A chondroitin sulfate ac lyase mutant with high catalytic activity and high thermal stability and application thereof

Abstract

The application discloses a chondroitin sulfate AC lyase mutant with high catalytic activity and high thermal stability and application thereof, and belongs to the technical field of bioengineering. Pedobacter xixiisoli The application carries out site-directed mutation on chondroitin sulfate AC lyase from Bacillus sp. V max and k cat / K m The catalytic activity and thermal stability of the mutant Y301E are most significantly improved, the catalytic activity and thermal stability of the mutant Y301E are 145.35% and 106.58% of M1 respectively, and the half-life at 37 o C is 1188.78 min, which is 1.94 times of M1. SAX-HPLC analysis shows that the mutation influences the recognition preference of the enzyme to the sulfation mode of the substrate under the premise of maintaining the enzyme-substrate recognition mechanism, enhances the cutting activity to the monosulfated region of the substrate, and increases the feasibility for the practical application of the chondroitin sulfate AC lyase.

Description

Technical Field

[0001] This invention relates to a chondroitin sulfate AC lyase mutant with both high catalytic activity and high thermal stability and its applications, belonging to the field of bioengineering technology. Background Technology

[0002] Chondroitin sulfate is one of the major types of glycosaminoglycans, widely distributed in animal connective tissues. Its molecular structure consists of alternating and different forms of sulfated D-glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) disaccharide units.

[0003] Chondroitin sulfate, as an important biomolecule, exhibits high biological activity, such as anti-inflammatory, antioxidant, and anticancer effects. In vivo, it can interact with various signaling molecules, thereby regulating cell behavior, participating in signal transduction, and promoting tissue repair. It is currently widely used as a symptom reliever in the field of osteoarthritis. However, due to its large molecular weight and low bioavailability, chondroitin sulfate's absorption efficiency and pharmacological activity are severely affected.

[0004] Recent studies have shown that low molecular weight chondroitin sulfate in the range of 3000–5000 Da possesses strong pharmacological activity; ultra-low molecular weight chondroitin sulfate, with a molecular weight further reduced to below 3000 Da, exhibits even more significant advantages in oral absorption and bioactivity. Therefore, the preparation of low molecular weight chondroitin sulfate is essential.

[0005] Currently, the main methods for preparing low molecular weight chondroitin sulfate are: (1) chemical degradation, which mainly includes acid degradation and oxidative degradation. These methods break the glycosidic chain by using strong acids such as hydrochloric acid or by using oxidants such as hydrogen peroxide to attack the polysaccharide chain. They have disadvantages such as long processing time, difficulty in product separation and purification, and easy loss of sulfate groups during degradation; (2) physical degradation, which includes ultrasonic degradation, microwave degradation, and radiation degradation. These methods require advanced experimental equipment and are more complex; (3) enzymatic hydrolysis, which uses chondroitin sulfate lyase to break the β(1,4) glycosidic bond between GalNAc and GlcA in chondroitin sulfate to obtain low molecular weight chondroitin sulfate. Compared with physical separation and chemical degradation methods, enzymatic hydrolysis has received widespread attention due to its advantages such as mild reaction conditions, high specificity, environmental friendliness, few side reactions, and easy monitoring of the reaction process.

[0006] Chondroitin sulfate lyases are a class of lyases that specifically degrade the β(1,4) glycosidic bonds between the disaccharide units of chondroitin sulfate, dermatan sulfate, and hyaluronic acid via a β-elimination mechanism, forming unsaturated oligosaccharides. Based on substrate specificity, chondroitin sulfate lyases are classified into chondroitin sulfate lyase AC, chondroitin sulfate lyase B, chondroitin sulfate lyase C, and chondroitin sulfate lyase ABC. Among them, chondroitin sulfate AC lyase can specifically cleave the glycosidic bonds between the repeating disaccharide units of chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid, playing an important role in the preparation and identification of low molecular weight chondroitin sulfate and the structural analysis of chondroitin sulfate. However, the application of chondroitin sulfate AC lyase has been limited by its low catalytic activity and low stability. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a chondroitin sulfate AC lyase mutant with improved catalytic activity and thermal stability, and its applications. Pedobacter xixiisoli The chondroitin sulfate AC lyase was used as the parent, and its amino acid sequence is shown in SEQ ID NO.1. Then, through site-directed mutagenesis, a chondroitin sulfate AC lyase mutant with significantly enhanced catalytic activity and thermostability was obtained.

[0008] The specific technical solution is as follows: The chondroitin sulfate AC lyase mutant, possessing both high catalytic activity and high thermal stability, is formed by any one or more of the following mutations based on the amino acid sequence shown in SEQ ID NO.1: The mutant is one of the following: (1) The lysine at position 295 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamic acid; (2) The tyrosine at position 301 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid; (3) The tyrosine at position 301 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamic acid; (4) The serine at position 551 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine; This invention provides the encoding gene for the chondroitin sulfate AC lyase mutant.

[0009] The present invention provides an expression vector containing the coding gene.

[0010] The present invention provides microbial cells containing the mutant, or containing the coding gene, or containing the expression vector.

[0011] The present invention provides a method for expressing the chondroitin sulfate AC lyase mutant, characterized in that the gene is ligated to an expression vector to obtain a recombinant plasmid, the recombinant plasmid is transformed into a host cell to obtain a recombinant microbial cell, and the recombinant microbial cell is cultured to produce the enzyme.

[0012] In one embodiment of the present invention, the recombinant microbial cells are first cultured to mid-logarithmic phase, and the mid-logarithmic bacterial culture is inoculated into the reaction system. After culturing at 37°C and 220 rpm for 2 h, IPTG with a final concentration of 0.2 mM is added for induction, and the culture is carried out at 25°C and 220 rpm for 15 h.

[0013] This invention provides the application of the chondroitin sulfate AC lyase mutant in the degradation of chondroitin sulfate and the preparation of low molecular weight chondroitin sulfate.

[0014] A method for preparing low molecular weight chondroitin sulfate, wherein chondroitin sulfate is cleaved using the chondroitin sulfate AC lysin mutant (preferably mutant Y301E); the amount of enzyme added in the reaction system is 5.80 × 10⁻⁶. 4 ~9.23×10 4 Ug -1 Substrate; reaction temperature: 37℃.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention, through site-directed and iterative mutagenesis of chondroitin sulfate AC lyase, yielded a series of chondroitin sulfate AC lyase mutants with significantly enhanced catalytic activity and thermostability. Among these, four single mutants—K295E, Y301D, Y301E, and S551T—showed significant improvements in thermostability. Of the four single mutants, Y301E exhibited the most significant improvement in catalytic activity and thermostability. V max and k cat / K m The values ​​were 1244.73 U mg. -1 and 2054.83 mL s -1 mg -1 It is 145.35% and 106.58% of M1. The half-life at 37°C increased from 612.79 min for M1 to 1188.78 min, which is 1.94 times that of the wild type.

[0016] Further experiments showed that the enzymatic hydrolysis products of chondroitin sulfate A by mutant Y301E were consistent with those of chondroitin sulfate AC lyase M1. Furthermore, the mutation, while maintaining the enzyme's substrate recognition mechanism, adjusted the enzyme's recognition preference for substrate sulfation patterns, enhancing its cleavage activity towards the substrate's single sulfation region. Other mutants also exhibited excellent catalytic activity and thermostability; mutants K296E, Y301D, and S551T showed... V max and k cat / K m The half-lives were 111.72%, 137.99%, 111.74% and 71.91%, 95.58% and 108.88% of M1, respectively, and the half-lives were 1.6 times, 1.84 times and 1.39 times that of M1, respectively. Attached Figure Description

[0017] Figure 1 The relative catalytic activities of chondroitin sulfate AC lyase M1 and its mutant are given.

[0018] Figure 2 Thermostability of chondroitin sulfate AC lyase M1 and its mutant at 37°C.

[0019] Figure 3 Comparison of catalytic energy production of chondroitin sulfate AC lyase M1 and its mutants.

[0020] Figure 4 This is a diagram showing the enzymatic hydrolysis products of chondroitin sulfate AC lyase M1 and Y301E. Detailed Implementation

[0021] SEQ ID NO.1 is the amino acid sequence encoding chondroitin sulfate AC lyase, and SEQ ID NO.2 is the nucleotide sequence encoding chondroitin sulfate AC lyase. The gene expression vector is pET28a. Pedobacter xixiisoli The chondroitin sulfate AC lyase from which this was derived is the parent, denoted as M1.

[0022] (1) Method for determining the activity of chondroitin sulfate AC lyase: Chondroitin sulfate A was used as the substrate at a concentration of 10 mg / mL. -1 Use Tris-HCl buffer (50 mM, pH 8.0, 20 mM Ca) 2+ Dissolve the substrate. The reaction system for enzyme activity assay is set to 1 mL, containing: 895 μL Tris-HCl buffer (50 mM, pH 8.0, 20 mM Ca2+). 2+ 5 μL enzyme solution (protein concentration 0.1 mg / mL) -1), 100 μL substrate solution (10 mg / mL) -1 The change in absorbance at 232 nm was measured using a spectrophotometer. Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of product per minute, which is 1 U.

[0023] (2) Method for determining the thermal stability of chondroitin sulfate AC lyase: Use Tris-HCl buffer (50 mM, pH 8.0, containing 20 mM Ca) 2+ The protein concentration of the purified chondroitin sulfate AC lyase was diluted to 1 mg / mL. -1 Wild-type and mutant samples of uniform concentration were placed in a water bath at 37°C. Every so often, 20 μL of the sample was taken and cooled on ice for 15 min before the residual enzyme activity was measured. The enzyme activity without water bath was taken as 100%, and the residual activity ratio was calculated.

[0024] Example 1: Construction and screening of chondroitin sulfate AC lyase mutants Using a recombinant plasmid containing the amino acid sequence shown in SEQ ID NO.1 as a template, site-directed mutagenesis was performed on the gene for chondroitin sulfate AC lyase. The PCR primer sequences are shown in Table 1. A circular plasmid with a nick was obtained by amplification of the whole plasmid. The template plasmid was digested with DpnI enzyme, and the digestion product was directly transformed. E. coli DH5α competent cells were transformed and plated on LB plates, then incubated at 37°C until single colonies grew. Plasmids were extracted from single colonies and sequenced for verification.

[0025] Table 1. Primers for site-directed mutagenesis of chondroitin sulfate AC lyase mutants S72V-F (SEQ ID NO. 3) <![CDATA[GAAAGGCATTGATTACACCGCAAAA GTC ATCGCTAAATGGGAAC]]> 67.0 S72V-R (SEQ ID NO. 4) <![CDATA[GTTCCCATTTAGCGAT GAC TTTTGCGGTGTAATCAATGCCTTTC]]> 67.0 S72N-F (SEQ ID NO. 5) <![CDATA[GAAAGGCATTGATTACACCGCAAAA AAC ATCGCTAAATGGGAAC]]> 69.8 S72N-R (SEQ ID NO. 6) <![CDATA[GTTCCCATTTAGCGATGTTTT TTG CCGGTGTAATCAATGCCTTTC]]> 69.8 S72T-F (SEQ ID NO. 7) <![CDATA[GCATTGATTACACCGCAAAA ACC ATCGCTAAATGGGAA]]> 66.2 S72T-R (SEQ ID NO. 8) <![CDATA[TTCCCATTTAGCGA TGGT TTTTGCGGTGTAATCAATGC]]> 66.2 S130G-F (SEQ ID NO. 9) <![CDATA[GCAACTGGTGGCATAAC GGC ATCGCGACCAGACA]]> 70.2 S130G-R (SEQ ID NO. 10) <![CDATA[TGTCTGGTCGCGAT GCC GTTATGCCACCAGTTGC]]> 70.2 S130A-F (SEQ ID NO. 11) <![CDATA[AACTGGTGGCATAAC GCC ATCGCGACCAGAC]]> 68.1 S130A-R (SEQ ID NO. 12) <![CDATA[GTCTGGTCGCGAT GGC GTTATGCCACCAGTT]]> 68.1 S130N-F (SEQ ID NO. 13) <![CDATA[AGCAACTGGTGGCATAAC AAC ATCGCGACCAGACAG]]> 71.5 S130N-R (SEQ ID NO. 14) <![CDATA[CTGTCTGGTCGCGAT GTT GTTATGCCACCAGTTGCT]]> 71.5 R134V-F (SEQ ID NO. 15) <![CDATA[CATAACTCCATCGCGACC GTA CAGGCTATCGGTGAACT]]> 70.0 R134V-R (SEQ ID NO. 16) <![CDATA[AGTTCACCGATAGCCTG TAC GGTCGCGATGGAGTTATG]]> 70.0 R134M-F (SEQ ID NO. 17) <![CDATA[ATAACTCCATCGCGACC ATG CAGGCTATCGGTGAACTG]]> 70.0 R134M-R (SEQ ID NO. 18) <![CDATA[CAGTTCACCGATAGCCTG CAT GGTCGCGATGGAGTTTAT]]> 70.0 R134A-F (SEQ ID NO. 19) <![CDATA[ATAACTCCATCGCGACC GCA CAGGCTATCGGTGAAC]]> 69.0 R134A-R (SEQ ID NO. 20) <![CDATA[GTTCACCGATAGCCTG TGC GGTCGCGATGGAGTTTAT]]> 69.0 R134L-F (SEQ ID NO. 21) <![CDATA[CATAACTCCATCGCGACC TTA CAGGCTATCGGTGAACT]]> 70.0 R134L-R (SEQ ID NO. 22) <![CDATA[AGTTCACCGATAGCCTG TAA GGTCGCGATGGAGTTATG]]> 70.0 M179I-F (SEQ ID NO. 23) <![CDATA[CCGGTGCAAATAAAACTGAC ATA GCGCTGCACTACTTC]]> 67.4 M179I-R (SEQ ID NO. 24) <![CDATA[GAAGTAGTGCAGCGC TAT GTCAGTTTTATTTGCACCGG]]> 67.4 M179K-F (SEQ ID NO. 25) <![CDATA[CCGGTGCAAATAAAACTGAC AAG GCGCTGCACT]]> 69.8 M179K-R (SEQ ID NO. 26) <![CDATA[AGTGCAGCGC CTT GTCAGTTTTATTTGCACCGG]]> 69.8 M179V-F (SEQ ID NO. 27) <![CDATA[GGTGCAAATAAAACTGAC GTG GCGCTGCACTACTT]]> 68.3 M179V-R (SEQ ID NO. 28) <![CDATA[AAGTAGTGCAGCGC CAC GTCAGTTTTATTTGCACC]]> 68.3 K291P-F (SEQ ID NO. 29) <![CDATA[CACCGAAGGCCGTGGCGTT CCA CGTCCGAACAA]]> 74.2 K291P-R (SEQ ID NO. 30) <![CDATA[TTGTTCGGACG TGG AACGCCACGGCCTTCGGTG]]> 74.2 K295D-F (SEQ ID NO. 31) <![CDATA[CGTGGCGTTAAACGTCCGAAC GAT CTGACTAAAAACACTTACAAAA]]> 68.6 K295D-R (SEQ ID NO. 32) <![CDATA[TTTTGTAAGTGTTTTTAGTCAG ATC GTTCGGACGTTTAACGCCACG]]> 68.6 K295E-F (SEQ ID NO. 33) <![CDATA[GGCGTTAAACGTCCGAAC GAG CTGACTAAAAACACTT]]> 67.1 K295E-R (SEQ ID NO. 34) <![CDATA[AAGTGTTTTTAGTCAG CTC GTTCGGACGTTTAACGCC]]> 67.1 T297S-F (SEQ ID NO. 35) <![CDATA[GTTAAACGTCCGAACAAGCTG TCT AAAAACACTTACAAAACCC]]> 65.5 T297S-R (SEQ ID NO. 36) <![CDATA[GGGTTTTGTAAGTGTTTTT AGA CAGCTTGTTCGGACGTTTAAC]]> 65.5 Y301K-F (SEQ ID NO. 37) <![CDATA[GAACAAGCTGACTAAAAACACT AAG AAAACCCGCCTGATCACC]]> 67.0 Y301K-R (SEQ ID NO. 38) <![CDATA[GGTGATCAGGCGGGTTTT CTT AGTGTTTTTAGTCAGCTTGTTC]]> 67.0 Y301S-F (SEQ ID NO. 39) <![CDATA[AACAAGCTGACTAAAAACACT AGC AAAACCCGCCTGATCACC]]> 68.9 Y301S-R (SEQ ID NO. 40) <![CDATA[GGTGATCAGGCGGGTTTT GCT AGTGTTTTTAGTCAGCTTGTT]]> 68.9 Y301D-F (SEQ ID NO. 41) <![CDATA[CGAACAAGCTGACTAAAAACACT GAC AAAACCCGCC]]> 67.9 Y301D-R (SEQ ID NO. 42) <![CDATA[GGCGGGTTTT GTC AGTGTTTTTAGTCAGCTTGTTCG]]> 67.9 Y301E-F (SEQ ID NO.43) <![CDATA[GAACAAGCTGACTAAAAACACT GAG AAAACCCGCCTGATCACC]]> 68.2 Y301E-R (SEQ ID NO.44) <![CDATA[GGTGATCAGGCGGGTTTT CTC AGTGTTTTTAGTCAGCTTGTTC]]> 68.2 S551A-F (SEQ ID NO.45) <![CDATA[GAAAGGTAGCTGGTACAAA GCA AACAACGCAACAGCAA]]> 67.9 S551A-R (SEQ ID NO.46) <![CDATA[TTGCTGTTTGCGTTGTT TGC TTTGTACCAGCTACCTTTC]]> 67.9 S551T-F (SEQ ID NO.47) <![CDATA[CAGAAAGGTAGCTGGTACAAA ACG AACAACGCAACAGCAAAAC]]> 68.7 S551T-R (SEQ ID NO.48) <![CDATA[GTTTTGCTGTTTGCGTTGTT CGT TTTGTACCAGCTACCTTTCTG]]> 68.7 Transfer the correctly sequenced mutant plasmid to E. coli BL21(DE3) competent cells were transformed and plated on LB agar plates, then incubated at 37°C until single colonies appeared, yielding cells containing the mutant plasmid. E. coli BL21 (DE3) engineered bacteria.

[0026] The plasmid containing chondroitin sulfate AC lyase M1 and mutant plasmids E. coli BL21(DE3) engineered bacteria were inoculated to a final concentration of 50 μg / mL. -1 Kanamycin was cultured overnight in LB liquid medium at 37°C and 220 rpm, and then transferred to the same LB liquid medium for scale-up culture until OD. 600 The concentration was increased to 0.8, and IPTG was added to the culture medium to a final concentration of 0.2 mM. The culture was then induced at 25°C and 220 rpm for 15 h.

[0027] The process of obtaining enzymes from recombinant *E. coli*: After induction culture, the fermentation broth was centrifuged at 2577 × g for 5 min at 4 °C, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended three times in Tris-HCl buffer (50 mM, pH 8.0), and the cells were sonicated for 10 min in an ice bath. Then, the cells were centrifuged at 6599 × g at 4 °C for 15 min to obtain crude enzyme solution. The crude enzyme solution was loaded onto a nickel affinity chromatography column and bound at 4 °C for 2 h. Non-specifically adsorbed contaminating proteins were washed with Tris-HCl buffer (50 mM, pH 8.0) containing 20 mM imidazole, and the target protein was eluted with Tris-HCl buffer (50 mM, pH 8.0) containing 250 mM imidazole to obtain purified enzyme solution.

[0028] The process of screening mutants with improved catalytic activity and thermal stability: First, the effect of point mutations on enzyme activity was analyzed, and the results are as follows: Figure 1 As shown, the relative catalytic activities of mutants S130G, R134L, R134V, M179V, K295E, T297S, Y301D, Y301E, S551T, and Y301S were significantly higher than those of M1, at 106.05%, 102.99%, 107.13%, 107.29%, 111.91%, 110.86%, 125.14%, 128.50%, 117.83%, and 112.58% of M1, respectively. Thermostability was studied for the single-point mutants with significantly enhanced catalytic activity. The enzyme solution was incubated in a 37°C water bath, and 20 μL samples were taken every 60 min and cooled on ice for 15 min before residual enzyme activity was measured. Figure 2 As shown, the mutants K295E, Y301D, Y301E and S551T exhibited significantly improved thermal stability, being 1.60, 1.84, 1.94 and 1.39 times that of M1, respectively.

[0029] Example 2: Determination of Kinetic Parameters of Dominant Mutants Kinetic parameters were determined for the superior mutants with enhanced catalytic activity obtained in Example 1, including 10 single mutants.

[0030] The specific steps are as follows: Use Tris-HCl buffer (50 mM, pH 8.0, 20 mM Ca). 2+ Prepare concentrations of 40.00, 20.00, 10.00, 5.00, 2.5, 1.25, 0.63, 0.31, and 0.16 mg / mL. -1The initial reaction rates of chondroitin sulfate A substrate solutions and chondroitin sulfate AC lyase M1 and mutants at various substrate concentrations were determined. The obtained reaction rate and substrate concentration data were fitted to the Michaelis-Menten equation using nonlinear regression to calculate... V max and K m The results are shown in Table 2. The mutants K295E, Y301D, Y301E, and S551T... V max , k cat / K m The values ​​were all significantly higher than M1, consistent with the results of relative catalytic activity. M1's... V max and k cat / K m The value was 856.36 U mg. -1 and 1928.06 mL s -1 mg -1 The mutant Y301E showed the most significant improvement in catalytic activity. V max and k cat / K m The values ​​increased by 145.35% and 106.58% respectively compared to M1.

[0031] Table 2 Kinetic parameters of chondroitin sulfate AC lysin M1 and its mutants M1 856.36±54.14 0.19±0.043 366.33 1928.06 S130G 864.00±49.28 0.22±0.047 369.60 1685.21 R134L 960.39±43.21 0.32±0.049 410.83 1296.78 R134V 920.90±55.96 0.28±0.060 393.94 1423.04 M179V 800.98±38.10 0.19±0.034 342.64 1793.27 K295E 956.73±41.32 0.30±0.044 409.27 1386.41 T297S 844.29±49.10 0.18±0.040 361.17 2010.08 Y301D 1181.66±39.10 0.27±0.051 505.49 1842.84 Y301E 1244.73±36.10 0.26±0.037 532.47 2054.83 Y301S 913.1±33.50 0.20±0.028 390.60 1972.75 S551T 956.9±37.00 0.20±0.029 409.34 2099.18 Example 3: Dominant Mutant T m Determination of value The 10 single mutants obtained in Example 1 (including four dominant mutants with enhanced catalytic activity) were measured. T m The values ​​are used to characterize thermal stability.

[0032] The midpoint temperature (also known as melting temperature) of chondroitin sulfate AC lyase M1 and catalytically dominant mutants was determined using a DSC25 instrument. T m The temperature range is set to 20 to 90°C, and increments are set at 5°C per minute. -1The temperature rises at a rate of [missing information]. By collecting data on the changes in heat flow during the heating and cooling cycles of the sample, and combining this with analysis of the thermodynamic curves using Trios v5.4.0.300 data processing software, the temperature parameter corresponding to the peak of the characteristic peak is the temperature parameter corresponding to each sample. T m Values. The results are shown in Table 3, for mutants K295E, Y301D, Y301E, and S551T. T m The values ​​are all higher than those of M1, consistent with the trend of enhanced thermal stability reflected in the half-life. M1's... T m The value was 74.87℃, and the mutant Y301E showed the most significant increase in half-life. T m The value is 1.38℃ higher than that of M1.

[0033] Table 3. Chondroitin sulfate AC lyase M1 and dominant mutants in catalytic activity. T m value M1 76.53 S130G 44.04 R134L 57.53 R134V 68.51 M179V 65.70 K295E 76.38 T297S 71.19 Y301D 77.32 Y301E 77.91 Y301S 61.55 S551T 76.61 Example 4: Comparison of catalytic production capacity of superior mutants The catalytic production capacity of chondroitin sulfate AC lyase and four mutants obtained in Example 1 with improved catalytic activity and thermal stability were compared.

[0034] The catalytic activity changes of chondroitin sulfate AC lyase M1 and the mutants K295E, Y301D, Y301E, and S551T, which all exhibited enhanced catalytic activity and thermostability, were integrated within 10 h of incubation at 37°C. The amount of product generated per unit mass of chondroitin sulfate AC lyase per unit hour was calculated. Figure 3 As shown, the catalytic capacity of the four mutants all showed varying degrees of improvement, with the mutant Y301E, which has both high catalytic activity and thermal stability, showing a 62.38% increase in catalytic capacity compared to M1.

[0035] Example 5: Analysis of Enzymatic Hydrolysis Products SAX-HPLC analysis was performed on chondroitin sulfate AC lyase and the mutant Y301E obtained in Example 1, which showed the most significant improvement in catalytic activity and thermal stability.

[0036] The specific steps are as follows: Add the purified enzyme solutions of M1 and mutant Y301E to a solution containing 10 mg / mL of [unclear text]. -1 In an aqueous substrate solution of chondroitin sulfate A, the reaction was carried out in a water bath at 37°C until the UV absorbance of the reaction system remained stable. The enzyme activities of M1 and Y301E were 5.80 × 10⁻⁶. 5 UL -1 and 9.23×10 5 UL-1 The thoroughly hydrolyzed solution was placed in a boiling water bath for 10 min to completely inactivate the enzyme. After cooling to room temperature, the solution was centrifuged at 12000×g for 10 min, and the supernatant was collected for SAX-HPLC analysis.

[0037] The enzymatic hydrolysis products of chondroitin A sulfate were separated using a ZORBAX SAX column. Mobile phase A was 2.33 mM sodium dihydrogen phosphate, and mobile phase B was 2.33 mM sodium dihydrogen phosphate + 1.14 M sodium perchlorate. The flow rate was 0.6 mL / min. -1 The column temperature was 40℃, and the detection wavelength was UV 232 nm. The sample injection volume was 3 μL, and the analysis was performed in the following order: chondroitin sulfate disaccharide standard, chondroitin sulfate A (treated with M1), and chondroitin sulfate A (treated with Y301E).

[0038] The possible disaccharide structures are shown in Table 4 and Figure 4 As shown, the retention times of disaccharide standards with different sulfation modes are different, and their elution order is as follows: DS1, DS3, DS2, DS8, DS4, DS6, DS5, and DS7. Figure 4 As shown, the depolymerization products of M1 and Y301E both have four identical main peaks, corresponding to DS3 (21.49 min), DS2 (22.79 min), DS4 / DS6 (32.14 min), and DS5 (35.25 min), respectively. Compared with M1, the peak area ratio of monosulfated disaccharides DS3 and DS2 in the enzymatic hydrolysis products of mutant Y301E is increased. This change indicates that the Y301E mutation enhances the enzyme's catalytic efficiency or substrate preference for the monosulfated region of the substrate. The substrate for the enzymatic hydrolysis reaction was commercially available chondroitin sulfate A, purchased from Merck, Germany, with a molecular weight of approximately 20-30 kDa. The molecular weights of the disaccharides with different sulfation modes detected by HPLC ranged from 459.4 to 605 Da, indicating that chondroitin sulfate AC lyase M1 and mutant Y301E can degrade the substrate chondroitin sulfate A to prepare low molecular weight chondroitin sulfate.

[0039] After elution for 50 min, no other characteristic peaks were detected in the enzymatic hydrolysates of M1 and Y301E, indicating that the mutation did not alter the enzyme's minimum recognition unit for the substrate. Combined with the results of Examples 2, 3, and 4, it can be concluded that compared to wild-type M1, the mutant Y301E, while retaining the minimum recognition unit for the substrate, improves the enzyme's catalytic activity and thermal stability, making it more promising for the preparation of low molecular weight chondroitin sulfate.

[0040] Table 4. Eight possible disaccharide structures ΔUA-GalNAC (DS1) H H H ΔUA-GalNAC6S (DS3) H <![CDATA[SO3H]]> H ΔUA-GalNAC4S (DS2) <![CDATA[SO3H]]> H H ΔUA2S-GalNAC (DS8) H H <![CDATA[SO3H]]> ΔUA2S-GalNAC6S (DS6) H <![CDATA[SO3H]]> <![CDATA[SO3H]]> ΔUA-GalNAC4S6S (DS4) <![CDATA[SO3H]]> <![CDATA[SO3H]]> H ΔUA2S-GalNAC4S (DS5) <![CDATA[SO3H]]> H <![CDATA[SO3H]]> ΔUA2S-GalNAC4S6S (DS7) <![CDATA[SO3H]]> <![CDATA[SO3H]]> <![CDATA[SO3H]]> Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0041] Sequence List:

[0042] .

[0043] Chondroitin sulfate AC lyase M1 nucleotide sequence SEQ ID NO.2:

Claims

1. A chondroitin sulfate AC lyase mutant possessing both high catalytic activity and high thermal stability, characterized in that, The chondroitin sulfate AC lyase mutant is based on the amino acid sequence shown in SEQ ID NO.1 and undergoes any one or more of the following mutations: (1) The lysine at position 295 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamic acid; (2) The tyrosine at position 301 of the amino acid sequence shown in SEQ ID NO.1 is mutated to aspartic acid; (3) The tyrosine at position 301 of the amino acid sequence shown in SEQ ID NO.1 is mutated to glutamic acid; (4) The serine at position 551 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine.

2. The gene encoding the chondroitin sulfate AC lyase mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. A microbial cell expressing the mutant of claim 1, or containing the gene of claim 2 or the expression vector of claim 3.

5. A method for expressing the chondroitin sulfate AC lyase mutant of claim 1, characterized in that, The gene described in claim 2 is ligated to an expression vector to obtain a recombinant plasmid. The recombinant plasmid is then transformed into a host cell to obtain a recombinant microbial cell. The recombinant microbial cell is then cultured and used to produce enzymes.

6. The method according to claim 5, characterized in that, The recombinant microbial cells were cultured at 37°C and 220 rpm until the OD600 reached 0.8, then an inducing agent was added, and the cells were induced and cultured at 25°C and 220 rpm for 15 h.

7. The method according to claim 6, characterized in that, The inducing agent is IPTG, and the final concentration of IPTG in the reaction system is 0.2 mM.

8. The application of the chondroitin sulfate AC lyase mutant of claim 1 in the preparation of low molecular weight chondroitin sulfate.

9. A method for preparing low molecular weight chondroitin sulfate, characterized in that, The method is as follows: using the mutant described in claim 1 to lyse chondroitin sulfate.

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