SCREENING, IDENTIFICATION AND OPTIMIZED EXPRESSION OF CHONDROSULFATASE

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of biological engineering, more particularly, relates to screening, identification and optimized expression of a Chondrosulphatase.BACKGROUND

[0002] Chondrosulphatase (ChSase), a lyase that can degrade glycosaminoglycans such as chondroitin sulfate, dermatin sulfate and hyaluronic acid into unsaturated disaccharides and oligosaccharides. According to different substrates, ChSase can be divided into ChSase ABC, ChSase AC, ChSase B and ChSase C, etc. ChSase has important application in the fields of biochemistry and medicine. In basic research, ChSase can be used as a tool enzyme for quality study of chondroitin sulfate and efficient preparation of chondroitin sulfate oligosaccharides with multiple biological activities. In the field of medicine, ChSase is used as a medicinal enzyme, which can degrade the mucus in cystic fibrosis, promotes the regeneration of nerve axis, relieves the symptoms of lumbar disc herniation, anti-tumor, enhances the adhesion between chondrocytes and cartilage and other medicinal functions. ChSase is mainly derived from microorganisms, and most of them are lyase. Some microorganisms, such as Flavobacterium heparin, Proteus penneri, Aeromonas sobria, Proteus vulgarisand so on, can produce chondroitin sulfate lyase. However, most of the ChSase derived from microorganisms is an intracellular enzyme with low enzyme activity. Tao Ke et al. (Study on the screening of chondroitinase ABC producing strain and their fermentation technology[J]. Chinese Journal of Antibiotics, 2004, (29)3:138-140.) reports that a strain of Proteus penneri is used to produce ChSase by fermentation. The process is complicated, and the enzyme activity of ChSase decreases significantly in the later stage of fermentation, and in the whole process the highest enzyme activity is only 322.17 U / L. Su Xin et al. (Study on the preparation of chondroitin sulfate lyase by fermentation and its enzyme separation and purification[J]. Journal of Microbiology, 2005, (25)4:64-67) reports that a strain of Aeromonas sobria is screened from fish belly. Although it has a high enzyme production capacity, it needs a purification process of one-step dialysis and four-step column chromatography, which is complicated, costly and not conducive to industrial amplification. Besides fermentation, Li et al. (Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli[J]. Protein Expression and Purification, 2016,(128):36-41.) also reports that ChSase derived from Proteus is heterologously expressed in E. coli. In order to increase the expression of ChSase, glyceraldehyde-3-phosphate dehydrogenase is co-expressed, however the enzyme activity after purification is reduced by 3.1 times, so it's not conducive to industrial amplification. At present, there is no domestic product supply, almost all rely on import, and the price is very expensive, which limits the application of ChSase in the preparation of low molecular weight chondroitin sulfateand medicine. Therefore, it is of great significance to screen ChSase with high enzyme activity.SUMMARY

[0003] The invention is defined in the appended claims.

[0004] The present disclosure screens and identifies a ChSase by collecting soil samples, sewage or sludge from coastal areas, river banks, farmers' markets, slaughterhouses and dining halls. Extract suitable amount of soil samples with physiological saline, after enrichment culture, gradient dilution and spread it on the screening plate. Compare and select the colony with the largest ratio of transparent circle to strain diameter, and inoculate the colony into the liquid fermentation medium for culture. The genome of the flora is extracted for metagenomic sequencing, and the sequence with the highest possibility of ChSase is analyzed and screened. Then the sequence is artificially synthesized and further identified and optimized for expression in E. coli and B. subtilis, the expression vector is the plasmid pBRSFDuet-1, pHT01, pHT43-His, pMA5 or pWB980. The present disclosure screens and identifies a ChSase from the nature, the highest similarity between the amino acid sequence of the ChSase and the known ChSase reported by NCBI (National Center of Biotechnology Information) is 85%. An expression of the ChSase is optimized in Escherichia coli and Bacillus subtilis, and the high-efficiency biosynthesis of high activity ChSase with the highest enzyme activity of 11976.5 U / L is achieved.

[0005] In one embodiment, the ChSase is screened and identified from soil samples, sewage or sludge from coastal areas, river banks, farmers' markets, slaughterhouses and dining halls, the highest similarity between its amino acid sequence and the ChSase reported by NCBI is 85%.

[0006] In one embodiment, the ChSase is optimally expressed in the engineered strain of Escherichia coli or Bacillus subtilis, including E. coli MG1655, E. coli DH5α, E. coli W3110, E. coli BL21, B. subtilis 168, B. subtilis WB600, B. subtilis WB800 and other hosts.

[0007] In one embodiment, the engineer strain is further preferred to be B. Subtilis, because the strain has high safety and wide range of applications.

[0008] In one embodiment, an expression vector of a ChSase gene is a plasmid of pBRSFDuet-1 or pHT01.

[0009] In one embodiment, the optimized expression of the ChSase gene included codon optimization, ribosomal binding site (RBS) optimization, promoter optimization and so on.

[0010] In one embodiment, the components of a fermentation medium are: yeast powder is 12-18 g / L, glucose is 32-48 g / L, potassium sulfate is 3.2-4.8 g / L, magnesium sulfate is 1.8-2.2 g / L, phosphate buffer is 40-60 mM, FeCl 2 ·6H 2 O is 10.8-16.2 mg / L; MnCl 2 ·4H 2 O is 0.8-1.2 mg / L; ZnCl 2 is 1.36-2.04 mg / L; CuCl 2 ·2H 2 O is 0.344-0.516 mg / L; and pH 5-9.

[0011] In one embodiment, a fermentation method is to inoculate a recombinant strain into a fermentation medium and ferment at 30-40 °C for 20-80 h.

[0012] In one embodiment, a determination method of an enzyme activity of the ChSase includes: sonicating the fermentation broth to disrupt the cells, and the supernatant is harvested as crude enzyme by centrifugation at 12000 rpm at 4 °C for 20 min. The crude enzyme solution is purified by MBP column which eluted with 10 mM maltose. 40 µL of diluted enzyme solution is added to 960 µL of substrate solution (2 g / L of C4S is dissolved in 20 mM Tris-HCl). The initial reaction rate within 1 min is determined by kinetic-based enzyme activity determination method. Definition of the enzyme activity: the amount of enzyme required to consume 1 micromole of substrate per unit time is defined as one unit of enzyme activity.

[0013] The present disclosure also provides a use of the ChSase in a preparation of products containing low molecular weight chondroitin sulfate in the fields of medicine, cosmetics and biology.

[0014] The beneficial effects of the present disclosure relative to the prior art are as follows: the present disclosure screens and identifies a ChSase from the nature, the highest similarity between its amino acid sequence and the ChSase reported by NCBI is 85%, then optimally expressed in E. coli and B. subtilis. The expression vector is the plasmid pBRSFDuet-1, pHT01, pHT43-His, pMA5 or pWB980, the efficient biosynthesis of ChSase with high enzyme activity is realized, and the highest enzyme activity is 11976.5 U / L. The whole process and post-treatment are simplified. The invention has potential and wide application value in the preparation of products containing low molecular weight chondroitin sulfate in the fields of medicine, cosmetics and biology.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the schematic diagram of the construction of vector for optimized expression of ChSase in E. coli. Figure 2 shows the schematic diagram of the construction of vector for optimized expression of ChSase in B. subtilis. Figure 3 shows the enzyme activity of optimized expression of ChSase in E. coli and B. subtilis cultured in shake flask for 50 h. DETAILED DESCRIPTION OF THE EMBODIMENTS Amino acid sequence information involved in the examples:

[0016] SEQ ID No.1 is the amino acid sequence of a novel ChSase screened and identified from the nature of the invention.Example 1: Screening and identification of ChSase

[0017] Collected soil samples, sewage or sludge from coastal areas, river banks, farmers' markets, slaughterhouses and dining halls. Extracted suitable amount of soil samples with physiological saline. The enrichment cultured samples were gradient diluted and spreaded on the screening plate, then cultured at 37 °C for 3 days. The colonies with transparent circles were selected for secondary screening. The selected culture medium (g / L) was: chondroitin sulfate 4.5, ammonium chloride 2.5, sodium chloride 0.95, magnesium sulfate 1.0, dipotassium hydrogen phosphate 1.0, agar 20.0, bovine serum albumin 5.0, pH 7.0. Compared and selected the colony with the largest ratio of transparent circle to strain diameter in the re-screening plate, and inoculated the colony into the liquid fermentation medium for culture. The genome of the flora was extracted for metagenomic sequencing, and the sequence with the highest possibility of ChSase was analyzed and screened. The amino acid sequence with the highest probability of ChSase was compared with the ChSase reported by NCBI. The final result was that the highest similarity between this enzyme and the ChSase reported by NCBI was 85%. The sequence was artificially synthesized and expressed in E. coli and B. subtilis, and the results of further identification proved the enzyme was ChSase.Example 2: Optimized expression of ChSase in E. coli and B. subtilisConstruction of expression system:

[0018] The fragment CS DNA was amplified by PCR with the primer CS(pBRSF)-F / CS(pBRSF)-R which used artificially synthesized CS DNA as the template. Recombinant plasmid pBRSFDuet-1-CS was constructed by splicing the CS DNA PCR product and skeleton plasmid pBRSFDuet-1 which used the primer pBRSF-F / pBRSF-R.

[0019] The fragment CS DNA was amplified by PCR with the primer CS(pHT)-F / CS(pHT)-R which used artificially synthesized CS DNA as the template. Recombinant plasmid pHT01-CS was constructed by splicing the CS DNA PCR product and skeleton plasmid pHT01 which used the primer pHT01-F / pHT01-R.Construction of recombinant bacteria:

[0020] The recombinant plasmid pBRSFDuet-1-CS was transferred into E. coli MG1655, E. coli DH5α, E. coli W3110, and E. coli BL21 to construct the recombinant strain CSmg, CSdh, CSw300, and CSbl, respectively.

[0021] The recombinant plasmid pHT01-CS was transferred into B. subtilis 168, B. subtilis WB600, and B. subtilis WB800 to construct the recombinant strain CS168, CS600, and CS800, respectively.

[0022] The construction of E. coli vector optimized for ChSase expression is shown in Figure 1; the construction of B. subtilis vector optimized for ChSase expression is shown in Figure 2.

[0023] The primer information (5'-3') is as follows: pBRSF-FpBRSF-RCS(pBRSF)-FCS(pBRSF)-RpHT01-FpHT01-RCS(pHT)-FCS(pHT)-R

[0024] Monoclone of seven recombinant strains and the control strains (pBRSFDuet-1 empty plasmid and pHT01 empty plasmid were transformed by E. coli and B. subtilis, respectively) were inoculated into 5mL LB culture containing appropriate antibiotics (the final concentration of 50 µg / mL kanamycin for E. coli and the final concentration of 25 µg / mL chloramphenicol for B. subtilis). The seed inoculum was cultured at 37 °C at 200 rpm for 10 h, and transferred to a 250 mL flask with a liquid volume of 25 mL according to the inoculation amount of 10%, and the medium was the fermentation medium. 1.5 mmol / L IPTG and appropriate antibiotics (the final concentration of 50 µg / mL kanamycin for E. coli and the final concentration of 25 µg / mL chloramphenicol for B. subtilis) were added as required. The cultures were incubated at 37 °C and 220 rpm. After an incubation time of 50 h, the enzyme activity was determined. The method of the ChSases enzyme activity was as follows: sonicating the fermentation broth to disrupt the cells, and the supernatant was harvested as crude enzyme by centrifugation at 12000 rpm and 4 °C for 20 min. The crude enzyme solution was purified by MBP column and eluted with 10 mM maltose. 40 µL of diluted enzyme solution was added to 960 µL of substrate solution (2 g / L of C4S dissolved in 20 mM Tris-HCl). The initial reaction rate within 1 min was determined by kinetic-based enzyme activity determination method. Definition of the enzyme activity: the amount of enzyme required to consume 1 micromole of substrate per unit time is defined as one unit of enzyme activity. The results of enzyme activity in Figure 3 showed that all of the recombinant strains could realize the high-efficiency biosynthesis of high activity ChSase, and the highest ChSase activity was obtained from CSdh as 11976.5 U / L.

Claims

1. A chondrosulphatase comprising the amino acid sequence of SEQ ID No.1.

2. A method for the efficient biosynthesis of the chondrosulphatase according to claim 1, wherein the chondrosulphatase gene is optimized for expression in engineered Escherichia coli or Bacillus subtilis strains.

3. The method according to claim 2, wherein the expression of the chondrosulphatase gene is optimized for expression in in Bacillus subtilis.

4. The method according to claim 2, wherein the chondrosulphatase gene is expressed in the plasmid pBRSFDuet-1, pHT01, pHT43-HIS, pMA5 or pWB980.

5. The method according to claim 2, wherein the expression of the chondrosulphatase gene is optimized by codon optimization, ribosomal binding site optimization or promoter optimization.

6. The method according to claim 2, wherein the components of the fermentation medium for said biosynthesis are: yeast powder 12-18 g / L, glucose 32-48 g / L, potassium sulfate 3.2-4.8 g / L, magnesium sulfate 1.8-2.2 g / L, phosphate buffer 40-60 mM, FeCl2·6H2O 10.8-16.2 mg / L; MnCl2·4H2O is 0.8-1.2 mg / L; ZnCl2 is 1.36-2.04 mg / L; CuCl2·2H2O is 0.344-0.516 mg / L; and pH 5-9.

7. The method according to claim 2, comprising inoculating said engineered strain into a fermentation medium and fermenting at 30-40 °C for 20-80 h.

8. A method of determining the enzyme activity of the chondrosulphatase according to claim 1, wherein said method includes: sonicating fermentation broth to disrupt cells therein expressing the chondrosulphatase of claim 1; harvesting the supernatant comprising the crude enzyme by centrifugation at 12000 rpm and 4°C; the crude enzyme solution is purified by MBP column, and eluted with 10 mM maltose; 40 µL diluted enzyme solution is added to 960 µL substrate solution, wherein the substrate solution is obtained by dissolving 2 g / L C4S in 20 mM Tris-HCl, and the initial reaction rate within 1 min is determined by kinetic-based enzyme activity determination method.

9. Use of the chondrosulphatase according to claim 1 in the preparation of products containing low molecular weight chondroitin sulfate in the fields of medicine, cosmetics and biology.