A hyaluronan lyase and its gene expression and application

Abstract

The present invention relates to the field of biochemical engineering technology, and specifically discloses a hyaluronate lyase and its gene expression and application. The amino acid sequence of the hyaluronate lyase is such as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is such as SEQ ID NO.2. The present invention also provides an expression vector and an expression bacterium comprising the above-mentioned encoding gene, as well as the application of the hyaluronate lyase and its encoding gene in the preparation of unsaturated hyaluronate disaccharides. The hyaluronate lyase provided by the present invention has the advantages of high enzyme activity, high catalytic efficiency, good temperature and pH stability, wide temperature and pH range required for maintaining enzymatic activity, direct conversion in pure water, and small and uniform molecular weight of the enzymatic hydrolysis product, and is suitable for large-scale industrial enzymatic hydrolysis of hyaluronic acid to produce unsaturated hyaluronate disaccharides.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to a hyaluronan lyase and its gene expression and application. Background Art

[0002] Hyaluronic acid is a high molecular weight polysaccharide widely found in the extracellular matrix of higher and lower animals. Its basic structure is a polysaccharide composed of D-glucuronic acid and N-acetylglucosamine as disaccharide units. It is widely used in cosmetics, food and medicine. Hyaluronic acid with different molecular weights has different effects and functions. High molecular weight hyaluronic acid (>1.6 million Da) forms a dense protective film on the surface of the skin, which has long-lasting moisturizing and good repair effects; medium molecular weight hyaluronic acid (200,000 Da ~ 1.6 million Da) has moisturizing, lubrication, sustained release and stable emulsification effects; low molecular weight hyaluronic acid (10,000 Da ~ 200,000 Da) has a long-lasting moisturizing effect on the skin; hyaluronic acid oligosaccharides (<10,000 Da) can be absorbed through the skin for deep moisturizing, and have anti-aging, after-sun repair and other effects. Ultra-low molecular weight hyaluronic acid (<2000Da) can penetrate into the dermis, replenish moisture in the dermis, remove wrinkles and improve skin elasticity; it can bind tightly to skin cells, remove free radicals in cells, repair damaged collagen, and promote wound healing. Therefore, ultra-low molecular weight hyaluronic acid has broad application prospects in food health care, cosmetics, and clinical medicine. In recent years, the market demand for ultra-low molecular weight hyaluronic acid has also increased year by year.

[0003] At present, the preparation of low molecular weight hyaluronic acid is mainly through physical, chemical and enzymatic degradation methods to degrade macromolecular hyaluronic acid into low molecular weight hyaluronic acid. Among them, physical degradation mainly causes the degradation of hyaluronic acid through physical factors such as heating, mechanical shearing, ultraviolet rays, ultrasound, γ-ray radiation and high-pressure homogenization, but the stability of the product obtained by this method is poor, the molecular weight distribution is uneven, and the degradation efficiency is low; chemical degradation methods mainly include alkaline hydrolysis, hydrolysis and oxidative degradation. Although it can achieve the purpose of controlling the molecular weight of the product to a certain extent, due to the complex degradation conditions of different chemical reagents, the product properties are easily affected and the product purification is difficult, and there is a problem of waste liquid pollution. Enzymatic degradation is an ideal method for preparing low molecular weight hyaluronic acid because of its mild reaction conditions, simple operation, and the structure of polysaccharides does not change. Ultra-low molecular weight hyaluronic acid can only be prepared by enzymatic hydrolysis of hyaluronic acid. However, hyaluronidase currently has problems such as uneven molecular weight of enzymatic hydrolysis products, low enzymatic conversion efficiency or narrow temperature range and pH range required for enzyme activity maintenance, and requires a specific buffer system, and the product separation steps are cumbersome. Therefore, it is of great application value to discover hyaluronidases (or lyases) with small and uniform molecular weight of enzymatic hydrolysis products, high catalytic efficiency, good temperature and pH stability, and simple conversion system and recovery method. Summary of the invention

[0004] In view of the above-mentioned problems existing in the existing enzymatic hydrolysis for preparing ultra-low molecular weight hyaluronic acid, the present invention provides a hyaluronan lyase and its gene expression and application. The hyaluronan lyase has the advantages of high enzymatic activity and catalytic efficiency, good stability, small and uniform molecular weight of enzymatic hydrolysis products, simple reaction and recovery in pure water, and no need to remove salt.

[0005] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution:

[0006] A hyaluronan lyase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Relative to the prior art, the hyaluronate lyase provided by the present invention has a high conversion efficiency. Under suitable enzymolysis conditions, the enzymolysis conversion rate in a short time can reach 100%; the enzyme activity is stable, and high enzymolysis activity and conversion efficiency can be maintained under the conditions of 20°C-60°C and pH4-10, and the enzymolysis reaction can be carried out in water. The molecular weight of the enzymolysis product is small and uniform, and they are all unsaturated hyaluronic acid disaccharides, and the molecular weight is less than <500Da, and the product purity is high. That is, the hyaluronate lyase provided by the present invention has a high conversion efficiency, good activity stability, a wide range of temperature and pH required for maintaining enzymolysis activity, a small and uniform molecular weight of the enzymolysis product, can react in pure water, and the product recovery is simple, without the advantage of desalination, and is suitable for large-scale industrial production.

[0008] The present invention also provides a gene encoding the hyaluronan lyase, and the nucleotide sequence of the gene encoding the hyaluronan lyase is as shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector, which is obtained by inserting the coding gene of the hyaluronan lyase into an expression vector.

[0010] Preferably, the expression vector is plasmid pET28a-SUMO.

[0011] The present invention also provides an expression bacterial body, which is obtained by transferring the recombinant expression vector into a host cell.

[0012] Preferably, the host cell is Escherichia coli BL21 (DE3).

[0013] The present invention also provides the use of the hyaluronan lyase, the coding gene of the hyaluronan lyase, the recombinant expression vector or the expression bacteria in the preparation of unsaturated hyaluronan disaccharide.

[0014] The hyaluronan lyase can prepare unsaturated hyaluronan disaccharide with uniform molecular weight, high purity and high yield.

[0015] The present invention also provides a method for preparing unsaturated hyaluronic acid disaccharide, which comprises: adding sodium hyaluronate and the hyaluronate lyase into an aqueous phase system, and performing an enzymatic hydrolysis reaction at a temperature of 20°C-60°C and a pH of 4-10 to obtain the unsaturated hyaluronic acid disaccharide.

[0016] Preferably, the aqueous phase system is pure water or a calcium chloride aqueous solution.

[0017] Preferably, the temperature of the enzymatic hydrolysis reaction is 40°C.

[0018] Preferably, the pH of the enzymatic hydrolysis reaction is 7. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the construction and structure of the plasmid pACYC Duet-1-ulp in Example 3 of the present invention;

[0020] Figure 2 This is a protein gel detection diagram of the purified hyaluronan lyase in Example 4 of the present invention; wherein, M: Maker; 1: SaHyaL crude enzyme with SUMO tag; 2: SaHyaL crude enzyme with SUMO tag removed; 3: purified recombinant enzyme;

[0021] Figure 3 is an enzyme activity curve diagram of the hyaluronate lyase detected in Example 5 of the present invention at different temperatures;

[0022] Figure 4 is an enzyme activity curve diagram of the hyaluronate lyase detected in Example 5 of the present invention at different pH values;

[0023] Figure 5 is a temperature stability curve of the hyaluronate lyase detected in Example 5 of the present invention;

[0024] Figure 6 is a pH stability curve of the hyaluronate lyase detected in Example 5 of the present invention;

[0025] Figure 7 is a TLC analysis chart of the enzymatic hydrolysis product in Example 8 of the present invention;

[0026] Figure 8 is a HPLC detection chart of the enzymatic hydrolysis product in Example 8 of the present invention;

[0027] Fig. 9 This is a graph showing the conversion rate of sodium hyaluronate hydrolyzed by hyaluronate lyase at different time points using HPLC in Example 8 of the present invention;

[0028] Fig.10This is a HPLC test chart of the enzymatic hydrolysis end product in Example 8 of the present invention;

[0029] Fig.11 It is the LC-MS / MS mass spectrometry analysis diagram of the enzymatic hydrolysis final product in Example 8 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Example 1

[0032] A hyaluronan lyase, the amino acid sequence of which is shown in SEQ ID NO. 1, is obtained from a strain of Streptomyces sp. B1010 screened in a laboratory.

[0033] SEQ ID NO.1

[0034] ARAAEGRARLLANTADVFAGTAASNARPETAGRLAAIDKAARANLKAMMDDAGDGELFAGLVLGADEANLNTAYRRLYEIALATRAPTPGATTGLYGDTAAQRRVIDGLEWLHERYYGDQSKGYYGNWFHWEIGLSQHISRTLVLLAGEVRAHRPDLARAYVASMDAYLRNGADGDVDLGSRFHTGANLADITTNRVLQGALLGADGDAR IRKALVDQLTVFVTIDPYHLSHGVTDGHYADGSFIQHASVAYTGSYGKGLLSRVVQTLKILQGTGFAHGEELVPTVFGWVRDGFAPVIFEGWMMEIVKGRAVARTDSGYTDAAVVVEAVVDLSSLAEGAAAAALKSYVKHLGATSPAPLDPARFVSPVSIVRHADIAADDSVVPADLNPGERSVAFNAMDRTVHRRPGYAFALARNSDRI SKYEYMNGENLMPWFQGEGAHYLYLTGQDQTQAYGVDYYTTVSPYGLAGVTAPVERRRTVPEAYGKPYYDNPDHPLRFTSSESQNTYVYFPRGTARHSGGAVLGAYGTAAMVQSSDVAHRDRKLLPDDFLVHRGASATKSWFLLDDEIVVLAAGVGDFEGGGSGRAVTTTVDARVAAPGDRVTLTGARADGGAWTGPGTADLRWLRYA NATRGASVGYVFLDVPPVRVSLDRVTRSRRIVRTANADTAVTRSVFGVTVDGAAGARPAHLAYALLPNATEEGLRRYGDGKGDRWGSRGSRGPLRVLANSTRLQAVAHSGLGLTGVNSFTAGRHETAGLRIDGPASVLVRGSGRRGGITAVAVSDPTMRRDTVTVLLRGRRMRQVSADRGVRVSVTPGGTRIEVATRHAYGRSFTVTLRG

[0035] The coded gene of the above-mentioned transparent transparent layer is obtained:

[0036] The genome of a strain of Streptomyces sp. B1010 screened above was used as a template (DNA template), upstream and downstream primers were designed and the primer annealing temperature was predicted, an EcoRI restriction enzyme site and a protective base were added to the 5' end of the upstream primer, and an XhoI restriction enzyme site and a protective base were introduced to the 5' end of the downstream primer. The primer sequences were as follows:

[0037] hyal-F: (EcoRI)5′-TTAAGAATTCGCCAGGGCCCGCCGAGG-3′ (SEQ IDNO.3);

[0038] hyal-R: (XhoI)5'-CCGCTCGAGGCCCCGCAGGGTCACC-3' (SEQ ID NO. 4).

[0039] A large amount of amplification was performed using a PCR instrument. The DNA polymerase kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd. The PCR reaction system is shown in Table 1, and the PCR amplification program is shown in Table 2. The target gene obtained after amplification was detected using 0.8% agarose gel, and then the gel was cut and recovered, and purified to obtain the above-mentioned hyaluronan lyase coding gene, whose nucleotide sequence is shown in SEQ ID NO.2.

[0040] SEQ ID NO.2

[0041]

[0042] Table 1 PCR system

[0043]

[0044] Table 2 PCR amplification program

[0045]

[0046]

[0047] Example 2

[0048] Construction of the recombinant expression vector of the above hyaluronan lyase:

[0049] 1) Double enzyme digestion of the hyaluronan lyase encoding gene amplified and purified in Example 1 and the vector pET28a-SUMO.

[0050] The restriction endonucleases EcoRI and XhoI were used to double-digest the coding gene of hyaluronan lyase and the vector pET28a-SUMO, respectively. The double-digestion system is shown in Table 3. The double-digestion systems were placed in a 37°C water bath for 3 hours. After the end, the above-mentioned digestion systems were added to 0.8% agarose gel for detection, and the digested gene fragments and pET28a-SUMO vector bands of the correct size were cut and recovered and purified.

[0051] Table 3 Enzyme digestion system

[0052]

[0053] 2) The digested gene fragment was ligated with the linearized pET28a-SUMO vector.

[0054] The purified restriction-digested gene fragment and the linearized vector pET28a-SUMO were ligated by T4 DNA ligase. The enzyme ligation system was shown in Table 4. The enzyme ligation system was ligated at 16° C. for 12 h.

[0055] Table 4 Enzyme-linked system

[0056]

[0057] 3) Transform competent E. coli cells DH5α.

[0058] 8 μL of the above enzyme-linked system and 80 μL of competent E. coli cells DH5α were mixed and added to the culture tube, and then placed on ice for 20 minutes. After heating at 42°C for 45 seconds, the tube was placed on ice for 2 minutes, and then 400 μL of liquid LB without resistance was added to the tube, and the tube was placed on a shaker at 37°C at 180 rpm for 60 minutes. 200 μL of the cultured bacterial solution was evenly spread on a solid LB plate with resistance of kan, and inverted and cultured at 37°C overnight.

[0059] 4) Screening of positive clones.

[0060] The single clones on the above plates were picked and cultured in LB liquid medium containing kan resistance at 37°C and 220rpm overnight, and the bacterial cells of each strain were collected and the plasmids were extracted. Double enzyme digestion verification was performed using restriction endonucleases EcoRI and XhoI. The double enzyme digestion verification system is shown in Table 5. The plasmids with correct sequencing were picked and preserved. The plasmids were the recombinant expression vectors (pET28a-SUMO-Sahyal) into which the coding gene of the above hyaluronan lyase was inserted.

[0061] Table 5 Double enzyme digestion verification system

[0062]

[0063]

[0064] Example 3

[0065] Obtaining expression bacteria and expressing hyaluronan lyase.

[0066] The plasmid pACYC Duet-1-ulp (used to express Ubiquitin-Like protein-specific Protease Ulp without His tag, which can specifically recognize and remove SUMO tag) was co-expressed by the dual plasmid method. The structure of the pACYC Duet-1-ulp plasmid is shown in the figure. Figure 1As shown, the gene sequence expressing Ulp was inserted into the pACYC Duet plasmid) and the recombinant plasmid pET28a-SUMO-Sahyal were simultaneously transformed into competent cells BL21 (DE3) by heat shock method, and 400 μL of non-antibiotic LB liquid medium was added, and cultured at 37°C, 180rpm for 60 minutes. Take 80 μL of the above bacterial solution and evenly spread it on a double-resistant (containing both kan and Cm resistance) solid LB plate, and invert and culture at 37°C overnight. Pick a single colony in 2mL of LB liquid medium (containing both kan and Cm resistance). The culture was cultured overnight at 37°C and 200 rpm, and then inoculated into 200 mL of LB liquid culture medium (containing both kan and Cm resistance) at a volume inoculation rate of 1%, and cultured at 20°C and 200 rpm until the bacterial liquid OD600 = 0.6, and 100 mM IPTG was added to the culture medium at a volume ratio of 1:1000 to induce protein expression, and the induction time was 24 h.

[0067] Example 4

[0068] Purification of hyaluronan lyase.

[0069] The IPTG-induced protein expression system in Example 3 was centrifuged at 6000 rpm for 10 min using a centrifuge to collect the cells, wash the cells with 10 mL of Ni-NTA equilibration buffer, and then vortex the cells with 8 mL of Ni-NTA equilibration buffer until there were no bacterial lumps to obtain a bacterial suspension. The bacterial suspension was ultrasonically disrupted for 3 s, with an interval of 5 s, and the total ultrasonic disruption time was 35 min to obtain a cell lysate. The cell lysate was centrifuged at 12000 rpm and 4 ° C for 15 min, and the supernatant was collected after centrifugation and placed on ice.

[0070] The supernatant (crude enzyme solution) was purified by nickel column affinity chromatography. First, the nickel column was pretreated with 5 column volumes of precooled Ni-NTA equilibrium buffer and placed in a 4°C refrigerator. The supernatant placed on ice was added to the pretreated nickel column, 2 mL of crude enzyme solution was added each time, and the flow-through was released after incubation at 4°C for 6 minutes. 2 mL of crude enzyme solution was added again for purification, and the above steps were repeated until all the supernatant was loaded on the column. Then, the impurities were washed with Ni-NTA washing buffer, 2 mL was added each time, and the total washing volume was 20 mL. Then, 5 mL of 250 mM Ni-NTA and 5 mL of 350 mM Ni-NTA elution buffer were added to elute and collect the target protein bound to the nickel column, and the purified protein was concentrated using a 50 kDa ultrafiltration column, and the elution buffer in the protein was replaced with Tris-HCL with a salt concentration of 50 mM and pH 7.0. Finally, the purified target protein was stored in 1 mL of 50 mM Tris-HCl buffer at pH 7.0 to obtain a purified enzyme solution. The purification effect was detected using a 10% SDS-PAGE protein gel, and the test results are shown in Figure 2. Figure 2 As shown, it can be seen that the target protein (hyaluronan lyase) was successfully expressed in a soluble form and completed the cleavage of the SUMO tag in vivo.

[0071] Example 5

[0072] The enzymatic properties of the hyaluronan lyase purified in Example 4 were detected.

[0073] 1) Determination of the optimal enzymatic hydrolysis temperature

[0074] Reaction system: 200 μL of 50 mM Tris-HCl 7.0 buffer, 200 μL of 0.5 wt.% sodium hyaluronate (800,000-1.5 million Da), 100 μL of the purified enzyme solution obtained in Example 4, mixed and reacted at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C, respectively, for 15 min, terminated the reaction with 500 μL of 20 mM HCl and performed enzyme activity assay, and the protein activity at the optimum temperature was defined as 100%, and the protein activity at other temperatures was calculated as relative activity. It was found that when the reaction temperature was 50°C, the catalytic activity was the highest, as shown in FIG. Figure 3 shown.

[0075] 2) Determination of the optimal enzymatic hydrolysis pH

[0076] Reaction system: 200 μL of 0.5wt.% sodium hyaluronate (800,000-1.5 million Da), 100 μL of purified enzyme solution, of which 200 μL of buffer in the reaction system is a buffer of different pH. The buffers used are: citric acid-Na2HPO4 buffer (pH4.0-6.0), NaH2PO4-Na2HPO4 buffer (pH6.0–8.0), Tris-HCl buffer (pH8.0-9.0) and Gly-NaOH buffer (pH9.0-10.0), and the concentration of the above buffers is 50 mM. The reaction systems of different pH values ​​were reacted at the optimal temperature for 15 minutes, and the reaction was terminated with 500 μL of 20 mM HCl and the enzyme activity was determined. The enzyme activity under the optimal pH condition was defined as 100%, and the enzyme activity under other pH conditions was calculated as relative enzyme activity. It was found that the catalytic activity was highest when the pH was 7.0, such as Figure 4 shown.

[0077] 3) Determination of temperature stability

[0078] Reaction system: 200 μL of optimal pH buffer, 200 μL of 0.5wt.% sodium hyaluronate (800,000-1.5 million Da), 100 μL of purified enzyme solution, wherein the enzyme solution is first incubated at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C and 80°C for 60 min, and then added to the above reaction system, reacted under the optimal conditions for 15 min, terminated the reaction with 500 μL of 20 mM HCl and performed enzyme activity determination, the protease activity without temperature treatment is defined as 100%, and the enzyme activity with temperature treatment is calculated as relative activity. It is measured that when the reaction temperature is 20°C-45°C, the enzyme has a relative activity of more than 90% after incubation for 60 min, and still has an activity of more than 80% at 45-50°C after incubation for 60 min. Figure 5 As shown, it is shown that the hyaluronate lyase exhibits good temperature stability.

[0079] 4) Determination of pH stability

[0080] Reaction system: 200 μL of optimal pH buffer, 200 μL of 0.5wt.% sodium hyaluronate (800,000-1.5 million Da), 100 μL of purified enzyme, and 2.5 μL of CaCl2 with a final concentration of 1 M. In order to determine the pH stability of the protein, the enzyme needs to be mixed with the above-mentioned buffers of different pH values ​​at a ratio of 1:1, and then incubated at 37°C for 1 hour. The above-treated enzymes were added to the above-mentioned reaction system respectively, reacted for 15 minutes under the optimal reaction conditions, and the reaction was terminated with 500 μL of 20 mM HCl and the enzyme activity was determined. The activity of the protease not treated with pH was defined as 100%, and the activity of the enzyme treated under other different pH conditions was calculated as relative activity. It was measured that the activity was still above 90% after incubation for 60 minutes at pH 5.0-10.0, and more than 60% activity was still maintained after incubation for 60 minutes in the pH range of 4.0-5.0. Figure 6 As shown, it is shown that the hyaluronate lyase has good pH stability.

[0081] Example 6

[0082] The substrate spectrum of the hyaluronan lyase purified in Example 4.

[0083] Reaction system: 0.2 mL of sodium hyaluronate (5 mg / mL, molecular weight 800,000-1.5 million Da) and 0.1 mL of purified enzyme solution were added to 0.2 mM Tris-HCl buffer (pH 7.0). After incubation at 50°C for 15 min, 0.5 mL of 20 mM HCl was added to terminate the reaction, and the same mixture containing 0.1 mL of heat-inactivated enzyme solution was used as a blank control. Under the same reaction conditions and system, the purified hyaluronate lyase was reacted with different substrates under the optimal reaction conditions (50°C, pH 7.0), and then the enzyme activity was measured at a wavelength of 232 nm. The results are shown in Table 6. The enzyme showed the highest activity against sodium hyaluronate, and lower protein activity against chondroitin sulfate A, chondroitin sulfate B, and chondroitin sulfate C, but had no enzymatic activity against sodium heparin, sodium alginate, and carboxymethyl cellulose, indicating that the substrate of the hyaluronate lyase was relatively specific, and the degradation activity against sodium hyaluronate reached the highest level (152.12±5.17U / mg).

[0084] Table 6 Substrate spectrum of hyaluronan lyase

[0085]

[0086] Note: “ / ” represents no enzymatic activity.

[0087] Example 7

[0088] Determination of the kinetic parameters of the hyaluronan lyase purified in Example 4.

[0089] Reaction system: 0.2 mL of sodium hyaluronate (5 mg / mL, 800,000-1.5 million Da) and 0.1 mL of purified enzyme solution were added to 0.2 mM Tris-HCl buffer (pH 7.0). After incubation at 50°C for 15 min, 0.5 mL of 20 mM HCl was added to terminate the reaction, and the same mixture containing 0.1 mL of heat-inactivated enzyme solution was used as a blank control. After incubating the purified hyaluronate lyase and sodium hyaluronate for different times under the optimal reaction environment, the amount of product generated was measured, and it was determined that when the reaction time of hyaluronate lyase to degrade sodium hyaluronate was 15 min, its reaction rate reached the maximum. And according to the Linewaver-Burk plotting method, the kinetic parameters of hyaluronate lyase on sodium hyaluronate were calculated, as shown in Table 7. The analysis results show that the Michaelis constant Km of hyaluronate lyase for degradation of sodium hyaluronate is 0.31 mg / Ml, the maximum reaction rate Vmax is 135.14 μmol / min / mg, and the conversion rate kcat is 201.36 s -1 , the catalytic efficiency kcat / Km is 647.87 mg / mL / s.

[0090] Table 7 Kinetic parameters of hyaluronan lyase

[0091]

[0092] Example 8

[0093] Product analysis of the hyaluronan lyase purified in Example 4.

[0094] 0.1 mL of hyaluronan lyase (purified enzyme solution) and sodium hyaluronate (0.2 mL, 5 mg / mL, 800,000-1.5 million Da) were added to the reaction system at the same time, and hydrolyzed in a shaker at 37°C and 200 rpm. 500 μL was taken out from the total reaction system at 0 min, 1 min, 2 min, 10 min, 30 min and 60 min of reaction, and heated at 100°C for 5 min to terminate the reaction. The product was detected and analyzed by thin layer chromatography (TLC). The results are as follows: Figure 7 As shown, the products at different time points were detected by HPLC. The results are as follows Figure 8 As shown in the figure, the TLC and HPLC test results show that the final product of sodium hyaluronate hydrolysis by hyaluronan lyase is always only one unsaturated hyaluronan disaccharide, and the hyaluronan lyase is an exoenzyme, which gradually hydrolyzes from the end of the substrate to produce unsaturated disaccharide products until the polysodium hyaluronate is completely degraded.

[0095] 45 g of sodium hyaluronate (800,000-1.5 million Da) was added to 900 mL of water, placed in a shaker at 37°C and 200 rpm, 100 mg of hyaluronate lyase (purified enzyme solution) was added, and the product of hyaluronate lyase hydrolyzing sodium hyaluronate for 24 h was detected and analyzed by HPLC. Fig. 9 As shown, it was found that sodium hyaluronate degraded rapidly at the beginning of the reaction, and the conversion rate reached the maximum at 6 hours. When the conversion rate reached the maximum, the product was collected and purified. The protein substance in the system was removed by 3kDa ultrafiltration membrane, and then heated at 100°C for 10 minutes for inactivation treatment. The product water was removed by spray drying. The final yield of unsaturated hyaluronic acid disaccharide was 97.4% and the purity was ≥98.5%. HPLC detection showed only a single product peak, and the peak time was consistent with the peak time of the unsaturated disaccharide standard product, as shown in Fig.10 As shown, it is shown that the final product of hyaluronan degradation by hyaluronan lyase is only hyaluronan disaccharide.

[0096] At the same time, LC-MS / MS was used to analyze the products of sodium hyaluronate degradation by hyaluronan lyase. Fig.11 As shown, there is only one main peak in the product detection peak, and its mass-to-charge ratio m / z is 378.10, which is consistent with the size of unsaturated hyaluronic acid disaccharide, and the mass spectrum shows that there are no product peaks such as unsaturated tetrasaccharide and unsaturated hexasaccharide, indicating that the final product of the hyaluronate degradation by the hyaluronan lyase is only a single product, which is unsaturated hyaluronic acid disaccharide.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a hyaluronan lyase having an amino acid sequence as shown in SEQ ID NO.1 in the preparation of unsaturated hyaluronan disaccharide.

2. A method for preparing unsaturated hyaluronic acid disaccharide, characterized in that: Sodium hyaluronate and hyaluronan lyase with an amino acid sequence as shown in SEQ ID NO.1 are added to the aqueous phase system, and enzymatic hydrolysis is carried out at a temperature of 20° C.-60° C. and a pH of 4-10 to obtain the unsaturated hyaluronic acid disaccharide.

3. The method for preparing unsaturated hyaluronic acid disaccharide according to claim 2, characterized in that: The aqueous phase system is pure water or a calcium chloride aqueous solution.

4. The method for preparing unsaturated hyaluronic acid disaccharide according to claim 2, characterized in that: The temperature of the enzymatic hydrolysis reaction is 40°C; And / or the pH of the enzymatic hydrolysis reaction is 7.

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