Chondroitin sulfate ABC I lyase mutant and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to chondroitin sulfate ABC I lyase as well as a mutant and application thereof. The present invention provides a chondroitin sulfate ABC I lyase CSABC I-NS1, and a mutant CSABC I-NS1-muout of the enzyme, and a preparation method of the chondroitin sulfate ABC I lyase CSABC I-NS1-muout. The chondroitin sulfate ABC I lyase CSABC I-NS1 is derived from Proteus mirabilis, a mutant CSABCI-NS1-E502F with high enzyme activity and high stability is obtained after site-specific mutagenesis, the specific enzyme activity of the mutant CSABCI-NS1-E502F is 72.53 U / mg, and the half-life period of the mutant CSABCI-NS1-E502F at 37 DEG C is 96.80 h; and the method has important significance for realizing biological industrial preparation of low-molecular-weight chondroitin sulfate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a chondroitin sulfate ABC I lyase mutant and application thereof. BACKGROUND

[0002] Chondroitin sulfate (CS) is a kind of glycosaminoglycan covalently connected to proteins to form proteoglycans. Chondroitin sulfate plays an important role in processes including neuronal development, blood clotting, inflammation, cell transport, cell proliferation and tumor occurrence. As a naturally occurring component in connective tissue, chondroitin sulfate can bind with water molecules to lubricate and support joints, allowing joints to move freely and reduce joint pain. It can also be used as a food additive for emulsification, antioxidant, moisture retention and odor removal in food. In addition, adding chondroitin sulfate in cosmetics can regulate skin cell metabolism, promote nutrient absorption and waste excretion, maintain skin moisture and improve hair quality. Currently, the US Food and Drug Administration (FDA) has approved non-animal extracted CS for food additives, and the European Union has also listed CS as a new resource food. Chondroitin sulfate needs to act in cells, but its large molecular volume affects its smooth passage through the thin membrane on the surface of cells, and it cannot exert its various good pharmacological functions in the body. Low molecular weight chondroitin sulfate (LMWCSs) with a relative molecular mass of less than 10 kDa prepared by degrading chondroitin sulfate with chondroitin sulfate lyase is relatively easy to absorb through the gastrointestinal tract and can effectively overcome the above shortcomings. At present, the chondroitin sulfate series products used at home and abroad mainly include the first generation product chondroitin sulfate sodium, the second generation product chondroitin calcium sulfate and the third generation product low molecular weight chondroitin sulfate. Compared with the first and second generation products, low molecular weight chondroitin sulfate is more easily absorbed by the body and has better efficacy in treating coronary heart disease and arthritis. Therefore, the third generation low molecular weight chondroitin sulfate may have a broader application prospect.

[0003] Chondroitin sulfate lyase (chondroitinase or chondroitin sulfate yase, referred to as "ChSase" for short) is a kind of glycosaminoglycan lyase from microorganisms, which can degrade various glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, chondroitin and hyaluronic acid into unsaturated disaccharides and oligosaccharides. According to the difference of substrate spectrum, chondroitin sulfate lyase is mainly divided into three types: chondroitin sulfate lyase AC, chondroitin sulfate lyase B and chondroitin sulfate lyase ABC. Among them, chondroitin sulfate ABC lyase is the most specific one. According to the lytic mechanism, it can be divided into endogenous elimination enzyme (ChABC I, which can depolymerize chondroitin sulfate into unsaturated tetrasaccharides and disaccharides) and exogenous elimination enzyme (ChABC II, which can degrade chondroitin sulfate hexatetrasaccharide into disaccharide). Among them, the application of ChABC I in chondroitin sulfate oligosaccharide degradation has good medical prospects.

[0004] At present, there are literatures and patents reporting chondroitin sulfate lyases from heparin flavobacterium, sphingomonas subrtilis, mild aeromonas, proteus penneri, pseudomonas, photobacterium, proteus vulgaris, bacteroides thetaiotaomicron, acinetobacter, microbacterium WS15 and other microorganisms. However, the wild strain fermentation cycle is long, the process is complex, the purification steps are many, the enzyme activity and yield are low, which is not conducive to industrial amplification. For example, patent CN112126606B discloses a chondroitin sulfate lyase derived from photobacterium sp. and its production, but the cell fermentation broth needs to be treated by solid-liquid separation, ammonium sulfate precipitation, dialysis and other series of treatments to obtain chondroitin sulfate lyase. The steps are very cumbersome and the industrialization cost is high.

[0005] Heterologous recombinant expression is a means to obtain chondroitin sulfate lyase efficiently and quickly. For example, patent application CN106148265B discloses a recombinant bacillus subtilis producing chondroitin sulfate lyase, and the enzyme activity is only 21.4 U / mL; patent application CN111518822B discloses a molecular modification of recombinant chondroitin sulfate lyase ABC I, and the enzyme activity of the mutant is 57.6 U / mg after modification; patent application CN112430589B discloses a chondroitin sulfate ABC lyase mutant with high thermal stability, and the half-life of mutant NΔ5 / E689P at 37℃ is 19h, which is 247 times higher than that of wild type, and the enzyme activity increases to 788 U / g after mutation, and the protein expression amount is 1.7 g / L.

[0006] At present, a large number of studies on chondroitin sulfate lyase show that the enzyme has problems of low activity, low thermal stability and high cost, and is difficult to be applied to industrial production of low molecular weight chondroitin sulfate. Therefore, it is an urgent problem to be solved to improve the enzyme activity and thermal stability of the enzyme by genetic engineering means. SUMMARY

[0007] In order to solve the problems of low activity and low thermal stability of chondroitin sulfate lyase in the prior art, the present application provides a recombinant chondroitin sulfate ABC I lyase mutant, a coding gene thereof, a recombinant vector containing the coding gene of the mutant, a recombinant genetically engineered bacterium obtained by transformation of the recombinant vector, and an application in the preparation of low molecular weight chondroitin sulfate.

[0008] The present application provides a chondroitin sulfate ABC I lyase mutant (named CSABC I-NS1-E502F), which is obtained by single mutation of the 502th amino acid sequence shown in SEQ ID NO. 1 based on chondroitin sulfate lyase ABC I (GenBank NO.: HDT2999984.1) derived from Proteus mirabilis sp. as a starting strain (named CSABC I-NS1), and the mutant amino acid sequence is shown in SEQ ID NO. 3, and the mutant nucleotide sequence is shown in SEQ ID NO. 2.

[0009] The present application provides a coding gene of the above-mentioned chondroitin sulfate ABC I lyase mutant.

[0010] The present application provides an expression vector into which the coding gene of the mutant is inserted.

[0011] In an embodiment of the present application, the expression vector is preferably an E. coli expression vector, and more preferably pET-28a(+).

[0012] The present application provides a host cell expressing the mutant.

[0013] In an embodiment of the present application, the host cell is preferably E. coli BL21 (DE3).

[0014] The present application provides a method for expressing the above-mentioned chondroitin sulfate ABC I lyase mutant or a microbial cell containing the coding gene.

[0015] Further, the method for expressing the above-mentioned chondroitin sulfate ABC I lyase mutant comprises linking the coding gene of the mutant with an expression vector to obtain a recombinant plasmid, transforming the recombinant plasmid into a host cell to obtain a recombinant genetically engineered bacterium, and culturing and fermenting the recombinant genetically engineered bacterium to produce the enzyme. Further, the method for expressing the above-mentioned chondroitin sulfate ABC I lyase mutant comprises linking the coding gene of the mutant with an expression vector to obtain a recombinant plasmid, transforming the recombinant plasmid into a host cell to obtain a recombinant genetically engineered bacterium, and culturing and fermenting the recombinant genetically engineered bacterium to produce the enzyme.

[0016] In one embodiment of the present application, the recombinant genetically engineered bacteria are cultured to a bacterial body OD 600 of 0.6-0.8, IPTG is added to a final concentration of 0.2 mM, and the culture is induced at 20°C for 20 h. After the induction is completed, the fermentation broth is subjected to solid-liquid separation by a low-temperature high-speed refrigerated centrifuge to obtain wet bacterial bodies.

[0017] The present application provides a method for expressing the chondroitin sulfate ABC I lyase mutant, which comprises subjecting the wet bacterial bodies obtained after fermentation to ultrasonic disruption, centrifuging to obtain a crude enzyme solution, and subjecting the crude enzyme solution to chromatography preparation using a Cytiva HisTrap HP column and a Cytiva HiPrep 26 / 10 to obtain pure enzyme.

[0018] The present application also provides a method for determining the kinetic parameters of the pure enzyme obtained by expressing the chondroitin sulfate ABC I lyase mutant.

[0019] The present application also provides a method for determining the thermal stability of the pure enzyme obtained by expressing the chondroitin sulfate ABC I lyase mutant.

[0020] The present application also provides a method for determining the half-life of the pure enzyme obtained by expressing the chondroitin sulfate ABC I lyase mutant.

[0021] The present application also provides the use of the chondroitin sulfate ABC I lyase mutant in the preparation of low molecular weight chondroitin sulfate.

[0022] The present application has the following beneficial effects: the recombinant chondroitin sulfate lyase gene is screened by gene mining technology, and the mutant is obtained by molecular modification through protein engineering technology; the half-life of the mutant at 50°C is increased from 1.7 h before mutation to 2.9 h, which is about 0.7 times higher than that of the wild type, and the half-life of the mutant at 37°C is 96.80 h, which is significantly higher than that of the wild type. The thermal stability and enzyme activity of the mutant are significantly improved. The mutant obtained by the present application can be used to prepare low molecular weight chondroitin sulfate to overcome the technical bottleneck of poor thermal stability of enzymes in the current biological catalytic synthesis of low molecular weight chondroitin sulfate. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a comparison chart of the whole cell enzyme activity of CSABC I-NS1 and each mutant in Example 2.

[0024] Figure 2 It is a chart for studying the optimum reaction temperature of CSABC I-NS1-E502F pure enzyme in Example 4.

[0025] Figure 3Figure for determination of Michaelis-Menten curve for CSABC I-NS1 and CSABC I-NS1-E502F pure enzymes in Example 5.

[0026] Figure 4 Figure for determination of T of CSABC I-NS1 and CSABC I-NS1-E502F in Example 6. 50 15 Figure for determination of values.

[0027] Figure 5 Figure for determination of half-life of CSABC I-NS1 and CSABC I-NS1-E502F at 50°C in Example 8.

[0028] Figure 6 Figure for determination of half-life of CSABC I-NS1-E502F at 37°C in Example 9. DETAILED DESCRIPTION

[0029] The present application is explained by specific embodiments below, and other advantages and effects of the present application can be easily understood by those skilled in the art from the contents disclosed in the present specification. The present application can also be implemented or applied by other different embodiments, and each detail in the present specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict. The methods used in the embodiments of the present application are conventional methods unless otherwise specified, and the reagents used can be obtained from commercial channels.

[0030] The relevant nucleotide sequence information in the sequence listing is as follows: SEQ ID NO. 1 is the amino acid sequence encoding chondroitin sulfate ABC I lyase, SEQ ID NO. 2 is the nucleotide sequence encoding the mutant of chondroitin sulfate ABC I lyase, and SEQ ID NO. 3 is the amino acid sequence encoding the mutant of chondroitin sulfate ABC I lyase.

[0031] Example 1 Preparation of mutant of chondroitin sulfate ABC I lyase

[0032] A chondroitin sulfate ABC I lyase from Proteus mirabilis sp. (named CSABC I-NS1, GenBank NO. HDT2999984.1), the amino acid sequence of which is shown in SEQ ID NO. 1, was used as a template for site-directed mutagenesis. The amino acid sequence shown in SEQ ID NO. 1 was ligated to pET-28a (+), and the sequences of the site-directed mutagenesis primers are shown in Table 1. Mutations were introduced by PCR, and the PCR reaction program was as follows: 98 °C for 30 s; 98 °C for 10 s, 65 °C for 5 s, 72 °C for 1 min, repeated for 30 cycles; and 72 °C for 1 min for further extension. The linearized plasmid obtained by PCR was treated with Dpn I at 37 °C for 15 min, inactivated at 80 °C for 20 min, and then transformed into E. coli BL21 (DE3) competent cells, which were spread on LB solid plates containing kanamycin (final concentration 50 mg / L) and incubated at 37 °C overnight.

[0033] Table 1 Primer sequences

[0034]

[0035] Note: NNK represents a degenerate codon that can encode 20 kinds of random amino acids. M represents an A or C base, and MNN is complementary to NNK.

[0036] Example 2 Whole-cell enzyme activity determination of chondroitin sulfate ABC I lyase and mutants and obtaining of superior mutants

[0037] Whole-cell enzyme activity determination method: Chondroitin sulfate was used as a substrate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using a buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). A 50 g / L whole-cell working solution (50 mM Tris-HCl-200 mM NaAc, pH 7.4) was prepared. The enzyme activity determination reaction system was 1 mL, containing 50 μL of the whole-cell working solution and 950 μL of the substrate working solution, with 2 parallel reactions for each reaction. The reaction system was preheated at 37 °C and 800 rpm for 10 min, 50 μL of the whole-cell working solution was added, the reaction was performed for 5 min, and 6 M HCl was added to terminate the reaction. Centrifugation was performed at 14000 rpm for 10 min, the supernatant was taken, diluted, and the absorbance value at 232 nm was determined.

[0038] Enzyme activity definition (U): 1 U is the amount of enzyme required to generate 1 μmoL of product 4, 5-unsaturated uronic acid per minute at 37 °C. The enzyme activity of the wild-type CSABC I-NS1 was defined as 100%,

[0039] Mutant primary screening: using the experimental method in Example 1, 54 single colonies were picked and inoculated in LB liquid containing kanamycin, cultured at 37°C, 220 rpm to an OD 600 0.6-0.8, 0.2 mM IPTG was added, and the culture was induced at 20°C for 20 h. After centrifugation at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain recombinant E. coli containing recombinant chondroitin sulfate lyase ABC I mutant. The whole-cell enzyme activity was detected by the above method. The results are shown in Table 1. Among them, 13 mutants with higher enzyme activity (numbered M-7, M-12, M-16, M-17, M-18, M-23, M-24, M-25, M-27, M-29, M-31, M-32, M-40) were selected for rescreening. Figure 1

[0040] Mutant rescreening: using the experimental method in Example 1, the above 13 mutants were inoculated in LB liquid containing kanamycin, cultured at 37°C, 220 rpm for 12 h, then inoculated in 100 mL LB liquid containing kanamycin at 2% inoculation amount (v / v), and cultured at 37°C, 180 rpm until the OD 600 0.6-0.8, 0.2 mM IPTG was added, and the culture was induced at 20°C for 20 h. After centrifugation at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain recombinant E. coli containing recombinant chondroitin sulfate lyase ABC I mutant. The whole-cell enzyme activity was detected by the above method. The results are shown in Table 2. The enzyme activity of strains M-18, M-23, M-31, and M-40 was significantly improved compared to the original strain. They were sent to a sequencing company for sequencing verification. Sequencing showed that mutants M-18, M-23, and M-31 were mutated from glutamic acid (E) at position 502 to asparagine (N), and M-40 was mutated from glutamic acid (E) at position 502 to phenylalanine (F).

[0041] Mutants E. coli BL21(DE3) / pET-28a-CSABC I-NS1-E502N (hereinafter referred to as CSABC I-NS1-E502N) and E. coli BL21(DE3) / pET-28a-CSABC I-NS1-E502F (hereinafter referred to as CSABC I-NS1-E502F) were taken for protein purification to further determine the dominant mutant.

[0042] Table 2: Comparison of whole-cell enzyme activity of CSABC I-NS1 and each mutant

[0043] ​

[0044] Preparation of pure enzyme of recombinant chondroitin sulfate lyase ABC I mutant and detection of enzyme activity

[0045] Preparation of pure enzyme: the wet bacteria of two single mutants CSABC I-NS1-E502N and CSABC I-NS1-E502F obtained by the method in Example 2 were resuspended in buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4) at 0.1 g / mL, and the crude enzyme solution was obtained by crushing at 120 W. The supernatant was obtained by centrifugation, and purified using a Cytiva HisTrap HP column. 50 mM Tris-HCl-200 mM NaAc-80 mM imidazole was used to elute impurities, and 50 mM Tris-HCl-200 mM NaAc-500 mM imidazole was used to elute the target protein. The Cytiva HiPrep 26 / 10 column was used to remove imidazole to obtain pure enzyme. The pure enzyme of E. coli BL21(DE3) / pET-28a-CSABC I-NS1 (hereinafter referred to as CSABC I-NS1) was prepared in the same way as a control.

[0046] Detection of enzyme activity of pure enzyme: chondroitin sulfate was used as the substrate, and the substrate working solution was prepared using buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). In a 1 mL reaction system, 5 μg of pure enzyme was added, and the reaction was carried out in a constant temperature shaking metal bath at 37°C and 800 rpm for 2 min. After the reaction was completed, 100 μL of 6 M HCl was added to terminate the reaction. After the catalytic reaction was completed, the supernatant was obtained by centrifugation, and the amount of product 4,5-unsaturated uronic acid was determined by spectrophotometry, i.e., the OD value was detected. 232 The absorbance value was calculated to calculate the enzyme activity. The enzyme activity U is defined as the amount of enzyme required to generate 1 μmol of 4,5-unsaturated uronic acid per minute (molar extinction coefficient 3800 L / mol·cm).

[0047] Calculation formula:

[0048] Table 3. Comparison of pure enzyme activity of CSABC I-NS1 and each mutant

[0049]

[0050] The enzyme activity results are shown in Table 3: the enzyme activity of the two single-point mutants is improved compared with the wild type, and the most obvious improvement is the mutant CSABC I-NS1-E502F. Therefore, CSABC I-NS1-E502F is used as the dominant mutant for subsequent research.

[0051] Example 4 Determination of optimal reaction temperature of CSABC I-NS1-E502F pure enzyme

[0052] The substrate was chondroitin sulfate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using a buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). In a 1 mL reaction system, 5 μg of the pure enzyme obtained in Example 3 was added, and the reaction was carried out in a constant temperature shaking metal bath at 25, 30, 37, 40, 45, 50, 55 °C, respectively, at 800 rpm for 1 min. After the reaction was completed, 100 μL of 6 M HCl was added to terminate the reaction. After the catalytic reaction was completed, the supernatant was separated by centrifugation, and the OD 232 absorbance value was detected and the enzyme activity was calculated. As shown in Figure 2 , the optimal reaction temperature of the mutant CSABC I-NS1-E502F pure enzyme was 30 °C, and therefore the reaction temperature for the determination of enzyme activity in the subsequent characterization of kinetic parameters and thermal stability was 30 °C.

[0053] Example 5 Determination of kinetic parameters of CSABC I-NS1-E502F pure enzyme

[0054] The substrate was chondroitin sulfate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using a buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). In a 1 mL reaction system, 5 μg of the pure enzyme obtained in Example 3 was added, and the reaction was carried out in a constant temperature shaking metal bath at 25, 30, 37, 40, 45, 50, 55 °C, respectively, at 800 rpm for 1 min. After the reaction was completed, 100 μL of 6 M HCl was added to terminate the reaction. After the catalytic reaction was completed, the supernatant was separated by centrifugation, and the OD 232 absorbance value was detected and the enzyme activity was calculated. By GraphPad Prism-function nonlinear fitting (Michaelis-Menten) curve, the fitting results are shown in Figure 3 , and the apparent kinetic parameters are shown in Table 4. The maximum reaction rate (V max ) of wild-type CSABC I-NS1 was 76.80 (mg / mL / min), and the maximum reaction rate (V max ) of CSABC I-NS1-E502F was 50.72 (mg / mL / min), which was significantly improved compared with the wild type. The turnover number (k cat ) of CSABC I-NS1-E502F was 9.22 x 10 5 s -1 which was significantly improved compared with the wild type, indicating that the mutant had a higher catalytic rate.

[0055] Table 4 Kinetic parameters of CSABC I-NS1 and mutants on chondroitin sulfate

[0056]

[0057] Example 6 Tm value determination of CSABC I-NS1-E502F pure enzyme 50 15 value determination

[0058] Chondroitin sulfate was used as substrate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). 5 μg of pure enzyme obtained in Example 3 was incubated at 4, 25, 30, 37, 40, 45, 50, 55, 60, and 65 °C for 15 min, and then immediately placed in ice water for 10 min. In a 1 mL reaction system, the enzyme solution after ice water bath was added, and the reaction was carried out in a constant temperature shaking metal bath at 30 °C and 800 rpm for 1 min. After the reaction, 100 μL of 6 M HCl was added to terminate the reaction. After the catalytic reaction, the supernatant was separated by centrifugation, and the OD 232 absorbance value was detected and the enzyme activity was calculated. The fitting results are shown in Figure 2, and the T Figure 4 value of wild-type CSABC I-NS1 is 51.30 °C, and the T 50 15 value of CSABC I-NS1-E502F is 51.87 °C, which is 0.57 °C higher than that of the wild type. 50 15

[0059] Example 7 Tm value determination of CSABC I-NS1-E502F pure enzyme

[0060] The pure enzyme obtained in Example 3 was used to detect the Tm value by qPCR system using Protein Thermal Shift TM dye kit. The reaction program was as follows: 25 °C for 120 s; 0.05 °C / s to 99 °C; 99 °C for 120 s. The results are shown in Table 5, the Tm value of CSABC I-NS1 is 55.15 °C, and the Tm value of CSABC I-NS1-E502F is 58.76 °C, which is 3.16 °C higher than that of the wild type, which is significantly improved, indicating that the structural stability of mutant CSABC I-NS1-E502F is greatly improved.

[0061] Table 5 Tm value determination of CSABC I-NS1-E502F pure enzyme

[0062] ​

[0063] Half-life determination of CSABC I-NS1-E502F pure enzyme at 50°C

[0064] Chondroitin sulfate was used as substrate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). 5 μg of the pure enzyme obtained in Example 3 was incubated at 50°C for 0-25 h, and samples were taken at 0 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 25 h, and immediately ice-bathed after sampling. The ice-bathed enzyme solution was added to a constant temperature shaking metal bath at 30°C, and reacted for 1 min at 800 rpm. After the reaction was completed, 100 μL of 6M HCl was added to terminate the reaction. After the catalytic reaction was completed, the supernatant was separated by centrifugation, and the OD 232 absorbance value was detected and the enzyme activity was calculated. The fitting result is shown in FIG. 6, and the fitting equation was obtained by Origin 9.1-function nonlinear fitting (ExpDec1). The half-life was calculated to be 1.7 h at 50°C for CSABC I-NS1, and 2.9 h at 50°C for CSABC I-NS1-E502F, indicating that the thermal stability of mutant CSABC I-NS1-E502F was greatly improved. Figure 5

[0065] Half-life determination of CSABC I-NS1-E502F pure enzyme at 37°C

[0066] Chondroitin sulfate was used as substrate, and the substrate concentration was 5 mg / mL. The substrate working solution was prepared using buffer (50 mM Tris-HCl-200 mM NaAc, pH 7.4). 5 μg of the pure enzyme obtained in Example 3 was incubated at 50°C for 0-312 h, and samples were taken at 0, 2, 4, 8, 24, 31.5, 49, 55.5, 72, 79, 100, 124, 197, 246, 312 h, and immediately ice-bathed after sampling. The ice-bathed enzyme solution was added to a constant temperature shaking metal bath at 37°C, and reacted for 2 min at 800 rpm. After the reaction was completed, 100 μL of 6M HCl was added to terminate the reaction. After the catalytic reaction was completed, the supernatant was separated by centrifugation, and the OD 232 absorbance value was detected and the enzyme activity was calculated. The fitting result is shown in FIG. 6, and the fitting equation was obtained by Origin 9.1-function nonlinear fitting (ExpDec1). The half-life was calculated to be 1.7 h at 50°C for CSABC I-NS1, and 2.9 h at 50°C for CSABC I-NS1-E502F, indicating that the thermal stability of mutant CSABC I-NS1-E502F was greatly improved. Figure 6 ​​

[0067] The above-described embodiments are merely intended to describe the preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements of the present application made by those skilled in the art are intended to fall within the scope of the present application.

Claims

1. A chondroitin sulfate lyase ABC I mutant, characterized in that, The mutant is a parent strain of chondroitin sulfate lyase ABC I with amino acid sequence as shown in SEQ ID NO. 1, and the amino acid sequence of the parent strain is mutated at position 502 to obtain.

2. A mutant of chondroitin sulfate lyase ABC I according to claim 1, characterized in that, The amino acid sequence is as shown in SEQ ID NO. 3, and the nucleotide sequence is as shown in SEQ ID NO.

2.

3. A gene encoding the chondroitin sulfate lyase ABC I mutant of claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector is inserted into the coding gene of the chondroitin sulfate lyase ABC I mutant of claim 3; further preferably, the expression vector is an E. coli expression vector.

5. A host cell, characterized in that, The host cell is inserted with the coding gene of the chondroitin sulfate lyase ABC I mutant of claim 3; further preferably, the host cell is E. coli BL21 (DE3).

6. A microbial cell expressing the chondroitin sulfate lyase ABC I mutant of claim 1 or 2, or containing the gene of claim 3.

7. A method of expressing the chondroitin sulfate lyase ABC I mutant of claim 1, characterized by, The coding gene of claim 3 is connected with an expression vector to obtain a recombinant plasmid, the recombinant plasmid is transformed into a host cell to obtain a recombinant genetically engineered bacterium, and the recombinant genetically engineered bacterium is subjected to fermentation culture and enzyme production.

8. The method of expressing a chondroitin sulfate lyase ABC I mutant according to claim 7, wherein, The genetically engineered bacteria are cultured to an OD 600 0.6-0.8, IPTG is added to a final concentration of 0.2 mM, and the culture is incubated at 20°C for 20 h.

9. The method of expressing a chondroitin sulfate lyase ABC I mutant according to claim 8, wherein, After induction culture, centrifugation is performed at 4℃ and 8000 rpm for 10 min, the supernatant is discarded, and the precipitate is collected to obtain a wet bacterium containing the recombinant chondroitin sulfate lyase ABC I mutant gene, which can be directly used as a biological catalyst or for protein purification.

10. A method of preparing low molecular weight chondroitin sulfate, characterized in that, The chondroitin sulfate lyase ABC I mutant of claim 1 or 2 is used to lyse chondroitin sulfate.

11. Use of the chondroitin sulfate lyase ABC I mutant of claim 1 or 2 in the preparation of low molecular chondroitin sulfate.

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