CN114457061A - Hyaluronic acid lyase and application thereof - Google Patents

Hyaluronic acid lyase and application thereof Download PDF

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CN114457061A
CN114457061A CN202210156112.0A CN202210156112A CN114457061A CN 114457061 A CN114457061 A CN 114457061A CN 202210156112 A CN202210156112 A CN 202210156112A CN 114457061 A CN114457061 A CN 114457061A
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hyaluronic acid
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enzyme
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lyase
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常耀光
孙孟辉
薛长湖
李嘉靖
陈广宁
张玉莹
薛勇
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Ocean University of China
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    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides

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Abstract

The invention relates to the technical field of biotechnology and biochemical detection, in particular to a hyaluronic acid lyase and application thereof. The invention provides a hyaluronic acid lyase Hya16A, and the specific hyaluronic acid lyase is utilized to degrade hyaluronic acid into unsaturated disaccharide specifically, the content of hyaluronic acid is determined by determining the light absorption value at 232nm, the rapid quantitative detection of hyaluronic acid by an enzyme method is realized, and the method has the advantages of high sensitivity, good accuracy, simple operation and the like.

Description

Hyaluronic acid lyase and application thereof
Technical Field
The invention relates to the technical field of biotechnology and biochemical detection, in particular to a hyaluronic acid lyase and application thereof.
Background
Hyaluronic acid (Hyaluronan or Hyaluronic acid,HA), a glycosaminoglycan (GAG) naturally occurring in the body of an organism. Hyaluronic acid is a polysaccharide formed by connecting D-glucuronic acid and N-acetyl-D-glucosamine by beta-1, 3 glycosidic bonds to form disaccharide units, and the disaccharide units are connected by beta-1, 4 glycosidic bonds, and the molecular formula is as follows: (C)14H21NO11)n. Hyaluronic acid is widely found in connective tissues such as joints, vitreous, synovial fluid, umbilical cord, cartilage, skin, cockscomb, and group a and group C hemolytic streptococci. Has the functions of improving toughness, strengthening structural support, participating in cell metabolism and the like in organisms. Hyaluronic acid is generally a colorless, odorless, amorphous solid, readily soluble in water, and insoluble in organic solvents. The hyaluronic acid molecule is in a rigid spiral column shape in a spatial structure, hydroxyl groups on the inner side of the column have high hydrophilicity, and meanwhile, the continuous and directional arrangement of the hydroxyl groups forms a high hydrophobic region on a hyaluronic acid molecular chain, so that the hyaluronic acid can form a three-dimensional network structure even at low concentration, has high hygroscopicity, water retention and viscoelasticity, is a good natural moisturizing factor, is commonly used as a cosmetic ingredient and is added into food as a new food raw material.
The quantitative detection of hyaluronic acid is a basic link in the quality control, function research and product development of hyaluronic acid. The conventional detection methods for hyaluronic acid include a carbazole chromogenic method, an HPLC method, a CTAB turbidimetry method and the like. The carbazole method requires strong acid and high temperature conditions, and is poor in reproducibility and complicated in operation. The HPLC method obtains the content of the hyaluronic acid by hydrolyzing, deriving and pretreating a sample and measuring the content of monosaccharide consisting of the hyaluronic acid, and the method has the advantages of high sensitivity, good accuracy, high instrument cost and long detection period. Therefore, a method for detecting a flow rapidly and simply is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problems that the commonly used hyaluronic acid quantitative detection method is a carbazole method and an HPLC method, the two methods have the problems of poor reproducibility, complex operation, time and labor waste, and a method for quickly, simply and conveniently detecting the hyaluronic acid flow is urgently needed.
In order to solve the problems, the invention provides a hyaluronic acid lyase Hya16A, which specifically degrades hyaluronic acid into unsaturated disaccharide by utilizing the specific hyaluronic acid lyase, and the content of hyaluronic acid is determined by determining the light absorption value at 232nm, so that the rapid quantitative detection of the hyaluronic acid by an enzyme method is realized, and the hyaluronic acid lyase has the advantages of high sensitivity, good accuracy, simple operation and the like.
In order to achieve the purpose, the invention is realized by the following technical scheme: a hyaluronidase whose amino acid sequence is SEQ ID NO. 1.
SEQ ID NO.1
MSVSRRLFLGGFTAGAVTVAAGAAATPAAAAEADGPTTTFDGPVVAEGFRTDSTVKSAFFKTTSTTEHAVTAYQAGTSGSGVALNVVSKNPGDSAMYLSGTEKAHGTLKISHTGHADGSDEKASALSIDLLTAGTAAQGIFVKAGNGPTTGNLICLRNNARDDFVVKGSGRVGIGMGVGGNPWSQLHVVQQPGTDSALMVEGTVRVVDVASAPTGVDSRGGGVLYAENGALKWRGSDNTVTTIAPA
The hyaluronic acid lyase is derived from pathogenic fungi, bacteria, bacteriophage and other microorganisms. The hyaluronic acid lyase is named Hya16A, has novel amino acid sequence and good enzymological property, specifically degrades hyaluronic acid into unsaturated disaccharide through a beta-elimination mechanism, takes 2-aminoacetyl-2-deoxy-3-O- (beta-D-glucose-4-alkene pyranouronic acid-) D-glucose as a main product, and takes C of a sugar unit at the non-reduction end of the product4-C5Unsaturated double bonds are formed among sites, and the double bonds have the maximum absorption value under 232nm ultraviolet light, so that the content of the hyaluronic acid can be determined by detecting the change of the content of the unsaturated double bonds after the hyaluronic acid lyase reacts with a substrate by using an ultraviolet spectrophotometer.
Furthermore, the optimal reaction temperature of the hyaluronic acid lyase is 45 ℃, and more than 80% of enzyme activity can be maintained within the range of 30-50 ℃. The optimum reaction pH value is 8.0, and the pH value is basically kept stable in a pH range of 7.0-9.0. The enzyme has good storage stability, can be stored stably for at least 3 months at 4 ℃, and has an enzyme kinetic constant Km of 0.03mg/mL and a Kcat of 13.41s-1Km/Kcat of 4.25. mu.M-1s-1。
The enzyme has good reaction specificity, high activity on hyaluronic acid, and no degradation effect on other glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, heparin, etc. In conclusion, the hyaluronan lyase Hya16A has good application potential in the aspect of quantitative detection of the hyaluronan, has the characteristics of simple and convenient operation, high accuracy and good stability, has short detection period, can complete detection within 20 minutes, and realizes rapid quantification of the specificity of the hyaluronan.
The nucleotide sequence of the gene for coding the hyaluronic acid lyase is SEQ ID NO.2 or all genes capable of being translated into SEQ ID NO. 1.
SEQ ID NO.2
ATGAGCGTGTCGCGGAGGTTGTTCCTCGGAGGGTTCACCGCGGGGGCGGTGACCGTGGCGGCGGGCGCCGCCGCGACGCCGGCGGCGGCCGCGGAGGCGGACGGCCCGACGACGACGTTCGACGGTCCGGTGGTGGCGGAGGGTTTCAGGACGGACTCCACCGTCAAGTCCGCCTTCTTCAAGACGACGTCGACGACCGAGCACGCGGTGACGGCCTATCAGGCCGGCACGTCCGGCAGCGGCGTGGCCCTGAACGTCGTATCGAAGAACCCGGGTGACTCGGCCATGTATCTCAGTGGCACGGAGAAGGCGCACGGCACGCTGAAGATCTCGCACACGGGCCACGCGGACGGCTCGGACGAGAAGGCGTCCGCTCTGTCGATCGACCTGCTGACGGCGGGGACGGCAGCCCAGGGCATCTTCGTGAAGGCGGGCAACGGGCCCACCACCGGCAACCTGATCTGCCTGCGCAACAACGCCCGAGACGACTTCGTCGTCAAGGGCAGCGGGCGGGTCGGTATCGGCATGGGCGTGGGCGGCAACCCCTGGTCGCAGCTCCATGTCGTGCAGCAGCCGGGCACCGACTCGGCGCTGATGGTCGAGGGCACGGTGCGGGTCGTCGACGTGGCCTCCGCGCCCACGGGCGTCGACTCGCGCGGCGGCGGCGTGCTGTACGCGGAGAACGGTGCGCTGAAGTGGCGCGGCTCCGACAACACGGTCACCACCATCGCCCCCGCCTGA
The application of the hyaluronic acid lyase Hya16A in the rapid quantitative detection of hyaluronic acid.
A method for rapidly and quantitatively detecting hyaluronic acid by an enzyme method specifically degrades hyaluronic acid into unsaturated disaccharide by utilizing hyaluronic acid lyase Hya 16A; and (3) inactivating the enzyme, centrifuging, measuring the light absorption value of the supernatant at 232nm, and comparing with a standard curve to obtain the content of the hyaluronic acid.
Further, the method for rapidly and quantitatively detecting hyaluronic acid by using the enzyme method specifically comprises the following steps:
(1) preparing a hyaluronic acid solution: weighing hyaluronic acid with chemical purity or purity above, and dissolving in buffer solution to prepare hyaluronic acid standard solution with concentration gradient;
(2) drawing a quantitative standard curve: mixing the hyaluronic acid solutions with different concentrations prepared in the step (1) with a proper amount of hyaluronic acid lyase liquid respectively for reaction; after the reaction, standing in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme, quickly cooling to room temperature, centrifuging to obtain supernatant, measuring the light absorption value of the supernatant, and detecting the wavelength of 240 nm; mixing the hyaluronic acid solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the hyaluronic acid solution as a control, and calculating the light absorption value increment corresponding to the hyaluronic acid solutions with different concentration gradients; taking the concentration of a hyaluronic acid standard solution as an abscissa and the light absorption value increment of hyaluronic acid with each concentration as an ordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;
(3) and (3) sample determination: adding a certain amount of hyaluronic acid lyase into the sample solution to repeat the reaction in the step (2); and substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating to obtain the concentration of the hyaluronic acid in the reaction system so as to obtain the content of the hyaluronic acid in the sample.
Further, the pH value of the buffer solution in the step (1) is 7.0-9.0, and the enzyme activity is higher in the pH range, thereby being beneficial to full enzymolysis.
Further, the addition amount of the enzyme in the step (2) is 1-1000U, the reaction time is 5-40min, the reaction temperature is 30-50 ℃, and Hya16A has higher enzymolysis activity and can keep stability within the range of 30-50 ℃; within the parameter range, the hyaluronic acid lyase, particularly Hya16A, has higher enzymolysis activity, so that the rapid enzymolysis reaction is ensured; the addition amount of the enzyme and the reaction time need to correspond to each other, and if the addition amount of the enzyme is small, the reaction time is increased, so that the complete enzymolysis of the sample is ensured.
The invention has the beneficial effects that: the invention provides a method for rapidly and quantitatively detecting hyaluronic acid by an enzyme method, which utilizes hyaluronic acid lyase to efficiently and specifically degrade hyaluronic acid in a sample to form unsaturated disaccharide, wherein a double bond of the hyaluronic acid lyase has characteristic ultraviolet absorption at 232nm, and a light absorption value is in direct proportion to the concentration of the unsaturated disaccharide, and the content of hyaluronic acid in the detected sample can be obtained by detecting the light absorption value increment of reaction liquid before and after reaction.
Drawings
FIG. 1: an electrophoretogram of purified hyaluronan lyase Hya16A of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: cloning, expressing and obtaining hyaluronic acid lyase in escherichia coli
Escherichia coli carrying Hya16A gene of interest was subcultured in LB liquid medium (kanamycin resistance), cultured at 37 ℃ at 160r/min to an OD600 of about 0.4, and induced at 17 ℃ for 12 hours with IPTG added to a final concentration of 0.5 mmol/L. The cells were resuspended in 20mM/L PBS (pH 7.5) and sonicated to obtain Hya16A crude protein. The crude protein was purified by affinity chromatography using HisTrpTM HP column, and eluted with a gradient of 0-0.5mol/L imidazole in a mobile phase of 20mM PBS (pH 7.5) containing 0.3mol/L NaCl, and fractions showing UV absorption signals were collected. The collected fractions were desalted using a HiPrepTM 26/10 desaling column with water as the mobile phase to give purified Hya 16A. The SDS-PAGE analysis result is shown in FIG. 1, and the purified enzyme protein is a single band, which shows that the purity is good.
Example 2: the method of the invention is subjected to accuracy verification
The method and the carbazole method are utilized to determine the content of the hyaluronic acid in the sample:
(1) preparing a hyaluronic acid solution: weighing chemically pure hyaluronic acid, dissolving in 20mM phosphate buffer solution with pH8, and preparing hyaluronic acid standard solutions with concentrations of 0.05mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL respectively;
(2) and (3) carrying out enzymolysis reaction: 375. mu.L of the hyaluronic acid solutions prepared above and having different concentrations were reacted with 1U Hya16A (pH8 phosphate buffer to 375. mu.L) at 45 ℃ for 10 min. Standing in 100 deg.C metal bath for 5min after reaction to inactivate enzyme, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 232 nm; mixing the inactivated enzyme solution with the same concentration gradient with the hyaluronic acid solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations.
(3) Drawing a quantitative standard curve: taking hyaluronic acid standard solution as an abscissa, taking corresponding absorbance value increment as an ordinate, and obtaining a standard curve y of 0.9524x +0.0284 under specific reaction conditions through linear fitting, wherein R is2The value was 0.9993.
(4) And (3) sample determination: preparing a 0.1mg/mL sample solution, taking 375 mu L of the sample solution, repeating the operation in the step (2), substituting the increment of the light absorption value into the standard curve y of 0.9524x +0.0284, and calculating the concentration of hyaluronic acid in the reaction system to obtain the content of hyaluronic acid in the sample.
(5) And (3) determining the content of hyaluronic acid in the sample according to a carbazole method in the Roche notation QB/T4576-2013.
(6) Triplicate determinations were made for each method and the results are shown in the following table:
Figure BDA0003512697270000051
from the above results, it can be seen that the method of the present invention has better accuracy than the carbazole method.
Example 3: the method of the invention is subjected to precision measurement
(1) Preparing a hyaluronic acid solution: a standard solution of hyaluronic acid was prepared at a concentration of 100. mu.g/mL by weighing chemically pure hyaluronic acid in 20mM phosphate buffer, pH 8.
(2) And (3) sample determination: the procedure of (2) in example 2 was repeated by taking 375. mu.L of each standard solution, measuring 6 parallel samples, and calculating the concentration of hyaluronic acid in the reaction system by substituting the increase in absorbance into the obtained standard curve, and further calculating the standard deviation to examine the precision of the measurement method.
(3) The results are shown in the following table, and the average of 6 measurements was 100.2. mu.g/mL, and the relative standard deviation was 3.27%, from which it can be seen that the method of the present invention has good precision.
Figure BDA0003512697270000052
Example 4: quantitative specificity verification of the method of the invention
The method of the invention is used for respectively quantifying hyaluronic acid in three mixed solutions
(1) Preparing a hyaluronic acid solution: weighing chemically pure hyaluronic acid, dissolving in 20mM phosphate buffer solution with pH of 8, preparing hyaluronic acid standard solution with concentration of 0.1mg/mL, and adding chondroitin sulfate, dermatan sulfate and heparin solution respectively to obtain labeling mixed solution with corresponding mass concentration.
(2) And (3) respectively taking 375 mu L of the standard-added mixed solution, repeating the operation in the step (2) of the example 2, substituting the increment of the light absorption value into the obtained standard curve, calculating the concentration of the hyaluronic acid in the reaction system, and further calculating the content of the hyaluronic acid in the mixed solution.
The results of the three parallel measurements showed that the recovery rates of the mixed solutions containing chondroitin sulfate, dermatan sulfate and heparin were 100.42 + -4.81%, 99.94 + -2.68% and 101.31 + -1.94%, respectively. As can be seen from the above results, the method of the present invention is substantially unaffected by glycosaminoglycans other than hyaluronic acid, and has good quantitative specificity.
Example 5: the recovery rate of the added standard of the method of the invention is measured
(1) Preparing a hyaluronic acid solution: weighing chemically pure hyaluronic acid, dissolving in 20mM phosphate buffer solution with pH8, preparing hyaluronic acid standard solutions with concentrations of 0.04, 0.2 and 0.4mg/mL as sample solutions with low, medium and high quality concentrations, and adding 0.1mg/mL hyaluronic acid to prepare standard sample solutions with corresponding mass concentrations.
(2) And (3) measuring a labeled sample: and (3) respectively taking 375 mu L of standard sample solution, repeating the operation in the step (2) of the example 2, substituting the increment of the light absorption value into the obtained standard curve, calculating the concentration of hyaluronic acid in the standard sample solution, further calculating the standard recovery rate, and evaluating the reliability of the method by using the average value of the standard recovery rates at all mass concentrations.
(3) The recovery rates of the low, medium and high mass concentrations are 100.78 +/-3.24%, 102.05 +/-2.51% and 100.04 +/-2.35% respectively. From the above results, it can be seen that the method of the present invention has good reliability.
Example 6: the precision in the day and the time of day of the method of the invention is measured
(1) Preparing a hyaluronic acid solution: chemically pure hyaluronic acid was weighed and dissolved in 20mM phosphate buffer solution of pH8 to prepare standard solutions of hyaluronic acid having concentrations of 0.04, 0.2, 0.4mg/mL as sample solutions of low, medium, and high quality concentrations, respectively.
(2) And (3) sample determination: and (3) respectively taking 375 mu L of low, medium and high concentration sample solutions, repeating the operation in the step (2) of the example 2 at different times on the same day and the same time on different days, substituting the increment of the light absorption value into the obtained standard curve, calculating the concentration of the hyaluronic acid in each group, further calculating the coefficient of variation, and evaluating the repeatability of the method by using the average value of the coefficient of variation at each mass concentration.
(3) The assay was performed in triplicate and the results were as follows:
Figure BDA0003512697270000061
from the above results, it can be seen that the method of the present invention has good reproducibility.
Example 7: measuring hyaluronic acid content in oral liquid
(1) Chemically pure hyaluronic acid was weighed and dissolved in 20mM phosphate buffer solution of pH8 to prepare hyaluronic acid standard solutions having concentrations of 0.10mg/mL, 0.20mg/mL, 0.30mg/mL, 0.40mg/mL, and 0.50mg/mL, respectively.
(2) And (3) carrying out enzymolysis reaction: 375. mu.L of the hyaluronic acid solutions prepared above and having different concentrations were reacted with 1U Hya16A (pH8 phosphate buffer to 375. mu.L) at 45 ℃ for 10 min. Standing in 100 deg.C metal bath for 5min after reaction to inactivate enzyme, rapidly cooling to room temperature, centrifuging to obtain supernatant, and measuring light absorption value of the supernatant at 232 nm; mixing the inactivated enzyme solution with the same concentration gradient with the hyaluronic acid solution, repeating the operation, and measuring the light absorption value of the inactivated enzyme solution as a control so as to calculate the light absorption value increment corresponding to the solutions with different concentrations.
(3) Drawing a quantitative standard curve: taking hyaluronic acid standard solution as an abscissa, taking corresponding light absorption value increment as an ordinate, and obtaining a standard curve under specific reaction conditions by linear fitting, wherein y is 1.0271x +0.02, and R is2The value was 0.9993.
(4) And (3) sample determination: weighing a proper amount of oral liquid sample and dissolving the oral liquid sample in a buffer solution. The procedure of example 2 (2) was repeated by taking 375 μ L of the sample solution, and the increase in absorbance was substituted into 1.0271x +0.02 as the standard curve y, and the concentration of hyaluronic acid in the reaction system was calculated, and the content of hyaluronic acid in the oral liquid was converted to 0.194 mg/mL. And adding 0.1mg of hyaluronic acid into the sample to be detected to prepare a standard sample solution, repeating the operation in the step (2) in the example 2, and performing parallel measurement for three times to obtain a recovery rate of 98.23 +/-3.49%, which indicates that the result is reliable.
Finally, it should be noted that the above embodiments describe specific embodiments of the present invention, but do not limit the present invention; it will be understood by those skilled in the art that these are by way of example only and that the scope of the invention is defined by the appended claims. All changes, modifications and equivalents that come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Figure BDA0003512697270000081
Figure BDA0003512697270000091
Figure BDA0003512697270000101
Figure BDA0003512697270000111
Sequence listing
<110> China oceanic university
<120> hyaluronic acid lyase and application thereof
<130> China oceanic university
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ttcgtgaagg cgggcaacgg gcccaccacc ggcaacctga tctgcctgcg caacaacgcc 480
cgagacgact tcgtcgtcaa gggcagcggg cgggtcggta tcggcatggg cgtgggcggc 540
aacccctggt cgcagctcca tgtcgtgcag cagccgggca ccgactcggc gctgatggtc 600
gagggcacgg tgcgggtcgt cgacgtggcc tccgcgccca cgggcgtcga ctcgcgcggc 660
ggcggcgtgc tgtacgcgga gaacggtgcg ctgaagtggc gcggctccga caacacggtc 720
accaccatcg cccccgcctg a 741

Claims (9)

1. A hyaluronidase whose amino acid sequence is SEQ ID NO. 1.
2. The hyaluronan lyase of claim 1, wherein: the reaction temperature of the hyaluronic acid lyase is 30-50 ℃, and the optimal reaction temperature is 45 ℃; the reaction pH is 7.0-9.0, and the optimal reaction is achievedThe pH value is 8.0; the enzyme can be stored stably for at least 3 months at 4 ℃, the kinetic constant Km of the enzyme is 0.03mg/mL, and the Kcat is 13.41s-1Km/Kcat of 4.25. mu.M- 1s-1。
3. The nucleotide sequence of the gene for coding the hyaluronic acid lyase is SEQ ID NO.2 or all genes capable of being translated into SEQ ID NO. 1.
4. Use of the hyaluronan lyase of claim 1 for the rapid quantitative determination of hyaluronic acid.
5. A method for rapidly and quantitatively detecting hyaluronic acid by an enzyme method is characterized by comprising the following steps: specifically degrading hyaluronic acid into unsaturated disaccharide by using the hyaluronic acid lyase of claim 1; and (3) inactivating the enzyme, centrifuging, measuring the light absorption value of the supernatant at 232nm, and comparing with a standard curve to obtain the content of the hyaluronic acid.
6. The method for rapid quantitative determination of hyaluronic acid by enzymatic method according to claim 5, characterized in that it comprises the following steps:
(1) preparing a hyaluronic acid solution: weighing hyaluronic acid with chemical purity or purity above, and dissolving in buffer solution to prepare hyaluronic acid standard solution with concentration gradient;
(2) drawing a quantitative standard curve: mixing the hyaluronic acid solutions with different concentrations prepared in the step (1) with a proper amount of hyaluronic acid lyase liquid respectively for reaction; after the reaction, placing the mixture in a metal bath at 100 ℃ for 5-10min to inactivate the enzyme, quickly cooling the mixture to room temperature, centrifuging the mixture to take supernatant, and measuring the light absorption value of the supernatant; mixing the hyaluronic acid solution with the same concentration gradient and the inactivated enzyme solution, repeating the reaction, measuring the light absorption value of the hyaluronic acid solution as a control, and calculating the light absorption value increment corresponding to the hyaluronic acid solutions with different concentration gradients; taking the concentration of the hyaluronic acid standard solution as a horizontal coordinate, taking the light absorption value increment of hyaluronic acid with each concentration as a vertical coordinate, and obtaining a standard curve under a specific reaction condition through linear fitting;
(3) and (3) sample determination: adding a certain amount of hyaluronic acid lyase into the sample solution to repeat the reaction in the step (2); and substituting the light absorption value increment into a standard curve under the conditions of corresponding enzyme adding amount, reaction time, reaction temperature, reaction pH and the like, and calculating to obtain the concentration of the hyaluronic acid in the reaction system so as to obtain the content of the hyaluronic acid in the sample.
7. The method for rapid quantitative detection of hyaluronic acid by enzymatic method according to claim 6, wherein: the pH value of the buffer solution in the step (1) is 7.0-9.0.
8. The method for rapid quantitative detection of hyaluronic acid by enzymatic method according to claim 6, wherein: in the step (2), the addition amount of the enzyme is 1-1000U, the reaction time is 5-40min, and the reaction temperature is 20-50 ℃.
9. The method for rapid quantitative detection of hyaluronic acid by enzymatic method according to claim 6, wherein: the wavelength of the light absorption value detected in the step (2) is in the range of 230-240 nm.
CN202210156112.0A 2022-02-21 2022-02-21 Hyaluronic acid lyase and application thereof Pending CN114457061A (en)

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