CN111394344B - A low-temperature sulfate-tolerant hyaluronan lyase YNLX-HYL and its application - Google Patents

Abstract

The invention discloses a low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL and an application thereof, the amino acid sequence of which is shown in SEQ NO.1. The hyaluronan lyase YNLX‑HYL has the following properties: optimum pH 5.5, optimum temperature 35°C, 10%, 39% and 55% enzymatic activity at 0°C, 10°C and 20°C, respectively, at high concentrations (NH ₄ ) ₂ SO ₄ and Na ₂ SO ₄ have good activity and stability, and can split sodium hyaluronate to prepare unsaturated hyaluronic acid oligosaccharides. Unsaturated hyaluronic acid oligosaccharides have good antioxidant properties It can scavenge hydroxyl radicals, nitrogen radicals, superoxide anion radicals and resist the oxidation of 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). Ability. The hyaluronan lyase of the present invention can be applied to industries such as beauty, medicine, and leather making.

Description

A low-temperature sulfate-tolerant hyaluronan lyase YNLX-HYL and its application

technical field

The invention belongs to the technical field of genetic engineering, in particular to a low-temperature sulfate-resistant hyaluronic acid lyase YNLX-HYL and an application thereof.

Background technique

Hyaluronic acid, also known as hyaluronic acid, is a naturally occurring glycosaminoglycan in living organisms. D-glucuronic acid and N-acetyl-D-glucosamine are connected by β-1,3 glycosidic bond to form a disaccharide unit, and the polysaccharide formed by the disaccharide unit connected by β-1,4 glycosidic bond is hyaluronic acid .

Hyaluronidase can degrade hyaluronic acid. The first class of hyaluronidases are hyaluronic acid 4-glycosyl hydrolases (EC 3.2.1.35), which degrade hyaluronic acid by hydrolyzing 1,4-glycosidic bonds, with tetrasaccharide molecules as the main product. The second class of hyaluronidases are hyaluronan 3-glycosyl hydrolases (EC 3.2.1.36), which degrade hyaluronic acid by hydrolyzing 1,3-glycosidic bonds, with tetrasaccharide and hexasaccharide molecules as the main products. The third class of hyaluronidases are hyaluronan lyases (EC 4.2.2.1), which degrade hyaluronic acid through a β-elimination mechanism to generate unsaturated hyaluronan disaccharides (El-Safory et al. Carbohydrate Polymers, 2010, 81 :165–181.).

Hyaluronidase can be used in beauty, medicine, leather and other industries. For example, hyaluronidase can be used as a drug injection additive to enhance the body's absorption efficiency of drugs; in ophthalmic surgery, hyaluronidase can be used as an adjuvant to reduce the use of anesthetics; hyaluronidase can be used as a drug for therapeutic injection Post-cosmetic complications of hyaluronic acid; hyaluronidase can be used as an enzyme for processing leather to improve the tanning process and reduce the environmental pollution caused by the tanning process.

Low-temperature enzymes can be used in the field of biotechnology under low-temperature environments, which can prevent microbial contamination and reduce by-products caused by substrate degradation at high temperatures. Low-temperature reactions have lower energy consumption than high-temperature reactions (Cavicchioli et al. Microbial Biotechnology, 2011 , 4(4):449–460.). Salt can have a dramatic effect on the properties of enzymes. In neutral salts, enzymes will undergo salting out, resulting in most enzymes not having good catalytic activity at high salt concentrations. Salt is widely present in nature and in various production practices, including sewage, washing, tanning, food, paper, etc. For example, in the process of leather softening, sodium sulfate needs to be added. Enzymes with salt tolerance can be better used in the field of biotechnology in high-salt environments, and reactions in high-salt environments can also prevent microbial contamination and save energy consumed by sterilization (Madern et al. Extremophiles, 2000, 4: 91–98). Therefore, low-temperature sulfate-resistant hyaluronan lyase has advantages in applications such as tanning.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL and its application.

The technical purpose of the present invention is specifically realized through the following technical solutions:

A low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL, the amino acid sequence of which is shown in SEQ ID NO.1.

The hyaluronan lyase YNLX-HYL of the present invention contains a total of 799 amino acids, the theoretical molecular weight is 88kDa, and the 20 amino acids at the N-terminal are the predicted signal peptide sequence "MKKTNLAFSLLCLSMGSVHA". Compared with the hyaluronan lyases whose functions have been studied experimentally, the hyaluronan lyase YNLX-HYL has the highest amino acid sequence identity with the hyaluronan lyase derived from Streptococcus pneumoniae (Genbank ID: CTD38391), which is only 30.9%.

The hyaluronan lyase YNLX-HYL has the following properties: the optimum pH is 5.5, the optimum temperature is 35°C, and the enzyme activities are respectively 10%, 39% and 55% at 0°C, 10°C and 20°C, It has good activity and stability in high concentrations of (NH4)2SO4 and Na2SO4, and can split sodium hyaluronate to prepare unsaturated hyaluronic acid oligosaccharides. The ability to scavenge hydroxyl radicals, nitrogen radicals, superoxide anion radicals and anti-oxidation of 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).

The encoding gene ynlx-hyl of the low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL, the nucleotide sequence of the encoding gene ynlx-hyl is shown in SEQ ID NO.2. Its full length is 2400bp, the start code is ATG, and the stop code is TAG.

In another aspect of the present invention, the hyaluronan lyase gene of the present invention is inserted into an expression vector, and its nucleotide sequence is linked to an expression control sequence. Provided is a recombinant vector comprising the hyaluronan lyase gene ynlx-hyl. Preferred is pEasy-E2-ynlx-hyl.

As a most preferred embodiment of the present invention, the hyaluronan lyase gene of the present invention is connected with the expression vector pEasy-E2 to obtain the recombinant E. coli expression plasmid pEasy-E2-ynlx-hyl.

In another aspect of the present invention, there is also provided a recombinant strain comprising the hyaluronan lyase gene ynlx-hyl.

Preferably, the recombinant strain is Escherichia coli, yeast, Bacillus or Lactobacillus, more preferably recombinant strain BL21(DE3)/ynlx-hyl.

In another aspect of the present invention, a method for preparing unsaturated hyaluronic acid oligosaccharide using hyaluronan lyase YNLX-HYL is also provided.

In another aspect of the present invention, there is also provided a method for preparing hyaluronan lyase YNLX-HYL, comprising the following steps:

1) transform host cell with above-mentioned recombinant vector, obtain recombinant strain;

2) Cultivate the recombinant strain to induce the expression of recombinant hyaluronan lyase YNLX-HYL;

3) Recovery and purification of the expressed hyaluronan lyase YNLX-HYL.

Wherein, the host cell is an Escherichia coli cell, preferably, the recombinant Escherichia coli expression plasmid is transformed into an Escherichia coli cell BL21(DE3) to obtain a recombinant strain BL21(DE3)/ynlx-hyl.

In another aspect of the present invention, the hyaluronic acid lyase YNLX-HYL can be used for degrading hyaluronic acid in the fields of beauty and medicine, and can also be used in the preparation of leather.

The beneficial effects of the present invention are:

The present invention provides a new hyaluronan lyase gene, the encoded hyaluronan lyase YNLX-HYL has the characteristics of low temperature sulfate resistance, and YNLX-HYL can cleave sodium hyaluronate to prepare unsaturated hyaluronan oligosaccharides Sugar, unsaturated hyaluronic acid oligosaccharide with excellent antioxidant properties. The hyaluronic acid lyase of the invention can be applied to industries such as beauty, medicine, leather making and the like.

Description of drawings

Figure 1 is the SDS-PAGE analysis of the hyaluronan lyase YNLX-HYL expressed in Escherichia coli, wherein, M: protein Marker; HYL: purified recombinant hyaluronan lyase YNLX-HYL;

Figure 2 is the pH activity of purified recombinant hyaluronan lyase YNLX-HYL;

Figure 3 is the pH stability of purified recombinant hyaluronan lyase YNLX-HYL;

Figure 4 is the thermal activity of purified recombinant hyaluronan lyase YNLX-HYL;

Figure 5 is the thermostability of purified recombinant hyaluronan lyase YNLX-HYL;

Figure 6 is the activity of purified recombinant hyaluronan lyase YNLX-HYL in (NH4)2SO4;

Figure 7 is the stability of purified recombinant hyaluronan lyase YNLX-HYL in (NH4)2SO4;

Figure 8 is the activity of purified recombinant hyaluronan lyase YNLX-HYL in Na2SO4;

Figure 9 is the stability of purified recombinant hyaluronan lyase YNLX-HYL in Na2SO4;

Figure 10 is the analysis of the product of purified recombinant hyaluronan lyase YNLX-HYL cleaving sodium hyaluronate, wherein, R: lactose; 4h: product of reaction 4h; 1h: product of reaction 1h; 20min: product of reaction 20min;

Figure 11 is the nitrogen free radical scavenging rate of purified recombinant hyaluronate lyase YNLX-HYL cleaving sodium hyaluronate product, wherein, HA: nitrogen free radical scavenging rate of sodium hyaluronate; HAM: nitrogen free radical of the product of reaction 1h Free radical scavenging rate; HAD: nitrogen free radical scavenging rate of the product of reaction 5h;

Figure 12 is the hydroxyl radical scavenging rate of purified recombinant hyaluronan lyase YNLX-HYL cleavage product of sodium hyaluronate, wherein HA: the hydroxyl radical scavenging rate of sodium hyaluronate; HAM: the hydroxyl radical scavenging rate of the product of reaction 1h Free radical scavenging rate; HAD: hydroxyl radical scavenging rate of the product of reaction 5h;

Figure 13 is the superoxide anion free radical scavenging rate of purified recombinant hyaluronan lyase YNLX-HYL cleavage of sodium hyaluronate, wherein, HA: superoxide anion free radical scavenging rate of sodium hyaluronate; HAM: reaction 1h The superoxide anion free radical scavenging rate of the product; HAD: the superoxide anion free radical scavenging rate of the product of the reaction for 5h;

Figure 14 is the relative total antioxidant rate of purified recombinant hyaluronate lyase YNLX-HYL cleavage of sodium hyaluronate products, wherein, HA: the relative total antioxidant rate of sodium hyaluronate; HAM: the relative total antioxidant rate of the product of reaction 1h Total antioxidant rate; HAD: the relative total antioxidant rate of the product of reaction 5h.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Test Materials and Reagents

1. Strain and vector: Citrobacter freundii has the same properties as reported in the literature, such as Citrobacter freundii CGMCC NO.1.12836 of China General Microorganism Culture Collection and Management Center; Escherichia coliBL21 (DE3) and expression vector pEasy- E2 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

II试剂盒购自南京诺维赞公司，氮自由基清除能力测试试剂盒购于上海聪羿生物公司，超氧阴离子和羟自由基清除能力检测试剂盒购于索莱宝生物科技有限公司，总抗氧化能力检测试剂盒购于南京建成生物，其它试剂均可从普通生化试剂公司购买得到。2. Reagents: DNA polymerase and dNTP were purchased from TaKaRa company, sodium hyaluronate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., Nickel-NTAAgarose was purchased from QIAGEN company,

The II kit was purchased from Nanjing Novizan Company, the nitrogen free radical scavenging ability test kit was purchased from Shanghai Congyi Biological Company, and the superoxide anion and hydroxyl free radical scavenging ability test kit was purchased from Soleibo Biotechnology Co., Ltd. Antioxidant ability detection kit was purchased from Nanjing Jiancheng Bio, and other reagents can be purchased from general biochemical reagent company.

3. Culture medium:

LB medium: Peptone 10g, Yeast extract 5g, NaCl 10g, add distilled water to 1000ml, pH is natural (about 7). The solid medium was supplemented with 2.0% (w/v) agar on this basis.

Note: The molecular biology experimental methods that are not specifically described in the following examples are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) by J. Sambrook, or according to the kit and product manual.

Example 1 Cloning of hyaluronan lyase gene ynlx-hyl

Extraction of the genomic DNA of Citrobacter freundii: centrifuge the 2-day-culturing liquid bacterial solution to take the bacterial cells, add 1 mL of lysozyme, treat at 37°C for 60 min, and then add the lysate. The lysate consists of: 50mM Tris, 20mM EDTA, NaCl 500mM, 2% SDS (w/v), pH 8.0, lysed in a water bath at 70°C for 60 min, mixed every 10 min, and centrifuged at 10000 rpm for 5 min at 4°C. The supernatant was extracted in phenol/chloroform to remove impurity proteins, and then the supernatant was added with an equal volume of isopropanol, and after standing at room temperature for 5 min, centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried under vacuum, dissolved by adding an appropriate amount of TE, and placed at -20°C for later use.

The genome DNA of Citrobacter freundii was sent to Wuhan Future Group Biotechnology Co., Ltd. for genome sequencing. After the data obtained by genome sequencing is predicted by the reading frame and the database sequence is compared, the genome of Citrobacter freundii is functionally annotated, and the hyaluronan lyase gene ynlx-hyl is obtained. The gene sequence is shown in SEQ ID NO.2 .

Example 2 Preparation of recombinant hyaluronan lyase YNLX-HYL

Using 5'TAAGAAGGAGATATACATATGCAGATCGCTACCGAAAATGTAAAT 3' and 5'GTGGTGGTGGTGGTGCTCGAGTTTATTTTTAGATAATTCAAAAGAATAACTACTG 3' as primer pairs and Citrobacter freundii genomic DNA as a template, PCR amplification was performed. The PCR reaction parameters were: denaturation at 95°C for 5 min; then denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min 30 sec, and incubation at 72°C for 10 min after 30 cycles. As a result of PCR, the hyaluronan lyase gene ynlx-hyl was obtained, and the recombination region with the vector pEasy-E2 was introduced into the 5' and 3' ends of the gene.

The plasmid pEasy-E2 was double digested with restriction enzymes Nde I and Xho I, and the product was recovered by agarose gel electrophoresis to obtain the linearized vector pEasy-E2.

II试剂盒，将透明质酸裂解酶基因ynlx-hyl和线性化载体pEasy-E2重组连接，获得含有ynlx-hyl的重组表达质粒pEasy-E2-ynlx-hyl。use

II kit, the hyaluronan lyase gene ynlx-hyl and the linearized vector pEasy-E2 were recombined to obtain the recombinant expression plasmid pEasy-E2-ynlx-hyl containing ynlx-hyl.

By heat shock, pEasy-E2-ynlx-hyl was transformed into E. coli BL21(DE3) to obtain recombinant E. coli strain BL21(DE3)/ynlx-hyl.

Take the recombinant Escherichia coli strain BL21(DE3)/ynlx-hyl containing the recombinant plasmid pEasy-E2-ynlx-hyl, inoculate it in LB (containing 100 μg mL-1Amp) medium with 0.1% inoculum, and shake rapidly at 37°C for 16h . Then the activated bacterial solution was inoculated into fresh LB (containing 100μg mL-1Amp) culture solution at 1% inoculum, and after rapid shaking culture for about 2-3h (OD600 reached 0.6-1.0), the final concentration of 0.7mM was added. IPTG was induced, and the shaking culture was continued at 20°C for about 20 hours or at 26°C for about 8 hours. The cells were collected by centrifugation at 12000 rpm for 5 min. After suspending the cells with an appropriate amount of pH7.0 McIlvaine buffer, the cells were disrupted by ultrasonic waves in a low temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 12,000 rpm for 10 min, the supernatant was aspirated and the target protein was affinity and eluted with Nickel-NTA Agarose and 0–500 mM imidazole, respectively. SDS-PAGE results (Fig. 1) showed that the recombinant hyaluronan lyase YNLX-HYL was purified and the product was a single band.

Example 3 Characterization of the purified recombinant hyaluronan lyase YNLX-HYL

1. Activity analysis of purified recombinant hyaluronan lyase YNLX-HYL:

Example 2 The method for measuring the activity of the purified recombinant hyaluronan lyase YNLX-HYL adopts the ultraviolet method: dissolving sodium hyaluronate in the buffer to make the final concentration 0.5% (w/v); the reaction system contains 50 μL Appropriate amount of enzyme solution, 450μL of 0.5% (w/v) sodium hyaluronate; after the substrate is preheated at the reaction temperature for 5min, add the enzyme solution and react for another 10min, then add 3.5mL of 0.2M HCl to stop the reaction, at a wavelength of 232nm Absorbance value was determined; 1 unit of enzyme activity (U) was defined as the amount of enzyme required to degrade sodium hyaluronate to form 1 μmol of unsaturated double bonds per minute. The unsaturated double bond extinction coefficient is 5.5/mM/cm.

2. Determination of pH activity and pH stability of purified recombinant hyaluronan lyase YNLX-HYL:

Enzyme pH activity assay: The hyaluronan lyase YNLX-HYL was enzymatically reacted at 37°C in buffer pH 3.0–10.0.

Determination of pH stability of the enzyme: The purified enzyme solution was placed in a buffer of pH 3.0–9.0, treated at 20 °C for 60 min, and then enzymatically reacted at pH 5.5 and 37 °C, and the untreated enzyme was used. liquid as a control.

The buffers were: McIlvaine buffer (pH3.0–8.0) and 0.1M glycine–NaOH (pH9.0–10.0). Using sodium hyaluronate as the substrate, the reaction was carried out for 10 min to determine the enzymatic properties of the purified hyaluronan lyase YNLX-HYL.

The results showed that the optimum pH of YNLX-HYL was 5.5 (Fig. 2); after 1 h of buffer treatment with pH 5.0-7.0, the enzyme activity remained over 59% (Fig. 3).

3. Determination of thermal activity and thermal stability of purified recombinant hyaluronan lyase YNLX-HYL:

Enzyme Thermal Activity Assay: Enzymatic reactions were performed at 0–70°C in pH 5.5 buffer.

Determination of the thermal stability of the enzyme: The enzyme solution with the same amount of enzyme was placed at 40 °C, 50 °C and 60 °C, respectively, and after treatment for 10–60 min, the enzymatic reaction was carried out at pH 5.5 and 37 °C, and the untreated enzyme was used. Enzyme solution served as a control.

Using sodium hyaluronate as the substrate, the reaction was carried out for 10 min, and the enzymatic properties of the purified YNLX-HYL were determined. The results show that YNLX-HYL is a typical low temperature enzyme. The optimum temperature of YNLX-HYL was 35°C, and the enzyme activities were 10%, 39%, and 55% at 0°C, 10°C, and 20°C, respectively (Fig. 4); the enzyme was treated at 40°C and 50°C for 60 min. , the enzyme activity remained 59% and 18%, respectively, and was rapidly inactivated at 60 °C (Figure 5).

4. Determination of activity and stability of purified recombinant hyaluronan lyase YNLX-HYL in (NH ₄ ) ₂ SO ₄ :

Determination of enzyme activity in (NH ₄ ) ₂ SO ₄ : add 1.0–30.0% (w/v) (NH ₄ ) ₂ SO ₄ to the enzymatic reaction system, and carry out the enzymatic reaction at pH 5.5 and 37°C .

Stability determination of enzyme in (NH ₄ ) ₂ SO ₄ : The purified enzyme solution was placed in 5.0–30.0% (w/v) aqueous (NH ₄ ) ₂ SO ₄ solution, treated at 37 °C for 60 min, and then The enzymatic reaction was carried out at pH 5.5 and 37°C, and the untreated enzyme solution was used as a control.

Using sodium hyaluronate as the substrate, the reaction was carried out for 10 min, and the enzymatic properties of the purified YNLX-HYL were determined. The results showed that: adding 1.0-30.0% (w/v) (NH ₄ ) ₂ SO ₄ to the reaction system, the activity of YNLX-HYL gradually increased to 276% (Fig. 6); after 5.0-30.0% (w/v) ) of (NH ₄ ) ₂ SO ₄ was treated at 37° C. for 60 min, and the activity of the enzyme gradually decreased from 143% to 18% ( FIG. 7 ).

5. Determination of activity and stability of purified recombinant hyaluronan lyase YNLX-HYL in Na ₂ SO ₄ :

Determination of enzyme activity in Na ₂ SO ₄ : 1.0–30.0% (w/v) Na 2 SO 4 was added to the enzymatic reaction system, and the enzymatic reaction was carried out at pH 5.5 and 37°C.

Determination of enzyme stability in Na ₂ SO ₄ : The purified enzyme solution was placed in a 5.0–30.0% (w/v) Na ₂ SO ₄ aqueous solution, treated at 37 °C for 60 min, and then at pH 5.5 and 37 The enzymatic reaction was carried out at ℃, and the untreated enzyme solution was used as a control.

Using sodium hyaluronate as the substrate, the reaction was carried out for 10 min, and the enzymatic properties of the purified YNLX-HYL were determined. The results show that: adding 1.0-20.0% (w/v) Na _{2 SO 4 to the reaction system, the activity of YNLX-HYL gradually increased to 220%, adding 20.0-30.0% (w/v) Na 2 SO 4 ,} The activity of YNLX-HYL gradually decreased from 220% to 130% (Fig. 8); after being treated with 5.0–30.0% (w/v) Na ₂ SO ₄ for 60 min at 37°C, the activity of the enzyme showed a decreasing trend overall, and the lowest dropped to 19% (Figure 9).

Example 4 Method and application of purified recombinant hyaluronan lyase YNLX-HYL in the preparation of unsaturated hyaluronan oligosaccharides

1. Preparation of unsaturated hyaluronic acid oligosaccharides:

The reaction system contains 450 μL of 0.5% (w/v) sodium hyaluronate, 50 μL of appropriately diluted YNLX-HYL (about 130 U enzyme solution), at pH 5.5 and 37 °C, after 20 min–4 h of reaction, boil for 5 min to terminate the reaction . The cleavage products were analyzed by thin-layer chromatography (TLC) (using a high-performance thin-layer chromatography silica gel plate type G from Qingdao Ocean Chemical Co., Ltd.).

The thin layer chromatography steps are as follows:

(1) Prepare a developing agent (20 mL of glacial acetic acid, 20 mL of double-distilled water, 40 mL of n-butanol, mix well), pour an appropriate amount into the developing tank, and let it stand for about 30 minutes;

(2) Put the silica gel plate in a 110°C oven for activation for 30min, draw a line after cooling, and spot the sample (0.5 μL each time, blow dry, spot 3 times in total);

(3) Put the silica gel plate at one end of the spotting down into the developing tank, and do not submerge the developing agent into the spotting spot;

(4) When the developing agent is 1.5cm away from the silica gel plate, take out the silica gel plate, blow it dry, and expand it again;

(5) After the second expansion, the silica gel plate is directly immersed in an appropriate amount of color developer (1 g of diphenylamine is dissolved in 50 mL of acetone, and after dissolving, 1 mL of aniline and 5 mL of 85% phosphoric acid are added, mixed, and used now and prepared);

(6) After a few seconds, immediately take out the silica gel plate and place it in an oven at 90°C for 10-15min to make the spots develop.

The results showed that the products formed by YNLX-HYL cleaving sodium hyaluronate for 20 min were mainly unsaturated hyaluronic acid disaccharides and two unsaturated hyaluronic acid oligosaccharides; The product is mainly unsaturated hyaluronic acid disaccharide, and there is also an unsaturated hyaluronic acid oligosaccharide; the product formed after YNLX-HYL cleaves sodium hyaluronate for 4h is unsaturated hyaluronic acid disaccharide (Figure 10).

2. Antioxidative analysis of unsaturated hyaluronic acid oligosaccharides:

The reaction system contains 5 mL of 5% (w/v) sodium hyaluronate, 1 mL of appropriately diluted YNLX-HYL (about 5000U enzyme solution), at pH 5.5 and 35 ℃, after 1h and 5h of reaction, boiled for 5min to terminate the reaction . The cleavage product was added to a 10kDa ultrafiltration tube, centrifuged at 5000 rpm to remove macromolecular substances such as enzymes, and the filtrate in the tube was collected to obtain unsaturated hyaluronic acid oligosaccharides.

The nitrogen free radical scavenging ability, hydroxyl radical scavenging ability, superoxide anion free radical scavenging ability and total antioxidant capacity of sodium hyaluronate and unsaturated hyaluronic acid oligosaccharides were detected by kits. The control substance for total antioxidant capacity is 1 mM water-soluble vitamin E (Trolox), which is detected by ABTS.

The results showed that the nitrogen radical scavenging rates of unsaturated hyaluronan oligosaccharides formed by YNLX-HYL cleaving sodium hyaluronate for 1h and 5h were 68% and 73%, respectively, while the nitrogen radical scavenging rate of uncleaved sodium hyaluronate was 68% and 73%, respectively. The hydroxyl radical scavenging rate of unsaturated hyaluronic acid oligosaccharides formed by YNLX-HYL cleaving sodium hyaluronate for 1h and 5h was 68% and 79%, respectively, while the uncleaved hyaluronic acid The hydroxyl radical scavenging rate of sodium is 41% (Fig. 12); the superoxide anion radical scavenging rate of unsaturated hyaluronic acid oligosaccharides formed by YNLX-HYL cleavage of sodium hyaluronate for 1h and 5h are 62% and 64%, respectively , while the superoxide anion radical scavenging rate of uncleaved sodium hyaluronate was 13% (Fig. 13); rates were 62% and 61%, respectively, while the relative total antioxidant rate of uncleaved sodium hyaluronate was 14% (Figure 14).

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principle and spirit of the invention Variations, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Yunnan Normal University

<120> A low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL and its application

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Ala Val Thr Thr Ile Ala Asn Gln Lys Ile Ser Gly Ser Ala Lys Val

580 585 590

Leu Val Asp Gly Ile Val Leu Leu Pro Gly Glu Glu Arg Lys Ala Thr

595 600 605

Gln Ser Val Val Leu Asn Asp Lys Gly Asn Asn Ile Ile Trp Lys Pro

610 615 620

Leu Ala Gly Ser Ser Ala Gln Val Ser Val Lys Gln Arg Gln Gly Asn

625 630 635 640

Trp Ala Asp Ile Gly Thr Ser Ser Gly Lys Val Ser Ala Gln Phe Leu

645 650 655

Thr Ile Ile Gln Pro His Ser Ala Glu Ser Asp Asn His Tyr Ala Trp

660 665 670

Val Val Phe Pro Ser Gly Ser Ala Ser Pro Ser Val Asn Ala Asp Ile

675 680 685

Thr Leu Leu Ala Asn Asp Ala Lys Val Gln Ala Val Ser Leu Pro Gly

690 695 700

Gln Gln Val Ile Tyr Ala Asn Phe Trp Arg Ser Ala Thr Val Gly Gly

705 710 715 720

Ile His Ala Leu Thr Pro Met Ser Leu Ile Met Thr Pro Thr Thr Gln

725 730 735

Gly Tyr Gln Ile Ala Val Ser Ser Pro Arg Arg Asp Ser Arg Val Ser

740 745 750

Phe Gln Leu Pro Asp Asn Ala Ile Pro Phe His Ile Ser Ser Asp Pro

755 760 765

Asp Lys Arg Val Ser Leu Asn Gly Glu Ile Val Ser Val Asn Met Thr

770 775 780

Asn Leu Arg Gly Ser Ser Tyr Ser Phe Glu Leu Ser Lys Asn Lys

785 790 795

<210> 2

<211> 2400

<212> DNA

<213> Hyaluronan lyase gene (ynlx-hyl)

<400> 2

atgaaaaaga caaatttagc attttcgcta ttgtgcttaa gtatgggcag cgtacatgca 60

cagatcgcta ccgaaaatgt aaatctgcca gtagtaaaat caactaccac aacgtcaacc 120

cagcaacagc atattattga gcgtatgcgt gatacctggc ggcagaattt tgtgccttca 180

ggcccagcgg cgccagaatt atcagcagag tatgtggcaa gtttaaacaa aacggccaat 240

atattctgga aaggaataga taaaaatacc cccgcaggcc agttatgggc agataccgta 300

ctggatagtg aaagcacatc aggacgcctg aaactgggca ctactcttta tacggtatac 360

caacgcctgt tcaccctggc aaaagcctgg gctacgccgg ggacagatct ttataaaaat 420

gctcagttga atactgtact taaatcagcg ctgatcaatc tgaaccagga ctattataac 480

gatcagaccc cagaatgggg aaactggtgg aactgggagt taggcatttc acgcagtgtt 540

aacaatactc tggtgatact ttatgatgat ctcccttcca cgctgattga taattataat 600

ctcgcgaccc gacattttgt tcgcgaccct cgttatttag ctgaaggaag cggagccccc 660

tactccacaa caaaaaatgc ctttacgtcg actggcggaa accgcattga cagcgcaatg 720

gtcgtttttg ttcggggtct cctggctaac gatcctggag aaattagcgc tgcagtaact 780

tcagtacctg aagtgcttaa caccgttcag tccggagatg gtttctacaa agacggatcg 840

tttatccagc ataaagattt accttatagc ggaacctatg gccaggtttt gctgaatggt 900

ctgggattaa ttaaaaacag cgtcgcaggt acaccatggg atttctcagt tgaagataat 960

cgccgtattt atgacgtgat cagacaagct tttttacctt tacttcatga gggaaaaatg 1020

cccgatgccg tcaatgggcg cagtatttca cgtaaaaatg ggcaggatca ggatgttggc 1080

gcatcagtta tgaatgccat tgcattgttt gttaatggtg cgccaccaga agaaaagcgc 1140

cacattgaac aggtattaaa agcccagcta aattcaaaaa caacggaata ttatcacact 1200

cacttgccag aaaacttaac ctcctggcag gttattacgc gtattcaaca gcatagccat 1260

ttgccaccgg ccccccgaac agcgggcggt aaactgtatg cagatatgga tcgtctgatt 1320

tatcagggta caaactatct ggctgttgta gcgatgcatt ccaatcgtac tggtagctac 1380

gaatgtatta ataacgagaa tctaaaaggg cagagaacat ctgatgggat gacctggctg 1440

tatttgccca atgacgatca atatcgtgat tactggcctg tggtcgacag tcggttttta 1500

ccaggcacca cctctgctgg cgagcagggt tggtgtgatg agcaataccg tgtgactcag 1560

ttaggtcggg caaatatcgc ctgggcgggt ggcaatactc tgaataaatg ggcaagcgcg 1620

agtatgcatt taaaagtgcc gacttattca ctcaaggcga aaaaatcctg gttcatggca 1680

ccgcatgaaa tgatcatgct tgggagccag atatccagta gtagcccggc ggtaacgacc 1740

attgctaatc agaaaatcag tggctcggca aaagtgcttg ttgatggcat cgtcttgctg 1800

cccggagaag agagaaaagc aacccaatct gtcgttttaa acgataaagg taataacatt 1860

atctggaagc cattagctgg ctcaagcgca caagttagtg tgaaacaacg tcagggtaac 1920

tgggccgata tcggcacctc atccggtaaa gtttcggcac aatttttaac cattatccag 1980

cctcatagcg cagaatcaga taatcattat gcctgggttg tcttcccctc ggggtccgca 2040

tccccttccg taaatgctga cataacgctc cttgcgaatg atgcaaaagt ccaggctgtg 2100

tcgctaccag gtcagcaggt tatttatgct aatttctggc gttctgcaac tgtgggaggc 2160

attcatgcat tgacgccaat gtccctgatt atgacgccaa caacacaagg ttatcagata 2220

gcagtatctt caccacgtcg tgatagtcgg gtgtcattcc aactgcccga taatgcaatc 2280

ccattccata tttccagtga ccctgataag cgcgtatctc ttaacgggga gatcgtcagc 2340

gtgaatatga ccaatctacg cggcagtagt tattcttttg aattatctaa aaataaatag 2400

<210> 3

<211> 20

<212> PRT

<213> Artificial Sequence

<400> 3

Met Lys Lys Thr Asn Leu Ala Phe Ser Leu Leu Cys Leu Ser Met Gly

1 5 10 15

Ser Val His Ala

20

<210> 4

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 4

taagaaggag atatacatat gcagatcgct accgaaaatg taaat 45

<210> 5

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 5

gtggtggtgg tggtgctcga gtttattttt agataattca aaagaataac tactg 55

Claims (6)

1. a low-temperature sulfate-resistant hyaluronan lyase YNLX-HYL, is characterized in that, the amino acid sequence of described hyaluronan lyase YNLX-HYL is as shown in SEQ ID NO.1. 2. The coding gene ynlx-hyl of the hyaluronan lyase YNLX-HYL according to claim 1, wherein the nucleotide sequence of the coding gene ynlx-hyl is as shown in SEQ ID NO.2. 3. A recombinant expression vector, characterized in that it comprises the encoding gene ynlx-hyl of claim 2. 4 . The recombinant expression vector according to claim 3 , wherein the recombinant expression vector is pEasy-E2-ynlx-hyl. 5 . 5. a recombinant bacteria, is characterized in that, comprises the described coding gene ynlx-hyl of claim 2. 6 . The recombinant bacteria according to claim 5 , wherein the recombinant bacteria is BL21(DE3)/ynlx-hyl. 7 .

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