Chondroitin sulfate lyase of pl35 family chha3, its coding gene and application

Abstract

The present application relates to a PL35 family chondroitin sulfate lyase CHa3, a coding gene and application thereof, and belongs to the technical field of enzyme engineering. The chondroitin sulfate lyase CHa3 has an amino acid sequence shown as SEQ ID NO. 2, is a novel chondroitin sulfate lyase, has high enzyme activity on chondroitin sulfate, and degrades chondroitin sulfate into an endo-enzyme mode; when degrading chondroitin sulfate polysaccharide, the end product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; when degrading heparin, the end product is mainly unsaturated disaccharides; and when degrading dermatan sulfate, the end product includes unsaturated tetrasaccharides to unsaturated octasaccharides. The chondroitin sulfate lyase CHa3 can be applied to the preparation of CS / DS oligosaccharides and the research on the structure and function of CS / DS, and has a wide application prospect in the fields of medicine, food and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a PL35 family chondroitin sulfate lyase CHa3, a coding gene thereof and an application thereof, and belongs to the technical field of enzyme engineering. BACKGROUND

[0002] Glycosaminoglycans (GAGs) are a class of polyanionic linear acidic polysaccharides, which are often distributed in the extracellular matrix (ECM) and cell surface, and are involved in a variety of physiological and pathological processes, such as cell growth, cell adhesion, cell proliferation, tissue morphogenesis, cell signal transduction, viral infection, etc. Glycosaminoglycans are composed of basic disaccharide units of hexuronic acid (D-glucuronic acid [GlcA] or L-iduronic acid [IdoA]) and hexosamine (D-galactosamine [GalNAc] or D-glucosamine [GlcNAc]) alternatingly, and these disaccharide repeating units can also be N- or O-type sulfated, forming a structurally diverse and functionally complex sulfated glycosaminoglycan. According to the difference of disaccharide composition, GAGs can be divided into four categories: hyaluronic acid (HA), chondroitin sulfate / dermatan sulfate (CS / DS), heparin / heparan sulfate (HP / HS) and keratin sulfate (KS) (the disaccharide unit of KS is composed of D-galactose and D-glucosamine).

[0003] The disaccharide units of CS / DS have complex sulfation patterns, which increase the structural and functional complexity of CS / DS. During or after the synthesis of the polysaccharide chain of CS / DS, sulfate groups are often introduced at the C-2 position of GlcA / IdoA and the C-4 and C-6 positions of GalNAc by the action of various enzymes, such as N-deacetylase-N-sulfotransferase, sulfotransferase. GlcA is changed into IdoA by the action of C5 epimerase. Based on these modifications, CS synthesized in vivo by organisms in nature is usually composed of six different sulfation patterns of disaccharides: non-sulfated O / iO unit (-GlcA / IdoA-GalNAc-), GalNAc four-hydroxyl sulfated A / iA unit (-GlcA / IdoA-GalNAc4S-), GalNAc six-hydroxyl sulfated C / iC unit (-GlcA / IdoA-GalNAc6S-), GlcA / IdoA two-hydroxyl and GalNAc four-hydroxyl double-sulfated D / iD unit (-GlcA / IdoA2S-GalNAc6S-), GalNAc four-, six-hydroxyl double-sulfated E / iE unit (-GlcA / IdoA-GalNAc4S,6S-), GlcA / IdoA two-hydroxyl and GalNAc four-, six-hydroxyl triple-sulfated T / iT unit (-GlcA / IdoA2S-GalNAc4S,6S-). In addition, iB unit (-IdoA2S-GalNAc4S-) is often found in DS. Due to the presence of various disaccharide units in CS / DS, the polysaccharide structure becomes more complex and exhibits many important biological functions.

[0004] The high complexity of GAGs structure and function brings challenges to the study of GAGs structure-activity relationship, and the use of tool enzymes with specific activity often solves the problems related to the study of GAGs structure-activity relationship. According to the degradation mechanism of the enzyme, GAGs degrading enzymes can be divided into hydrolytic enzymes and cleavage enzymes, which exist widely in animals and microorganisms. GAGs hydrolytic enzymes cut glycosidic bonds and introduce a molecule of water, and GAGs cleavage enzymes cut glycosidic bonds and introduce an unsaturated double bond at the C4 and C5 positions of the uronic acid. Common GAGs cleavage enzymes include HA cleavage enzymes (degrading HA), chondroitin sulfate cleavage enzymes (degrading HA and CS / DS), and heparinases (degrading HP / HS), each of which exhibits different substrate preferences. The GAGs cleavage enzyme family includes the HA cleavage enzyme and chondroitin sulfate cleavage enzyme (CSase) family, such as PL6, PL8, PL16, PL23, PL29, PL30, PL33, and PL35, and the heparinase family, such as PL12, PL13, PL21, and PL15. Generally, cleavage enzymes (including HA cleavage enzymes, CSases, and heparinases) exhibit relatively conserved substrate selectivity. For example, CSases can degrade HA and CS / DS, but cannot degrade HP / HS, while heparinases can degrade HP / HS, but cannot degrade HA or CS / DS. The PL35 family is a recently established polysaccharide cleavage enzyme family, which belongs to the chondroitin sulfate cleavage enzyme family, but detailed studies and related applications of this family are very few. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a PL35 family chondroitin sulfate cleavage enzyme CHa3, an encoding gene thereof and applications, which has high efficient chondroitin sulfate degrading enzyme activity.

[0006] The technical scheme of the present application is as follows:

[0007] A chondroitin sulfate cleavage enzyme CHa3, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0008] The chondroitin sulfate cleavage enzyme CHa3 of the present application is an endo-enzyme, and when the chondroitin sulfate cleavage enzyme CHa3 degrades chondroitin sulfate polysaccharide, the end product is a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; when the chondroitin sulfate cleavage enzyme CHa3 degrades heparin, the end product is unsaturated disaccharide; when the chondroitin sulfate cleavage enzyme CHa3 degrades dermatan sulfate, the end product is unsaturated tetrasaccharide to unsaturated octasaccharide; the recombinant chondroitin sulfate cleavage enzyme CHa3 cannot degrade hyaluronic acid and heparan sulfate.

[0009] An encoding gene can encode the above-mentioned chondroitin sulfate cleavage enzyme CHa3.

[0010] According to the application, the nucleotide sequence encoding the gene is shown as SEQ ID NO. 1.

[0011] A recombinant expression vector, wherein the gene encoding the chondroitin sulfate lyase CHa3 is inserted into the expression vector.

[0012] According to the application, the expression vector is an E. coli expression vector, a yeast expression vector, a B. subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

[0013] A recombinant cell, wherein the gene encoding the chondroitin sulfate lyase CHa3 or the recombinant expression vector is inserted into the host cell.

[0014] According to the application, the host cell is an E. coli host cell, a yeast host cell, a B. subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell or a mammalian cell.

[0015] Further preferably, the E. coli host cell is Escherichia coli BL 21, Escherichia coli JM109 or Escherichia coli DH 5α; the yeast host cell is Saccharomyces cerevisiae, Pichia pastoris or Kluyveromyces lactis; the B. subtilis host cell is Bacillus subtilis R25 or Bacillus subtilis 9920; the lactic acid bacteria host cell is Lactic acid bacteria COCC101; the actinomycete host cell is Streptomyces spp.; the filamentous fungus host cell is Trichoderma viride, Trichoderma reesei, Aspergillus niger or Aspergillus nidulans; the insect cell is Bombyx mori or Antharaea eucalypti; and the mammalian cell is Chinese hamster ovary cell CHO, baby hamster kidney cell BHK or Chinese hamster lung cell CHL.

[0016] The recombinant cell is used for expressing the chondroitin sulfate lyase CHa3.

[0017] The chondroitin sulfate lyase CHa3 is used for preparing CS / DS oligosaccharide.

[0018] The application of the above-mentioned chondroitin sulfate lyase CHa3 in studying the structure and function of CS / DS.

[0019] Beneficial effects:

[0020] 1. The chondroitin sulfate lyase CHa3 in the present invention is isolated from a marine mud metagenomic library, is easy to heterologously express and purify, has stable physicochemical properties, high degradation activity, and has the potential for industrial application.

[0021] 2. The chondroitin sulfate lyase CHa3 in the present invention is a new type of chondroitin sulfate lyase with high enzymatic activity towards chondroitin sulfate. The enzymatic activities towards chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D and chondroitin sulfate E are 1.11 U / mg, 4.78 U / mg, 2.63 U / mg and 1.91 U / mg respectively.

[0022] 3. The chondroitin sulfate lyase CHa3 of the present invention degrades chondroitin sulfate in an endoenzymatic mode. When degrading chondroitin sulfate polysaccharides, the final products are a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides. When degrading heparin, the final products are primarily unsaturated disaccharides. When degrading dermatan sulfate, the final products range from unsaturated tetrasaccharides to unsaturated octasaccharides. The chondroitin sulfate lyase CHa3 of the present invention can be used for the preparation of CS / DS oligosaccharides and the study of CS / DS structure and function, and has broad application prospects in the fields of medicine and food.

[0023] 4. The chondroitin sulfate lyase CHa3 of the present invention produces a series of unsaturated oligosaccharides when degrading chondroitin sulfate A, including unsaturated disaccharides, unsaturated tetrasaccharides, unsaturated hexasaccharides, unsaturated octasaccharides, unsaturated decasaccharides, and unsaturated dodecasaccharides. The disaccharide composition analysis results of the above unsaturated oligosaccharides show that CHa3 is a typical chondroitin sulfate lyase C, and the oligosaccharides of the final product of CSA degradation are all composed of O, A and C units, and the number of A and C units decreases with the increase of oligosaccharide size. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a functional module analysis diagram of chondroitin sulfate lyase CHa3.

[0025] Figure 2 This is the evolutionary tree analysis diagram of chondroitin sulfate lyase CHa3.

[0026] Figure 3 This is the three-dimensional structure model of the protein chondroitin sulfate lyase CHa3.

[0027] Figure 4Polyacrylamide gel electrophoresis map of the expression and purification sample of chondroitin sulfate lyase CHa3; wherein: lane 1, protein molecular weight marker, 5 μL was loaded, and the sizes of the bands from top to bottom were 180 kDa, 130 kDa, 100 kDa, 70 kDa, 55 kDa, 40 kDa, 35 kDa, 25 kDa, and 15 kDa; lane 2, the bacterial liquid after the control strain was broken, 8 μL was loaded; lane 3, the bacterial liquid after the recombinant strain was broken, 5 μL was loaded; lane 4, the supernatant after the recombinant strain was broken, 4 μL was loaded; lane 5, the CHa3 enzyme liquid purified by a nickel column, 5 μL was loaded.

[0028] Figure 5 Temperature effect curve on the activity of chondroitin sulfate lyase CHa3.

[0029] Figure 6 pH effect curve on the activity of chondroitin sulfate lyase CHa3.

[0030] Figure 7 Temperature effect curve on the stability of chondroitin sulfate lyase CHa3.

[0031] Figure 8 Bar chart of the effect of metal ions and other compounds on the activity of chondroitin sulfate lyase CHa3.

[0032] Figure 9 NaCl concentration effect curve on the activity of chondroitin sulfate lyase CHa3.

[0033] Figure 10 High performance liquid chromatogram of the degradation products of chondroitin sulfate lyase CHa3 degrading different substrates; in the figure: Figure A is the HPLC chromatogram of hyaluronic acid (HA) as the substrate, Figure B is the HPLC chromatogram of chondroitin sulfate A (CSA) as the substrate, Figure C is the HPLC chromatogram of chondroitin sulfate C (CSC) as the substrate, Figure D is the HPLC chromatogram of chondroitin sulfate D (CSD) as the substrate, Figure E is the HPLC chromatogram of chondroitin sulfate E (CSE) as the substrate, Figure F is the HPLC chromatogram of dermatan sulfate (DS) as the substrate, Figure G is the HPLC chromatogram of heparin (HP) as the substrate, and Figure H is the HPLC chromatogram of heparan sulfate (HS) as the substrate; wherein, Di-1S, monosulfated GAGs disaccharide; Tetra, GAGs tetrasaccharide; Hexa, GAGs hexasaccharide; Octa, GAGs octasaccharide; Deca, GAGs deca-saccharide; Dodeca, GAGs dodeca-saccharide.

[0034] Figure 11HPLC profile of degradation products of CSD degraded by chondroitin sulfate lyase CHa3 for different times; in the figure: Di-1S, monosulfated CSD unsaturated disaccharide; Tetra, CSD unsaturated tetrasaccharide; Hexa, CSD unsaturated hexasaccharide; Octa, CSD unsaturated octasaccharide; Deca, CSD unsaturated decasaccharide; Dodeca, CSD unsaturated dodecasaccharide.

[0035] Figure 12 HPLC profile of disaccharide composition of the end product of chondroitin sulfate lyase CHa3 degradation of CSA: Figure A is the HPLC profile of the end product unsaturated disaccharide; Figure B is the HPLC profile of the disaccharide composition of the end product unsaturated tetrasaccharide; Figure C is the HPLC profile of the disaccharide composition of the end product unsaturated hexasaccharide; Figure D is the HPLC profile of the disaccharide composition of the end product unsaturated octasaccharide; Figure E is the HPLC profile of the disaccharide composition of the end product unsaturated decasaccharide; Figure F is the HPLC profile of the disaccharide composition of the end product unsaturated dodecasaccharide. DETAILED DESCRIPTION

[0036] The following examples are set forth to provide some usual techniques of how to practice the present application, but not to limit the scope of the application. The inventors have made utmost efforts to ensure the accuracy of various parameters (e.g., amount, temperature, etc.) in the examples, but some experimental errors and deviations should also be considered. Unless otherwise specified, the molecular weight in the present application refers to the weight average molecular weight, and the temperature refers to the Celsius degree. The reagents and materials involved in the examples are all ordinary commercially available products unless otherwise specified.

[0037] Biological material source:

[0038] The sea mud sample was collected from the intertidal zone of Hutouya in Yantai, Shandong Province (N37°18'31", E119°09'27"), and the sample was collected on July 8, 2021.

[0039] Example 1: Construction of metagenomic library

[0040] The intertidal zone sea mud sample was added to the sole carbon source medium and cultured at 28°C and 200 rpm for 3 days. 1 milliliter of bacterial liquid was transferred to fresh sole carbon source medium, and then cultured at 28°C, 200 rpm for 3 days. The culture was centrifuged at 8000 x g for 20 minutes to collect the cultured bacteria, and the metagenomic DNA was sequenced and annotated by Huada Gene Technology Service Co., Ltd. (Guangdong, China) to construct the metagenomic library.

[0041] The above-mentioned sole carbon source medium contains the following components per liter:

[0042] K2HPO41 g, MgSO4-7H2O 0.5 g, NaCl 30 g, CaCl2-2H2O 0.1 g, FeSO4 0.001 g, NH4Cl 1 g, Chondroitin sulfate C 5 g, water 1000 mL, pH 7.0.

[0043] Example 2: Extraction of bacterial DNA

[0044] 40 mL of the culture solution of Example 1 was centrifuged at 12,000 rpm for 25 min, and the bacterial precipitate was collected and washed with 20 mL of lysozyme buffer (10 mM Tris-HCl, pH 8.0), and then centrifuged at 12,000 rpm for 25 min to collect the bacterial precipitate;

[0045] To the bacterial precipitate, 12.0 mL of lysozyme buffer (10 mM Tris-HCl, pH 8.0) was added to obtain about 14.0 mL of bacterial solution, and 560 μL of lysozyme solution having a concentration of 20 mg / mL was added to give a final concentration of about 800 μg / mL. After ice-bath for 1.0 h and warm-bath at 37°C for 2 h, the solution became viscous. Then, 0.82 mL of 10 wt% SDS (sodium dodecyl sulfate) solution and 60 μL of 100 mg / mL protease K solution were added, and the mixture was subjected to water-bath at 52°C for 1.0 h. Then, 15 mL of Tris-equilibrated phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) was added, and the mixture was mixed well by inverting gently until it was fully emulsified. The mixture was centrifuged at 10,000 x g at 4°C for 10 min, and the supernatant was transferred. Then, 2.0 mL of NaAc-HAc buffer (pH 5.2, 3.0 M) and 17.0 mL of absolute ethanol were added, and the mixture was mixed well. Then, 1.0 mL of syringe tip was used to pick up the filamentous DNA, and the DNA was transferred to a 1.5 mL EP tube. The DNA was washed twice with 70 wt% ethanol (stored at -20°C) by microcentrifugation, and the supernatant was discarded. The DNA was centrifuged at 10,000 x g at 4°C for 3 min, and the supernatant was completely discarded. The sample was dried in a sterile workbench under the wind of an alcohol lamp. The DNA sample was resuspended in sterile deionized water, and the mixture was left to stand at 4°C overnight to obtain the genomic DNA having a large molecular weight.

[0046] Example 3: Sequence analysis of chondroitin sulfate lyase CHa3

[0047] The metagenomic library prepared in Example 1 was subjected to analysis of sequencing results using software on NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ). The NCBI analysis software used was Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0048] The NCBI analysis results showed that the chondroitin sulfate lyase gene cha3 in the metagenomic library had a coding region of 1884 bp in length, and its nucleotide sequence is shown in SEQ ID NO. 1. The chondroitin sulfate lyase gene cha3 encoded a chondroitin sulfate lyase CHa3 consisting of 627 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2. The theoretical molecular weight of the protein was about 69.25 kDa. Online analysis using BLAST software showed that the chondroitin sulfate lyase CHa3 had 28.84% homology with the chondroitin sulfate lyase chondroitinase encoded by the heparinase II / III family protein gene in the whole genome sequence of Victivallis vadensis that had been subjected to activity studies (NCBI sequence number: WP_116885688.1), which consisted of 792 amino acids.

[0049] Analysis of the structural information of the chondroitin sulfate lyase CHa3 using Simple Modular Architecture Research Tool (SMART, http: / / smart.embl-heidelberg.de / ) showed that the first to 35th amino acids at the N-terminus were signal peptide sequences, and the 416-555th amino acid sequences belonged to the Hepar-II-III family, as shown in Figure 1 By performing a neighbor-joining tree analysis of CHa3 and other types of chondroitin sulfate lyases that had been identified, CHa3 was found to be in the same evolutionary branch as the PL35 family, as shown in Figure 2The protein three-dimensional structure model of chondroitin sulfate lyase CHa3 was finally obtained by homology modeling of the protein three-dimensional structure of chondroitin sulfate lyase CHa3 using the SWISS-MODEL homology modeling server (http: / / swissmodel.expasy.org), as shown in Figure 1. Figure 3

[0050] Example 4: Recombinant expression of cha3 gene in E. coli

[0051] The macromolecular weight genomic DNA prepared in Example 2 was used as a template for PCR amplification, and the primers were as follows:

[0052] Forward primer cha3-F: 5'-ATCTGTATACATATGGCGATGGATACGGTGAAATTGGAG-3' (SEQ ID NO. 3);

[0053] Reverse primer cha3-R: 5'-GTGGTGGTGCTCGAGCAATGCTTTTAATCTAACCTTGATTTTC-3' (SEQ ID NO. 4);

[0054] PrimeSTAR HS DNA polymerase was purchased from Baobio Company, and the PCR reaction system was operated according to the product instructions provided by the company.

[0055] The reaction conditions of PCR amplification were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 40 s, 60°C annealing for 30 s, 72°C extension for 2 min, 35 cycles; 72°C extension for 10 min, 4°C stabilization for 15 min.

[0056] ​After the cha3 gene fragment was amplified by PCR, the PCR amplification product, pET-30a expression vector (the vector was linearized by double enzyme digestion of Nde I and Xho I) and recombinase Exnase II were prepared into a recombination reaction system, and the recombination reaction was completed at 37°C for 30 min to realize the in vitro circularization of the two linear DNAs. The recombinant product was directly transformed into Escherichia coli DH5α (purchased from Nanjing Novagen Company) strain, spread on LB solid medium containing 50 μg / mL kanamycin, and cultured at 37°C for 12-14 h. A single colony was picked into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm for 12-14 h. Bacterial liquid PCR was performed using the forward primer cha3-F and the reverse primer cha3-R. The result showed that the correct size of the amplification product was obtained, which preliminarily proved that the constructed recombinant plasmid was correct. 20 μL of the recombinant plasmid was taken to GenScript Biotech Co., Ltd. for sequencing. The sequencing result showed that the cha3 gene fragment (SEQ ID NO. 1) was successfully inserted between the enzyme digestion sites of the pET-30a expression vector, the insertion direction was correct, and no base mutation, deletion or addition occurred. Therefore, it was further proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET30a-cha3. The recombinase Exnase II was purchased from Baobio Company, the pET-30a expression vector was purchased from Novagen Company, USA, and the reaction system of the enzyme and the substrate, the reaction temperature and the reaction time all followed the instructions.

[0057] The recombinant plasmid pET30a-cha3 was transformed into Escherichia coli BL21 (DE3) (purchased from Nanjing Novagen Company), and then the induction expression of recombinant chondroitin sulfate lyase CHa3 was performed according to the operation steps provided by the company. The target protein was purified by NiSepharose 6Fast Flow (GE) gel to obtain CHa3 enzyme solution, and the purified target protein was detected by polyacrylamide gel electrophoresis. The detection result is shown in Figure 4 The purified chondroitin sulfate lyase CHa3 showed a single band on the electrophoresis gel, and the position was consistent with the predicted molecular weight, and the purity reached 95%.

[0058] Example 5: Enzymatic property analysis of recombinant chondroitin sulfate lyase CHa3

[0059] 1. Effect of temperature on enzyme activity

[0060] First, 10 mg / mL chondroitin sulfate C solution, 150 mM NaH2PO4-Na2HPO4 buffer solution (pH 8.0), CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio) and reacted at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C and 70°C for 30 min, respectively. After the reaction, the enzyme activity was determined by UV spectrophotometry, and the optimal enzyme activity was defined as 100% relative activity. The detection results are shown in Table 1. Figure 5 As shown in Table 1, the relative enzyme activity of CHa3 on chondroitin sulfate C is more than 90% at 20-30°C, and the enzyme activity of CHa3 rapidly decreases when the reaction temperature is above 40°C. Among them, the maximum activity of CHa3 on chondroitin sulfate C is achieved at 20°C, indicating that the optimal reaction temperature of recombinant chondroitin lyase CHa3 on chondroitin sulfate C is 20°C.

[0061] The method for determining enzyme activity by UV spectrophotometry is based on the prior art (Yamagata, T., et al., Purification and properties of bacterial chondroitinases and chondrosulfatases. The Journal of biological chemistry, 1968. 243(7): p. 1523-35), which uses inactivated enzyme as a negative control and determines the production of reaction products by spectrophotometry at 232 nm to determine the amount of enzyme activity.

[0062] 2. Effect of pH on enzyme activity

[0063] 10 mg / mL chondroitin sulfate C solution, reaction buffer, CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio), and the reaction buffer included 150 mM NaAc-Hac buffer (pH 5.0-6.0), 150 mM NaH2PO4-Na2HPO4 buffer (pH 6.0-8.0) and 150 mM Tris-HCl buffer (pH 7.0-10.0). The reaction was carried out at the optimal temperature for 30 min, and after the reaction, the enzyme activity was determined by UV spectrophotometry, and the optimal enzyme activity was defined as 100% relative activity. The detection results are shown in Table 2. Figure 6 As shown in Table 2, the enzyme activity of recombinant chondroitin lyase CHa3 on chondroitin sulfate C is the highest in NaH2PO4-Na2HPO4 buffer (pH 8.0).

[0064] 3. Effect of temperature on enzyme stability

[0065] The CHa3 enzyme solution after incubation treatment at different temperatures (0-70°C) for 1, 2, 4, 8, 12, 24h was mixed with 10mg / mL chondroitin sulfate C solution, and the residual enzyme activity was determined at the optimum temperature and pH, and the enzyme activity of CHa3 enzyme solution before incubation treatment was defined as 100% relative activity, and the detection results are shown in Figure 7 As shown in the table, the relative enzyme activity of recombinant chondroitinase CHa3 to chondroitin sulfate C remained more than 80% after 24h treatment at 0-20°C; the relative enzyme activity of recombinant chondroitinase CHa3 to chondroitin sulfate C sharply decreased after 24h treatment at more than 30°C; it is indicated that temperature has a greater impact on the stability of CHa3 enzyme, and it is not resistant to high temperature.

[0066] 4、Metal ion effect on enzyme activity

[0067] 10mg / mL chondroitin sulfate C solution, 150mM pH8.0 NaH2PO4-Na2HPO4 buffer, CHa3 enzyme solution and deionized water were mixed in the ratio of 10:10:3:4 (volume ratio), then different metal ions or compounds were added to the reaction system, the final concentration was 5mM, and the reaction was carried out at the optimum temperature for 30min, after the reaction, the residual enzyme activity was detected by ultraviolet spectrophotometry, and the enzyme activity without metal ions was defined as 100% relative activity, and the detection results are shown in Figure 8 As shown in the table, no metal ion showed strong promotion effect on the enzyme activity of CHa3, Fe 3+ , Zn 2+ , β-mercaptoethanol, DTT, Ni 2+ showed weak inhibition effect on the enzyme activity of CHa3, Ag + , Cd 2+ , Cu 2+ , Hg 3+ , Cr 3+ , Fe 2 + , EDTA and SDS showed strong inhibition effect on the enzyme activity of CHa3.

[0068] 5、NaCl concentration effect on enzyme activity

[0069] A 10 mg / mL chondroitin sulfate C solution, 150 mM pH 8.0 NaH2PO4-Na2HPO4 buffer, CHa3 enzyme solution, and deionized water were mixed in a ratio of 10:10:3:4 (volume ratio). Then, different final concentrations of NaCl were added to the reaction system. The final concentrations of NaCl added were 0 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 250 mM, 500 mM, and 1000 mM. The reaction was carried out at the optimal temperature for 30 minutes. After the reaction, the residual enzyme activity was detected by UV spectrophotometry. The enzyme activity at a final NaCl concentration of 0 mM was defined as 100% relative activity. The test results are shown in FIG. Figure 9 As shown in the figure, with the increase of NaCl concentration, the enzyme activity of CHa3 gradually decreased, indicating that NaCl has an inhibitory effect on the enzyme activity of CHa3.

[0070] Example 6: Determination of enzyme activity of recombinant chondroitin sulfate lyase CHa3

[0071] A 10 mg / mL chondroitin sulfate C solution, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CHa3 enzyme solution, and deionized water were mixed in a volume ratio of 10:10:3:7. The reaction was carried out at the optimal temperature for 1-10 minutes. An equal amount of inactivated enzyme was added to the negative control. After the reaction, the enzyme activity was determined using the UV spectrophotometric method described above.

[0072] The enzyme activities of chondroitin sulfate A, chondroitin sulfate D, and chondroitin sulfate E were determined according to the above method.

[0073] Definition of enzyme activity unit (U): the amount of enzyme required to generate 1 μmol of product containing unsaturated double bonds per minute.

[0074] At the same time, the protein content of CHa3 enzyme solution was determined using a protein quantification kit purchased from Kangwei Century Company. The results showed that the enzymatic activities of recombinant chondroitin sulfate lyase CHa3 for chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E were 1.11U / mg, 4.78U / mg, 2.63U / mg, and 1.91U / mg, respectively.

[0075] Example 7: High Performance Liquid Chromatography (HPLC) Analysis of Degradation Products of Different Substrates by Recombinant Chondroitin Sulfate Lyase CHa3

[0076] A 10 mg / mL substrate (chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, dermatan sulfate, heparin, heparan sulfate or hyaluronic acid) solution, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CHa3 enzyme solution and deionized water were mixed in a ratio of 10:10:3:7 (volume ratio) and reacted overnight at the optimum temperature. The degradation products were analyzed by HPLC under the following conditions: gel column: Superdex peptide 10 / 300 GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection condition: UV 232 nm.

[0077] The results are shown in Table 1. Figure 10 As shown in Table 1, the end products of the degradation of chondroitin sulfate polysaccharides by the recombinant chondroitin sulfate lyase CHa3 were a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides; the end product of the degradation of heparin by the recombinant chondroitin sulfate lyase CHa3 was unsaturated disaccharide; the end product of the degradation of dermatan sulfate by the recombinant chondroitin sulfate lyase CHa3 included unsaturated tetrasaccharides to unsaturated octasaccharides; the recombinant chondroitin sulfate lyase CHa3 could not degrade hyaluronic acid and heparan sulfate.

[0078] Example 8: Degradation mode of the recombinant chondroitin sulfate lyase CHa3

[0079] A 10 mg / mL CSD solution, a CHa3 enzyme solution, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0) and deionized water were mixed in a ratio of 10:3:10:7 (volume ratio) and reacted at the optimum temperature. The degradation products at different enzyme hydrolysis times were selected for HPLC analysis under the following conditions: gel column: Superdex peptide 10 / 300 GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection condition: UV 232 nm.

[0080] The results are shown in Table 2. Figure 11 As shown in Table 2, when CHa3 degraded CSD, a large amount of macromolecular unsaturated oligosaccharides was first generated, then the macromolecular oligosaccharides were gradually degraded into smaller molecular weight oligosaccharides, and finally a series of unsaturated oligosaccharides, including but not limited to unsaturated disaccharides to unsaturated dodecasaccharides, were generated. This result indicated that the recombinant chondroitin sulfate lyase CHa3 was an endo-enzyme.

[0081] Example 9: Analysis of the composition of the end product oligosaccharides of the degradation of CSA by the recombinant chondroitin sulfate lyase CHa3

[0082] A solution of 10 mg / mL chondroitin sulfate A, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CHa3 enzyme solution and deionized water were mixed in a ratio of 10:2:3:15 (volume ratio) and reacted overnight at the optimum temperature, and the degradation products were analyzed by HPLC. The HPLC analysis conditions were as follows: gel column: Superdex peptide 10 / 300 GL (GE); mobile phase: 0.2 M ammonium bicarbonate; flow rate: 0.4 mL / min; detection condition: UV 232 nm. The unsaturated disaccharide, unsaturated tetrasaccharide, unsaturated hexasaccharide, unsaturated octasaccharide, unsaturated decasaccharide and unsaturated dodecasaccharide components of the final product were recovered and freeze-dried three times to remove salts.

[0083] A solution of 2 μg / mL of each component of the final product, 150 mM NaH2PO4-Na2HPO4 buffer (pH 8.0), CSaseABC enzyme solution and deionized water were mixed in a ratio of 10:2:3:15 (volume ratio) and reacted overnight under optimum conditions, and the degradation products were analyzed by HPLC after 2-AB labeling. The HPLC analysis conditions were as follows: ion column: CarboPac PA1; mobile phase: 0.05-2 M sodium chloride; flow rate: 1 mL / min; detection condition: fluorescence detector, excitation light 330 nm, emission light 420 nm.

[0084] The results of the detection showed that the disaccharide composition of the unsaturated disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide and dodecasaccharide oligosaccharide end products of CSA degraded by the recombinant chondroitin sulfate lyase CHa3 were ΔC, ΔO / ΔA / ΔC (molar ratio 1:3:12), ΔO / ΔA / ΔC (molar ratio 1:1.62:3.5), ΔO / ΔA / ΔC (molar ratio 1:1.6:2.1), ΔO / ΔA / ΔC (molar ratio 1:1.9:1.5) and ΔO / ΔA / ΔC (molar ratio 1:2:1.3), respectively. Figure 12 These results showed that the disaccharide product produced by CHa3 when degrading the substrate was only ΔC, which is a characteristic of a typical chondroitin sulfate lyase C (mainly degrading the C unit section in CS). In addition, the oligosaccharide products were all composed of ΔO / ΔA / ΔC three disaccharide units, and the proportion of ΔA gradually increased as the size of the oligosaccharide increased, meaning that CHa3 tended to degrade the ΔC aggregation section and could not degrade other regions containing ΔC.

Claims

1. A chondroitin sulfate lyase CHa3, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. A coding gene, characterized in that Encodes the chondroitin sulfate lyase Cha3 according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that: The encoding gene according to claim 2 is inserted into the expression vector.

5. The recombinant expression vector according to claim 4, wherein The expression vector is an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

6. A recombinant cell, characterized in that The encoding gene according to claim 2 or the recombinant expression vector according to claim 4 is inserted into a host cell, and the host cell is an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell or a filamentous fungus host cell.

7. Use of the recombinant cell according to claim 6 in recombinantly expressing the chondroitin sulfate lyase CHa3 according to claim 1.

8. Use of the chondroitin sulfate lyase CHa3 according to claim 1 in the preparation of chondroitin sulfate oligosaccharides and / or dermatan sulfate oligosaccharides.

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