is the elution peak of collagenase purified by Q column in Example 3 of the present invention;

[0026] Figure 6 It is the SDS-PAGE detection of collagenase purification in Example 3 of the present invention; wherein, M: protein maker; 1: fermentation supernatant; 2: ammonium sulfate precipitation; 3: Q column;

[0027] Figure 7 It is the morphology of adipose-derived cells extracted by collagenase in Example 4 of the present invention after stable passage. Detailed implementation manners

[0028] The following further elaborates on the present invention patent in combination with specific examples. These implementation cases are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0029] Materials:

[0030] The components of the washing solution and low-glucose culture medium in Example 4 are as follows:

[0031] Washing solution: 50 mL PBS + 0.5 mL P / S

[0032] Low-glucose complete culture medium: 44.5 mL DMEM low-glucose culture medium + 5 mL FBS + 0.5 mL P / S.

[0033] Commercially available collagenase, purchased from Shanghai Guchen Biotech, product number GCB0506-100mg, enzyme activity 125 U / mg.

[0034] Detection method:

[0035] Determination of collagenase enzyme activity, ninhydrin colorimetric method: In the experimental group, collagen was used as the substrate (4 mg / ml), and the reaction system was: 250 μL substrate solution, 200 μL Tris-HCl (0.1 mol / L, pH 7.5, containing 50 mmol / L CaCl2), 250 μL strain fermentation supernatant (2 mg / mL), react at 37 °C for 5 h, and add an equal volume of 10% trichloroacetic acid to terminate the reaction; control group: use an equal volume (i.e., 700 μL) of 10% trichloroacetic acid termination solution as the control first.

[0036] Add 500 μL of the above-mentioned sample after the reaction, 500 μL of acetic acid buffer, and 500 μL of ninhydrin color-developing solution into a test tube in sequence. Heat in a water bath at 80 °C for 20 min. After the ninhydrin color-developing reaction, cool in an ice bath until completely cooled, and then add 1500 μL of 60% ethanol. Centrifuge at 12000 r for 1 min, take 200 μL and transfer it to an ELISA plate, and measure the absorbance at 570 nm. The obtained absorbance value is used to calculate the enzyme activity according to the glycine standard curve.

[0037] Definition of collagenase activity unit U: Under the conditions of 37 °C and pH 7.5, the amount of glycine equivalent to 1 μmol hydrolyzed from collagen by 1 mL of enzyme solution within 5 h is defined as 1 enzyme activity unit.

[0038] The following combines specific examples to elaborate on the technical solutions of the present invention. In the following examples, unless otherwise specified, the reagents, materials, and equipment used can be obtained from commercial channels, or prepared by conventional methods, or commonly used in this industry.

[0039] Example 1: Construction of recombinant expression vector and recombinant strain

[0040] According to the Clostridium histolyticum collagenase ColH gene sequence in the GeneBank database, specific primers were designed using primer design software Oligo and Snapgene. The collagenase ColH gene (SEQ ID NO.3) was obtained by PCR, and the PCR product was recovered using agarose gel.

[0041] The primer sequences are as follows:

[0042] F: 5’-tgaaatcagggggatccatgaagcgcaaatgtctg-3’

[0043] R: 5’-gtacgtaccaagctagcttaacggccaacgctg-3’

[0044] The obtained collagenase ColH gene was ligated to the vector pMA5 through homologous recombination technology to obtain the PMA5-H recombinant plasmid. Then, using the Bacillus subtilis genome as a template, 10 promoter fragments and 11 signal peptide fragments were amplified. The promoters and signal peptides of the PMA5-H recombinant plasmid were replaced, with enzyme activity as the screening criterion ( Figure 2 and Figure 3) Finally, the signal peptide mdh (SEQ ID NO.2) with the most significant improvement in enzyme activity and the promoter npre (SEQ ID NO.1) obtained based on the signal peptide were selected to obtain the plasmid PMA5-ColH. The synthesized recombinant plasmid PMA5-ColH was transformed into competent E. coli JM109 cells, spread on an LB plate containing ampicillin resistance, and single colonies were picked for PCR sequencing verification. The plasmid in the correctly sequenced positive transformant was transformed into Bacillus subtilis to obtain the recombinant Bacillus subtilis B. subtilis WB600 / PMA5-ColH. The recombinant strain B. subtilis WB600 / PMA5-ColH was picked and inoculated into LB medium supplemented with kanamycin and cultured at 37 °C and 220 rpm for 8 - 12 h, and then inoculated into 50 mL / 250 mL of TB medium containing kanamycin at an inoculation amount of 2%, and fermented and cultured at 37 °C and 220 rpm. Sampling started from 8 h, and samples were taken every 4 h. The target protein was successfully secreted and expressed extracellularly in the recombinant strain B. subtilis WB600 / PMA5-ColH by SDS-PAGE identification ( Figure 1 ).

[0045] Example 2: Fermentation of the recombinant strain B. subtilis WB600 / PMA5-ColH in a fermenter

[0046] The recombinant strain B. subtilis WB600 / PMA5-ColH was picked and inoculated into LB medium supplemented with kanamycin and cultured at 37 °C and 220 rpm for 8 - 12 h as the primary seed solution. Then, it was inoculated into 50 mL / 250 mL of TB medium containing kanamycin at an inoculation amount of 2% and fermented at 37 °C and 220 rpm for 12 h to obtain the secondary seed solution. The secondary seed solution was inoculated into the fermenter at an inoculation amount of 3 - 10%, and fermented and produced under the conditions of a fermentation temperature of 30 - 40 °C, a rotation speed of 100 - 500 rpm, an aeration rate of 1 - 3 vvm, and a pH of 6.5 - 7.5 to obtain a fermentation broth containing collagenase. The results of measuring the growth enzyme activity curve are as Figure 4 shown, and the maximum extracellular enzyme activity can reach 669 U / mL.

[0047] Example 3: Purification of collagenase

[0048] Centrifuge the fermentation broth to remove the precipitate. Then, add 20% ammonium sulfate to the supernatant and centrifuge at 8000 rpm for 10 min. Collect the supernatant. Subsequently, continue to add ammonium sulfate to the remaining supernatant to 30% to precipitate more proteins, and then centrifuge at 8000 rpm for 10 min and collect the precipitate. Dissolve the collected precipitate in phosphate buffer and desalt it using an ultrafiltration centrifuge tube. Further purify the collected reconstituted solution using AKTA with an anion exchange column. First, wash and flush the pipeline with deionized water; after connecting the column, wash it with phosphate buffer until the column is balanced; then slowly pass the reconstituted solution through the pipeline to bind the target protein to the column; finally, elute it linearly with phosphate buffer containing 1 M NaCl, and detect the eluate with a UV spectrophotometer at 280 nm, showing a total of five peaks. Collagenase activity was identified in the first peak( Figure 5 ). Figure 6 The results of SDS-PAGE analysis showed that a single distinct band was obtained between 110 kDa and 130 kDa.

[0049] Example 4: Evaluation of the effect of collagenase on the isolation of adipose stem cells

[0050] After sacrificing the mice by dislocation, use surgical instruments to isolate the mouse adipose tissue and soak it in the washing solution for rinsing. Use 10 mice as a group and wash three times respectively. Use sterile forceps to place the isolated adipose tissue on the lid of the dish, and use clean ophthalmic surgical scissors to cut the adipose tissue into a paste, about 1 mm 3 small particle blocks.

[0051] Put the processed adipose particle blocks into a 15 mL digestion tube, add collagenase solution according to the volume ratio of adipose tissue 1:1 to each tube, and then add 8 mL of lactated Ringer's buffer. Seal it with a sealing film and mix well. Place the sealed digestion tube in a metal bath and digest it at 37 °C and 300 rpm for 1 h, then centrifuge at 800 rcf for 10 min. Retain the precipitate at the bottom layer, resuspend it thoroughly with the washing solution and filter it through a 40 μm cell filter, transfer it to a 15 mL centrifuge tube, centrifuge at 800 rcf for 10 min, and discard the supernatant. After washing, resuspend it with 2 mL of low-glucose complete medium, mix well and pipette 10 μL to a hemocytometer for counting.

[0052] Table 1 Comparison of the number of mouse adipose stem cells digested by the collagenase produced by the screened strain in Example 1 and commercially available collagenase

[0053]

[0054] Prepare a digestion solution with a commercially available collagenase concentration of 2 mg / mL; the enzyme activity of the commercially available collagenase is greater than 125 U / mg. Since over-digestion will damage the cells, the digestion time is limited to 120 min.

[0055] The digested primary cells were further cultured in a cell incubator at 37 °C and 5.0% CO2 concentration. Depending on the cell attachment status, the cell culture medium was changed every 2 - 3 days.

[0056] Cell passage: Observed under an inverted microscope. When the cells grew to 80 - 90%, after aspirating the original medium, gently wash with a washing solution and then aspirate. Add 1 ml of trypsin to digest the adherent cells, culture at 37 °C and 5.0% CO2 concentration for 3 - 5 min. Immediately add an equal volume of low - sugar complete medium to terminate digestion when the cells become round and start to migrate under the inverted microscope. Transfer the terminated liquid into a 5 - ml tube, centrifuge at 1000 rpm for 5 min, pour off the supernatant, and resuspend the precipitate in the lower layer with the medium. The cell culture status is as Figure 7 shown.

[0057] Comparative Example 1:

[0058] Recombinant bacteria were prepared according to the following groups:

[0059] WB - SPmdh: The optimized signal peptide of the PMA5 - H recombinant plasmid is mdh;

[0060] WB - Pnpre: The promoter of the PMA5 - H recombinant plasmid is npre;

[0061] WB - SPmdh - Ptrnq: The optimized signal peptide of the PMA5 - H recombinant plasmid is mdh and the promoter is trnq;

[0062] WB - H: The PMA5 - H recombinant plasmid was directly transferred into B. subtilis WB600;

[0063] WB - K: The plasmid of PMA5 without the collagenase ColH gene was directly transferred into B. subtilis WB600.

[0064] In Table 2 below, WB - SPmdh - Pnpre is the recombinant bacterium constructed in Example 1 of the present invention, that is, the optimized signal peptide of the PMA5 - H recombinant plasmid is mdh and the promoter is npre, and it was transferred into the B. subtilis WB600 host for expression.

[0065] According to the fermentation method and purification method of Examples 2 and 3, collagenase was prepared and extracted. Among them, the enzyme activity and yield data in the fermentation broth are shown in Table 2.

[0066] Table 2 Enzyme activity, yield of the modified strains and adipocyte digestion

[0067]

[0068] Comparative Example 2:

[0069] Recombinant bacteria were prepared separately according to the following groups:

[0070] BL21-SPmdh-Pnpre: The signal peptide of the PMA5-H recombinant plasmid was optimized to mdh and the promoter was npre, and it was transferred into the E. coli BL21 host for expression;

[0071] WB-col: The PMA5 plasmid carried the gene reported in the literature "Expression, Enzymatic Properties and Fermentation Optimization of Collagenase in Bacillus subtilis";

[0072] WB-K: The PMA5 plasmid did not carry the collagenase ColH gene plasmid and was transferred into the B. subtilis WB600 host for expression.

[0073] WB-SPmdh-Pnpre in Table 3 below was the recombinant bacterium constructed in Example 1 of the present invention, that is, the signal peptide of the PMA5-H recombinant plasmid was optimized to mdh and the promoter was npre, and it was transferred into the B. subtilis WB600 host for expression.

[0074] The preparation and purification of collagenase were carried out according to the fermentation method and purification method of Examples 2 and 3, and the substrate specificity of the purified collagenase was analyzed. The results are shown in Table 3.

[0075] Table 3 Substrate Specificity

[0076]

[0077] The examples provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit their execution order. Obvious improvements made by those skilled in the art in combination with the existing common general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A Bacillus subtilis that secretes and expresses collagenase, characterized in that, The Bacillus subtilis heterologously expresses a collagenase derived from Clostridium histolyticum using the pMA5 vector in a host bacterium.

2. The Bacillus subtilis according to claim 1, characterized in that, The nucleotide sequence of the collagenase is as shown in SEQ ID NO.

3.

3. The Bacillus subtilis according to claim 1, characterized in that, The host bacterium is Bacillus subtilis WB600.

4. The Bacillus subtilis according to claim 1, characterized in that, The collagenase is expressed through the signal peptide mdh and the promoter nprE. The nucleotide sequence of the signal peptide mdh is as shown in SEQ ID NO. 2, and the nucleotide sequence of the promoter nprE is as shown in SEQ ID NO.

1.

5. The Bacillus subtilis according to claim 1, characterized in that, The coding gene of the collagenase is cloned between the Nde I and BamH I restriction enzyme cleavage sites of the pMA5 vector.

6. A microbial inoculum comprising the Bacillus subtilis according to any one of claims 1 to 5.

7. Use of the Bacillus subtilis according to any one of claims 1 to 5 or the above-mentioned microbial inoculum in the preparation of collagenase.

8. The application according to claim 7, wherein The above-mentioned Bacillus subtilis is inoculated into a fermenter at an inoculation amount of 3% - 10%, and fermented and produced under the conditions of a fermentation temperature of 30 - 40 °C, a rotation speed of 100 - 500 rpm, an aeration rate of 1 - 3 vvm, and a pH of 6.5 - 7.5 to obtain a fermentation broth containing collagenase.

9. The application according to claim 8, wherein The said use further includes precipitating the fermentation broth containing collagenase with 20% - 30% ammonium sulfate and purifying it through an anion chromatography column to obtain purified collagenase.

10. Use of the Bacillus subtilis according to any one of claims 1 to 5 in the extraction of fat-derived cells.

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