Method for preparing L-ascorbic acid

Abstract

The invention relates to a method for preparing L-ascorbic acid, and belongs to the field of ascorbic acid production. According to the method disclosed by the invention, 2-keto-L-gulonic acid and / or a metal salt thereof are / is taken as a substrate, under an acidic condition, 2-keto-L-gulonic acid and / or a derivative thereof are / is converted into L-ascorbic acid through a one-step enzyme catalysis reaction of an enzyme catalyst, lactonization ring formation is carried out without a large amount of acid, alkali and organic solvent methanol, and the yield is high. The substrate conversion rate is high, the process is simple and green, the later product separation and purification steps can be simplified, and the production cost is reduced.

Description

Technical Field The present invention belongs to the field of ascorbic acid production and relates to a method for preparing L-ascorbic acid, and in particular to a method for converting 2-keto-L-gulonic acid and / or its derivatives into L-ascorbic acid and / or its derivatives by in vitro enzyme catalysis. Background Art Vitamin C (Vitamin C, referred to as Vc), also known as L-ascorbic acid (L-ascorbic acid) is a water-soluble vitamin that maintains the normal physiological functions of the body and is widely found in plants and animals. The U.S. Food and Drug Administration (FDA) allows L-ascorbic acid to be used as a nutritional supplement and chemical preservative, and lists it in the FDA's list of substances generally regarded as safe. Because it contains reducing protons and has antioxidant properties, L-ascorbic acid can be used as a flavoring antioxidant in soft drinks, in smoked or pickled meat or meat products, in flour to improve baking quality, in beer as a stabilizer, in fats or oils as an antioxidant, and added to many foods to provide rich ascorbic acid. It can also be used in detergents, hair care products, plastic products, photography and water treatment. Therefore, L-ascorbic acid is widely used in the fields of health care, food, cosmetics and feed. At present, the main production methods of L-ascorbic acid in industry are the Reitman method (chemical method) and the two-step fermentation method, among which the two-step fermentation method is the method commonly used by L-ascorbic acid production enterprises in my country. The two-step fermentation method includes three steps: fermentation, extraction, and esterification conversion: first, D-sorbitol is converted into L-sorbose by Gluconobacter oxyans; then, a mixed bacterial system composed of Ketogulonigenium vulgare (commonly known as small bacteria) and Bacillus megaterium (commonly known as large bacteria) converts L-sorbose into 2-keto-L-gulonic acid (2-keto-L-Gulonic acid, 2-KLG), and finally, the 2-keto-L-gulonic acid obtained after extraction is converted into L-ascorbic acid by chemical method. The esterification process of L-ascorbic acid generally includes the following steps: an esterification step, in which 2-keto-L-gulonic acid is esterified in methanol using concentrated sulfuric acid to generate 2-keto-L-gulonic acid methyl ester; a conversion step, in which NaHCO 3 2-keto-L-gulonic acid methyl ester is converted into L-ascorbic acid sodium salt; in the acidification reaction step, H 2 SO 4 , acidified into L-ascorbic acid. In this process, a large amount of acid, alkali and organic solvent methanol are required. The treatment of acid, alkali and organic solvent increases the difficulty and cost of subsequent separation and purification of the product. As the market demand for L-ascorbic acid continues to expand and market competition becomes increasingly fierce, it is crucial to explore new methods for producing L-ascorbic acid. Therefore, it is urgent to develop a green, low-cost method for preparing L-ascorbic acid. Summary of the invention Problem that the invention aims to solve Currently, the preparation process of L-ascorbic acid is complicated and requires a large amount of acid, alkali and organic solvent, which is not conducive to the separation of subsequent products and is also not conducive to the sustainable development of the environment. Solutions for solving problems

[0001] . A method for preparing L-ascorbic acid, wherein 2-keto-L-gulonic acid and / or its metal salt is used as a substrate and reacted with an enzyme catalyst under acidic conditions to produce L-ascorbic acid; Optionally, the acidic condition is pH ≤ 6.0.

[0002] . The method according to [1], wherein the enzyme catalyst comprises one or more hydrolases with an EC number of EC 3; Optionally, the hydrolase comprises a carboxylate hydrolase with an EC number of EC 3.1.1. Preferably, the carboxylate hydrolase is a lactonase that can hydrolyze lactone. [3]. The method according to [2], wherein the lactonase is selected from at least one of L-arabinonolactonase with an EC number of EC 3.1.1.15, gluconolactonase with an EC number of EC 3.1.1.17, 1,4-lactonase with an EC number of EC 3.1.1.25, D-arabinonolactonase with an EC number of EC 3.1.1.30, 6-phosphogluconolactonase with an EC number of EC 3.1.1.31 and xylon-1,4-lactonase with an EC number of EC 3.1.1.68. [4] The method according to [2] or [3], wherein the lactonase is derived from Tannerella forsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp., Aspergillus niger, Acetobacter senegalensis, Acidovoraxsoli, Luteitalea pratensis, Gluconobacter oxydans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp. [5] The method according to any one of [2] to [4], wherein the lactonase is selected from the following: 1 )~(a 3 ) any of: (a 1 ) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 2 ) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 3 ) is encoded by (a 1 ) or (a 2 ) is a polypeptide encoded by a polynucleotide of the polypeptide shown in FIG. [6] The method according to any one of [1] to [5], wherein L-ascorbic acid is produced under acidic conditions, wherein the acidic conditions are pH ≤ 5.5, preferably pH 2.0 to 5.5. [7]. The method according to any one of [1] to [6], wherein the 2-keto-L-gulonic acid metal salt is a soluble salt of 2-keto-L-gulonic acid; preferably, the soluble salt is selected from potassium salt, sodium salt or calcium salt. [8] The method according to any one of [1] to [7], wherein the 2-keto-L-gulonic acid and / or its metal salt comprises 2-keto-L-gulonic acid, 2-keto-L-gulonic acid hydrate, 2-keto-L-gulonic acid sodium, 2-keto-L-gulonic acid potassium and / or 2-keto-L-gulonic acid calcium. [9] The method according to any one of [1] to [8], wherein the enzyme catalyst is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cells; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cells are in free or immobilized form.

[10] A method according to any one of [1] to [9], wherein: In the step of synthesizing L-ascorbic acid, the reaction system contains calcium ions and reacts under anaerobic conditions at 0 to 100° C.; Optionally, the calcium ions are provided in the form of nitrate, sulfate or chloride.

[11] . At least one of the following uses of lactonase: (A) Use in the preparation of L-ascorbic acid; (B) Use as a catalyst for preparing L-ascorbic acid; Alternatively, the lactonase is derived from Tannerella forsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp., Aspergillus niger, Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis, Gluconobacter oxydans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp; Preferably, the lactonase is selected from the following: 1 )~(a 3 ) any of: (a 1 ) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 2) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 3 ) is encoded by (a 1 ) or (a 2 ) is a polypeptide encoded by a polynucleotide of the polypeptide shown in FIG.

[12] A catalyst for preparing L-ascorbic acid, comprising a lactonase derived from Tannerella forsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp., Aspergillus niger, Acetobactersenegalensis, Acidovorax soli, Luteitalea pratensis, Gluconobacter oxydans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp.; Optionally, the lactonase is selected from the following: 1 )~(a 3 ) any of: (a 1 ) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 2 ) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 3 ) is encoded by (a 1 ) or (a 2 ) is a polypeptide encoded by a polynucleotide of the polypeptide shown in FIG. Effects of the Invention 1. The present invention converts 2-keto-L-gulonic acid and / or its metal salt into L-ascorbic acid through an enzyme-catalyzed one-step reaction, without the need for a large amount of acid, alkali and organic solvent methanol to achieve lactonization and cyclization, and the process is simple and green. 2. The preparation of L-ascorbic acid in the present disclosure is carried out under acidic conditions. After the reaction is completed, the enzyme catalyst is removed and the product can be concentrated and crystallized without going through an acidification reaction step, which simplifies the separation and purification of subsequent products and reduces production costs. 3. The preparation method of L-ascorbic acid provided by the present disclosure has a high substrate conversion rate, mild reaction conditions, improved production efficiency, and further reduced production costs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A one-step conversion pathway for 2-keto-L-gulonic acid to L-ascorbic acid is shown. Figure 2 The HPLC detection chart of the one-step conversion of 2-keto-L-gulonic acid into L-ascorbic acid catalyzed by lactonase is shown. DETAILED DESCRIPTION Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below. The word "exemplary" used herein means "used as an example, embodiment or illustrative". Any embodiment described herein as "exemplary" is not necessarily to be interpreted as being superior or better than other embodiments. In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present disclosure. Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in this disclosure should be understood to include the inevitable systematic errors in industrial production. In this specification, the word "may" includes both performing a certain process and not performing a certain process. In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner. Although the disclosure supports the definition of the term "or" as only alternatives and "and / or", the term "or" in the claims means "and / or" unless it is explicitly stated that it is only alternatives or the alternatives are mutually exclusive. In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options. In order to enable those skilled in the art to understand the characteristics and effects of the present disclosure, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the common meanings understood by those skilled in the art for the present disclosure. In the event of a conflict, the definitions in this specification shall prevail. The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present disclosure in any way, i.e., the present disclosure can be implemented without being limited by any specific theory or mechanism. In the present disclosure, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution. In the present disclosure, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution. The use of "one", "an", "a" or similar expressions to describe the components and technical features of the present disclosure is merely for the convenience of expression and to provide a general meaning to the scope of the present disclosure. Therefore, such description should be understood to include one or at least one, and the singular also includes the plural, unless it is obvious that it refers to another meaning. The "plurality" and "multiple" mentioned in this disclosure refer to two (two kinds) or more than two (two kinds). "And / or" describes the association relationship of the associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B. In this document, "or its combination" means "or any combination thereof", and "any one", "any one" means "any one", "any one" or "any one". The terms "preferably" and "preferred" as used herein do not limit the scope of the disclosure claimed for protection, or imply that certain features are critical, necessary, or even important to the structure or function of the disclosure claimed for protection. Instead, these terms are merely intended to emphasize alternative or additional features that can or cannot be used in a particular embodiment of the disclosure. In the present disclosure, unless otherwise specified, the term "comprising" as used herein may be open-ended or closed-ended. For example, the term "comprising" may mean that other components not listed may also be included, or may only include the listed components. As used herein, the term "derivative" refers to a compound having a structure derived from a parent compound, whose structure is sufficiently similar to those disclosed herein, and based on the similarity, a person skilled in the art would expect to have the same or similar activity and utility as the claimed compound. Exemplary derivatives include salts, hydrates, etc. of the parent compound. As used herein, the term "suitable reaction conditions" refers to those conditions under which an enzyme catalyst catalyzes the lactonization of 2-keto-L-gulonic acid to produce L-ascorbic acid, such as the range of enzyme catalyst addition, substrate addition, temperature, pH, buffer, metal ions, etc. Some exemplary "suitable reaction conditions" are provided herein. As used herein, the term "dosage", such as in "enzyme dosing" or "substrate dosing", refers to the concentration or amount of a component in the reaction mixture at the start of the reaction. As used herein, the terms "polypeptide", "enzyme", "polypeptide or enzyme" or "polypeptide / enzyme" have the same meaning and are interchangeable in the present disclosure. The aforementioned terms refer to a polymer composed of a plurality of amino acids through peptide bonds, which may or may not contain modifications such as phosphate groups and formyl groups. As used herein, "EC number" or "EC number" is a classification system for enzymes created by the Enzyme Commission, based on the chemical reaction catalyzed by each enzyme. This classification system also gives a suggested name to each enzyme, so it is also called the Enzyme Commission Nomenclature. As used herein, "hydrolase" is an enzyme classified into enzyme class EC 3 according to the Enzyme Commission Nomenclature. Further, hydrolases include enzymes classified into enzyme class EC 3.1.1.x, in which the name "EC 3.1.1.x" represents any number after "EC 3.1.1.", which represents an enzyme of the carboxylic acid ester hydrolase class, and in particular, refers to a lactonase having a lactone hydrolyzing function in the carboxylic acid ester hydrolase class. Lactonase catalyzes a reversible reaction, specifically hydrolyzing a lactone (a cyclic ester formed by an intramolecular reaction between a carboxyl group and a hydroxyl group) and reforming the lactone. As used herein, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is performed when the two sequences are aligned to obtain the maximum percent identity. As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. As used herein, the term "expression vector" refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. <Method for producing L-ascorbic acid> The method for preparing L-ascorbic acid provided by the present disclosure is to use 2-keto-L-gulonic acid and / or its metal salt as substrate, and react it with an enzyme catalyst under acidic conditions to generate L-ascorbic acid. Furthermore, the reaction system also contains metal ions, preferably calcium ions, which are provided in the form of soluble salts such as nitrates, sulfates, chlorides, etc. In some specific embodiments, the method for preparing L-ascorbic acid is carried out under anaerobic conditions, and the reaction time is not less than 20 hours, preferably, the reaction time is not less than 24 hours, 30 hours, 40 hours, 50 hours, 60 hours or 70 hours. In some specific embodiments, the method for preparing L-ascorbic acid is carried out at a temperature of 0 to 100°C, and the temperature depends on factors such as the nature and amount of the enzyme catalyst and the amount of the substrate 2-keto-L-gulonic acid and / or its metal salt. In some preferred embodiments of the present disclosure, the method for preparing L-ascorbic acid is carried out at a temperature of 25 to 80°C. In some more preferred embodiments of the present disclosure, the method for preparing L-ascorbic acid is carried out at a temperature of 37 to 60°C. (Enzyme Catalyst) In the present disclosure, the enzyme catalyst has the function of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step, and has the ability to produce L-ascorbic acid without other auxiliary factors. All proteins of organisms such as microorganisms, plants, and animals that have the function of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step are also included. That is, as long as the newly discovered enzyme has the function of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step, it is included. In some optional embodiments, the enzyme catalyst that can be used in the present disclosure is a wild-type enzyme or an enzyme that has undergone certain genetic modifications, and the genetic modifications improve some properties of the enzyme catalyst, such as activity, such as acid resistance, such as thermal stability, etc. Enzymes with desired properties derived from wild-type enzymes by genetic engineering techniques are called "mutants", "mutant derivatives" or "mutants from the corresponding wild-type enzymes". In the present disclosure, the enzyme catalysts used to implement the present disclosure include these "mutants" with improved properties. According to some embodiments of the present disclosure, the enzyme catalyst includes one or more hydrolases with an EC number of EC 3; optionally, the hydrolase includes a carboxylate hydrolase with an EC number of EC 3.1.1, preferably a lactonase that can hydrolyze lactone; optionally, the lactonase includes L-arabinon lactonase (EC 3.1.1.15), gluconolactonase (EC3.1.1.17), 1,4-lactonase (EC 3.1.1.25), D-arabinon lactonase (EC 3.1.1.30), 6-phosphogluconolactonase (EC 3.1.1.31), xylon-1,4-lactonase (EC 3.1.1.68) or other enzymes that are not labeled with enzyme numbers but have lactonase activity. The disclosed method can also be realized by using the functional derivatives of the lytic enzyme. These functional derivatives are different from the lytic enzyme itself due to adding, inserting, deleting and / or replacing one or more amino acid residues in the normal sequence of the lytic enzyme (unmodified), but these derivatives still have hydrolase activity with experimental determinations well known in the art. These functional derivatives can be prepared with peptide chemical synthesis methods well known in the art or chemical modification methods of proteins, or with recombinant methods well known in the art according to DNA sequences disclosed herein. Amino acid replacement can refer to the amino acid at a certain position of the amino acid sequence being replaced by other amino acids, as long as the sequence after the change retains the activity of the original amino acid sequence in whole or in part. Amino acid replacement can be a conservative amino acid replacement, which refers to that compared with the original amino acid sequence, several amino acids are replaced by similar or close amino acids in nature to form peptides (conservative variant peptides). For example, these conservative variant peptides can be generated according to the following amino acid substitutions: substitution of Ala by Val, Leu or Ile, substitution of Arg by Lys, Gln, Asn or His, substitution of Asn by Gln, His, Lys or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln or Arg, substitution of Met, Ala, Val, Phe or norleucine by Ser or Ala, substitution of Cys by Asn or Glu, substitution of Gln ... Asn, Lys, Gln or Arg, substitution of Met, Ala, Val, Phe or norleucine by Ser or Ala, substitution of Cys by Asn or Glu, substitution of Met, Ala, Val, Phe or norleucine by Ser or Ala. The amino acid substitutions may also be non-conservative amino acid substitutions. In some embodiments of the present disclosure, the enzyme catalyst may be selected from any one or more of all enzymes belonging to the "EC3 (hydrolase)" group derived from acidophilic bacteria. The acidophilic bacteria include, but are not limited to: Acidimicrobium, Acidothermus, Alicyclobacillus, Acidiphilium, Acidithiobacillus, Acidomonas, Acidocella, Acetobacter, Gluconobacter, Leptospirillum, Sulfurococcus, Sulfobacillus, Thiobacillus. Exemplarily, the lactonase comprises an L-arabinonolactonase (EC 3.1.1.15) from Tannerella forsythia; a gluconolactonase (EC 3.1.1.17) from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp.; a 1,4-lactonase (EC 3.1.1.25) from Aspergillus niger; a 6-phosphogluconolactonase (EC 3.1.1.31) from Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis, Gluconobacter oxydans; a xylon-1,4-lactonase (EC 3.1.1.68) from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans, Thiobacillus sp., etc. lactonase. Exemplarily, the L-arabinonolactonase comprises a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues compared to the L-arabinonolactonase of the sequence shown in SEQ ID NO: 1. The judgment / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides). Exemplarily, the gluconolactonase comprises a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues compared to the gluconolactonase of the sequence shown in any one of SEQ ID NOs: 3 to 6. The determination / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or both of the compared biopolymers (i.e., nucleic acids or polypeptides). Exemplarily, the 1,4-lactonase comprises a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues compared to the 1,4-lactonase of the sequence shown in SEQ ID NO:7. The determination / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides). Exemplarily, the 6-phosphogluconolactonase comprises a "sequence identity" or "percentage identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues compared to the 6-phosphogluconolactonase of the sequence shown in any one of SEQ ID NOs: 8 to 11. The judgment / calculation of "sequence identity" or "percentage identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequence is substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides). Exemplarily, the lactonase comprises a "sequence identity" or "percent identity" of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid residues compared to the lactonase of the sequence shown in any one of SEQ ID NOs: 12 to 14. The determination / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides). [Enzyme form] According to some embodiments of the present disclosure, the enzyme catalyst can be used directly in the form of pure enzyme (i.e., free enzyme), or the pure enzyme can be immobilized (i.e., immobilized enzyme) to maintain stability and recyclability. It is usually best to use immobilized enzymes. Immobilized enzymes are generally more stable and durable. Immobilized enzymes are also easier to recover and use in multiple catalytic cycles, thereby reducing the cost of the production process. In the art, many methods for enzyme immobilization are known. Enzymes can also be cross-linked to form cross-linked enzyme aggregates (CLEA), which are generally more stable and easier to recover and reuse. In other embodiments of the present disclosure, the whole cell microorganism or the culture of the whole cell microorganism (i.e., fermentation product) containing the enzyme catalyst is used directly, or the whole cell is broken (i.e., cell lysate is obtained) and then used, or the whole cell is immobilized (i.e., immobilized cell) to maintain stability and recyclable reuse. In some specific embodiments of the present disclosure, in order to obtain a fast reaction rate, the whole cell can be permeable treated, illustratively, using heat treatment to promote cell membrane permeability. (2-Keto-L-gulonic acid and / or its metal salt) According to some embodiments of the present disclosure, the 2-keto-L-gulonic acid metal salt is a soluble salt of 2-keto-L-gulonic acid, and the soluble salt is selected from a potassium salt, a sodium salt or a calcium salt. In some specific embodiments, the 2-keto-L-gulonic acid and / or its metal salt comprises 2-keto-L-gulonic acid, 2-keto-L-gulonic acid hydrate, 2-keto-L-gulonic acid sodium, 2-keto-L-gulonic acid potassium and / or 2-keto-L-gulonic acid calcium. In some alternative embodiments, the 2-keto-L-gulonic acid and / or its metal salt is commercially available, or can be prepared from commercially available compounds according to techniques described in the literature or known to those skilled in the art. (Acidic conditions) In the present disclosure, the catalytic reaction is carried out under acidic conditions, which refers to conditions of pH ≤ 6.0, conditions of pH ≤ 5.5, conditions of pH ≤ 5.0, conditions of pH ≤ 4.5, conditions of pH ≤ 4.0 or conditions of pH ≤ 3.5. In some optional embodiments of the present disclosure, the acidic conditions are pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH 4.3, pH 4.2, pH 4.1, pH 4.0, pH 3.9, pH 3.8, pH 3.7, pH 3.6, pH 3.5, pH 3.4, pH 3.3, pH 3.2, pH 3.1, pH 3.0, pH 2.9, pH 2.8, pH 2.7, pH 2.6, pH 2.5, pH 2.4, pH 2.3, pH 2.2, pH 2.1 or pH 2.0 (or any value between any adjacent values). In some preferred embodiments of the present disclosure, the acidic condition is pH 1.0-6.0, preferably pH 2.0-5.5, more preferably pH 3.0-5.0, and further preferably pH 3.5-5.0. (Buffer) According to some embodiments of the present disclosure, the reaction system further contains a buffer to assist the catalytic reaction. The buffer and the buffer concentration are determined according to the pH of the reaction, the nature and amount of the enzyme catalyst, and the amount of the substrate 2-keto-L-gulonic acid and / or its metal salt. In some embodiments of the present disclosure, any buffer suitable for the aforementioned pH value or pH range, such as acetate buffer, citrate buffer, can be used in the method for preparing L-ascorbic acid. In some embodiments of the present disclosure, the buffer concentration can be determined according to the substrate concentration used, and is preferably in the range of 1 mM to 200 mM, such as 50 mM to 200 mM, 100 mM to 200 mM. <Uses of lactonase> According to some embodiments of the present disclosure, the present disclosure provides at least one of the following uses of a lactonase with an EC number of EC 3.1.1.x, in particular EC 3.1.1.15, EC 3.1.1.17, EC 3.1.1.25, EC 3.1.1.30, EC 3.1.1.31 and EC 3.1.1.68: (A) Use in the preparation of L-ascorbic acid; (B) Use as a catalyst for preparing L-ascorbic acid. In some optional embodiments, the lactonase is L-arabinonolactonase (EC 3.1.1.15) from Tannerella forsythia; gluconolactonase (EC 3.1.1.17) from Acetobacter pasteurianus, Acidobacteriumcapsulatum, Bryobacteraceae bacterium, Leptospirillum sp.; 1,4-lactonase (EC 3.1.1.25) from Aspergillus niger; 6-phosphogluconolactonase (EC 3.1.1.31) from Acetobactersenegalensis, Acidovorax soli, Luteitalea pratensis, Gluconobacter oxydans; xylon-1,4-lactonase (EC 3.1.1.68) from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans, Thiobacillus sp. and other lactonase. In some preferred embodiments, the lactonase is selected from the following: 1 )~(a 3 ) any of: (a 1 ) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 2 ) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 3 ) is encoded by (a 1 ) or (a 2 ) is a polypeptide encoded by a polynucleotide of the polypeptide shown in FIG. <Catalyst for producing L-ascorbic acid> According to some embodiments of the present disclosure, the present disclosure provides a catalyst for preparing L-ascorbic acid, wherein the catalyst comprises a lactonase with an EC number of EC 3.1.1.x, and further, the lactonase is an L-arabinonolactonase (EC 3.1.1.15) derived from Tannerella forsythia; a gluconolactonase (EC 3.1.1.17) derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp.; a 1,4-lactonase (EC 3.1.1.25) derived from Aspergillus niger; a 6-phosphogluconolactonase (EC 3.1.1.31) derived from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis, Gluconobacter oxydans; a xylon-1,4-lactonase (EC 3.1.1.26); 3.1.1.68), or lactonase from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans, Thiobacillus sp. In some optional embodiments, the lactonase is selected from the following (a 1 )~(a 3 ) any of: (a 1 ) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 2 ) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a 3 ) is encoded by (a 1 ) or (a 2 ) is a polypeptide encoded by a polynucleotide of the polypeptide shown in FIG. Example Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art. The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Materials and Methods Drugs and materials Unless otherwise noted, all drugs were of analytical grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Sinopharm (Shanghai, China). E. coli TOP10 and DH5α (Thermo Fisher Scientific, Waltham, MA, USA) were used for DNA manipulation and plasmid amplification. E. coli BL21 (DE3) (Invitrogen Life Sciences, Carlsbad, CA, USA) was used for recombinant protein expression. Preparation and purification of plasmids and recombinant proteins Those skilled in the art can prepare recombinant engineered bacteria using technical means known in the art according to their actual needs, and can refer to "Molecular Cloning Laboratory Guide (3rd Edition)" (Science Press). Those skilled in the art can culture engineered microbial cells to produce the enzyme. Guidelines and procedures for the production of recombinant enzymes by microbial cells can be found in the following publications, such as "Handbook of Fermentation and Biochemical Engineering: Principles, Process Design and Instruments" (2nd Edition, Henry C. Vogel and Celeste L. Todaro, Noyes Publications 1997) and "Principles of Fermentation Technology" (2nd Edition, PF Stanbury et.al., Butterworth Heineman, 2003). The vectors, plasmids, and hosts used in the experiment are all conventional vectors, plasmids, and host series used for expression of bacteria (such as Escherichia coli), such as PET series vectors and plasmids, and BL21 series host bacteria; the culture medium used is a culture medium for conventional bacteria (such as Escherichia coli) engineering bacteria, such as LB culture medium; the culture method used is a conventional bacteria (such as Escherichia coli) engineering bacteria culture method. In the technical solution disclosed in the present invention, the meanings represented by the numbers in the amino acid sequence table of the specification are as follows: SEQ ID NO: 1 shows the amino acid sequence of L-arabinonolactonase from Tannerella forsythia (Uniprot ID: A0A1D3UGI3); SEQ ID NO: 2 shows the amino acid sequence of gluconolactonase from Zymomonas mobilis (Uniprot ID: Q01578); SEQ ID NO: 3 shows the amino acid sequence of gluconolactonase from Acetobacter pasteurianus (UniprotID: S6CYH2); SEQ ID NO: 4 shows the amino acid sequence of gluconolactonase from Acidobacterium capsulatum (Uniprot ID: C1F8R6); SEQ ID NO: 5 shows the amino acid sequence of gluconolactonase from Bryobacteraceae bacterium (Uniprot ID: A0A949VV58); SEQ ID NO: 6 shows the amino acid sequence of gluconolactonase from Leptospirillum sp. (Uniprot ID: B6AQ12); SEQ ID NO: 7 shows the amino acid sequence of 1,4-lactonase from Aspergillus niger (Uniprot ID: A2R8C2); SEQ ID NO: 8 shows the amino acid sequence of 6-phosphogluconolactonase from Acetobacter senegalensis (Uniprot ID: A0A0U5F503); SEQ ID NO: 9 shows the amino acid sequence of 6-phosphogluconolactonase from Acidovorax soli (UniprotID: A0A7X0PKY7); SEQ ID NO: 10 shows the amino acid sequence of 6-phosphogluconolactonase from Luteitalea pratensis (Uniprot ID: A0A143PK71); SEQ ID NO: 11 shows the amino acid sequence of 6-phosphogluconolactonase from Gluconobacter oxydans (Uniprot ID: A0A067Z318); SEQ ID NO: 12 shows the amino acid sequence of a lactonase from Sulfobacillus thermosulfidooxidans (Uniprot ID: A0A1W1WCN5); SEQ ID NO: 13 shows the amino acid sequence of lactonase from Leptospirillum ferrooxidans (UniprotID: 10INF7); SEQ ID NO: 14 shows the amino acid sequence of lactonase from Thiobacillus sp. (Uniprot ID: A0A9D5J2S7). Exemplarily, the preparation method of the pET plasmid carrying the corresponding protein encoding gene is as follows: The corresponding coding genes of the enzymes from different sources in Table 1 were amplified from their corresponding genomes or obtained by gene synthesis. The obtained coding gene sequence was inserted into the pET28a plasmid by a simple cloning technique based on extended overlap extension PCR (POE-PCR) (You, C., X.-Z. Zhang and Y.-HP Zhang (2012). "Simple Cloning: direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis." Appl. Environ. Microbiol. 78: 1593-1595.), and a plasmid containing a nucleotide sequence encoding the corresponding target protein was constructed. Table 1 The plasmid containing the nucleotide sequence encoding the corresponding target protein was transformed into E. coli BL21 (DE3) competent cells, and the single colony obtained by overnight culture was added to a 5mL LB test tube containing the corresponding resistance, and cultured overnight at 37℃ constant temperature shaker 200rpm to obtain a culture solution. The culture solution was inoculated into a triangular flask containing the corresponding resistance and 200mL LB medium at a 1% (v / v) inoculation volume, and expanded culture was carried out at 37℃ constant temperature shaker 200rpm. When OD 600 When the concentration reaches 0.8-1.0, add the inducer IPTG to a final concentration of 0.1mM, and culture at 200rpm in a 16°C constant temperature shaker for 16-18h to express the target protein and obtain a cell culture fluid. The cell culture fluid is collected by centrifugation at 4°C, and the collected cells are washed once with a 0.9% (w / v) sodium chloride solution. The cell pellet is resuspended in a 20mM HEPES (pH 7.0) buffer solution and lysed by ultrasound. After centrifugation, the supernatant is collected for protein purification. The target protein is purified by Ni-NTA affinity chromatography. Enzyme activity assay used Principle of determination: In the presence of hydrochloric acid, silicate and molybdate react to generate silicomolybdate yellow, and L-ascorbic acid reduces silicomolybdate yellow to generate silicomolybdate blue. The concentration of L-ascorbic acid and the absorbance of silicomolybdate blue obey Beer's law within a certain range. By measuring the absorbance of silicomolybdate blue, the amount of L-ascorbic acid generated in the reaction system can be determined. Reaction system: In a 1 mL reaction system, 200 mM acetate buffer (pH 5.0), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 0.1-1g / L enzyme catalyst. Anaerobic reaction was carried out at 37°C for 16 hours, 120μL of the sample was taken, 10μL of 4M HCl was added to terminate the reaction, the sample was allowed to stand for 10 minutes, centrifuged at 12000rpm for 10 minutes, the supernatant was taken, and the amount of L-ascorbic acid generated in the supernatant was determined. Definition of enzyme activity: The amount of enzyme required to generate 1 μM L-ascorbic acid within 1 minute is one unit of enzyme activity. Determination system: In a 1mL reaction system, add 750μL of water, 90μL of 28.25g / L ammonium molybdate solution, 35μL of 4.293g / L sodium silicate solution, and 5μL of 4M hydrochloric acid solution in sequence, mix well, place in a 30℃ water bath to react for 20min, then add 120μL of the treated sample, place in a 30℃ water bath to react for 30min, and measure the absorbance at a wavelength of 735nm. Calculate the amount of L-ascorbic acid generated according to the standard curve. The experimental results are shown in Table 1. The experimental results show that the enzyme catalysts in Table 1 are all active in converting 2-keto-L-gulonic acid into L-ascorbic acid. Example 1 In a 1 mL reaction system, 200 mM acetate buffer (pH 5.0), 200 mM sodium 2-keto-L-gulonic acid, 1 mM CaCl 2 and 0.14 g / L ZmGL. After anaerobic reaction at 37°C for 40 hours, 120 μL of sample was taken, 10 μL of 4M HCl was added to terminate the reaction, and the mixture was allowed to stand for 10 minutes. The mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken. The amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as follows: the chromatographic column is ZORBAX SB-C18; the mobile phase is methanol / phosphate buffer (v:v, 2:98), in which the phosphate buffer is 10 mM potassium dihydrogen phosphate (pH 2.5); the flow rate is 0.6 mL / min; the column temperature is 30°C; the detector is an ultraviolet detector (detection wavelength is 254 nm); and the injection volume is 10 μL. The HPLC detection results are as follows: Figure 2 The HPLC test results showed that the amount of L-ascorbic acid produced was 5.1 mM. Example 2 In a 1 mL reaction system, 200 mM acetate buffer (pH 4.0), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 2g / L APGL. After anaerobic reaction at 30°C for 60h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 20.1mM. Example 3 In a 1 mL reaction system, 500 mM 2-keto-L-gulonic acid hydrate, 2 mM CaCl 2 and 5g / LAcGL, and the pH was adjusted to 3.5. After anaerobic reaction at 37°C for 72h, 120μL of the sample was taken, 10μL of 4M HCl was added, and the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 50.2mM. Example 4 In a 1 mL reaction system, 100 mM acetate buffer (pH 5.0), 100 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 0.2 g / L BbGL. After anaerobic reaction at 37°C for 60 h, 120 μL of the sample was taken, 10 μL of 4M HCl was added, the mixture was allowed to stand for 10 min, centrifuged at 12000 rpm for 10 min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 4.2 mM. Example 5 In a 1 mL reaction system, 500 mM 2-keto-L-gulonic acid hydrate, 5 mM CaCl 2 and 6g / LLsGL, and the pH was adjusted to 4.0. After anaerobic reaction at 45°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, and the mixture was allowed to stand for 10min. The mixture was centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 55.7mM. Example 6 In a 1 mL reaction system, 200 mM acetate buffer (pH 5.0), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 0.5 g / L TfAL. After anaerobic reaction at 30°C for 24 hours, 120 μL of the sample was taken, 10 μL of 4M HCl was added, the mixture was allowed to stand for 10 minutes, and the mixture was centrifuged at 12000 rpm for 10 minutes. The supernatant was taken and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 6.9 mM. Example 7 In a 1 mL reaction system, 200 mM acetate buffer (pH 5.0), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 1g / L AnL. After anaerobic reaction at 37°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 16.4mM. Example 8 In a 1 mL reaction system, 200 mM acetate buffer (pH 3.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 2g / L Abs6PGL. After anaerobic reaction at 37°C for 60h, 120μL of sample was taken, 10μL of 4M HCl was added, and the mixture was allowed to stand for 10min. The mixture was centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 18.8mM. Example 9 In a 1 mL reaction system, 200 mM acetate buffer (pH 3.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 2g / L As6PGL. After anaerobic reaction at 30°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 15.6mM. Example 10 In a 1 mL reaction system, 200 mM acetate buffer (pH 4.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 2g / L Lp6PGL. After anaerobic reaction at 50°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 19.7mM. Embodiment 11 In a 1 mL reaction system, 200 mM acetate buffer (pH 3.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2and 1g / L Go6PGL. After anaerobic reaction at 40°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 16.5mM. Example 12 In a 1 mL reaction system, 200 mM acetate buffer (pH 4.0), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 1g / L StLac. After anaerobic reaction at 60°C for 60h, 120μL of sample was taken, 10μL of 4M HCl was added, and the mixture was allowed to stand for 10min. The mixture was centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 23.2mM. Embodiment 13 In a 1 mL reaction system, 200 mM acetate buffer (pH 3.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 2g / L LfLac. After anaerobic reaction at 50°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 20.5mM. Embodiment 14 In a 1 mL reaction system, 200 mM acetate buffer (pH 3.5), 200 mM 2-keto-L-gulonic acid hydrate, 1 mM CaCl 2 and 1g / L ThsLac. After anaerobic reaction at 30°C for 24h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of L-ascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC detection conditions are as shown in Example 1. The HPLC detection results showed that the amount of L-ascorbic acid produced was 13.7mM. SEQ ID NO:1 (Tannerella forsythia) MRGFGKIGIILLCAVMLTGMAVSCSQVEHINAEVVYQTEAQLGEGAIWHPDRQSLFWVDIESKTLYEFLPRKKKCRSWKFDRKVTTVVPENEETMVVALENGIIRLNLKSKAKEEIAPIETRGGSLRCNDGKCSPNGWLWVGTMSNDGEKGKATLYCVRPDGRVDAMVKGVSISNGIVWSRNRKYIYYNDTPTGKIKRYRYDARSGDIIANGIAVNIPQGSGMPDGMAIDRNDNLWVAQWGGFGVYCYNAYTGELITKVDVPAPNVTSCTFGGRNMDTLYITTAREGLTDEELAAYPLSGCIFACKPGAVGLRPNYFGQELKKENNED SEQ ID NO:2(Zymomonas mobilis subsp.mobilis) MTTGRMSRRECLSAAVMVPIAAMTATATITGSAQAAKNNMNGSTIGKITKFSPRLDAILDVSTPIEVIASDIQWSEGPVWVKNGNFLLFSDPPANIMRKWTPDAGVSIFLKPSGHAEPIPAGQFREPGSNGMKVGPDGKIWVADSGTRAIMKVDPVTRQRSVVVDNYKGKRFNSPNDLFFSKSGAVYFTDPPYGLTNLDESDIKEMNYNGVFRLSPDGRLDLIEAGLSRPNGLALSPDETKLYVSNSDRASPNIWVYSLDSNGLPTSRTLLRNFRKEYFDQGLAGLPDGMNIDKQGNLFASAPGGIYIFAPDGECLGLISGNPGQPLSNCCFGEKGQTLFISASHNVVRVRTKTFG SEQ ID NO:3(Acetobacter pasteurianus) MAPPRSRLARRAMLKGITALSATVAAPVLARGADEVLPDTVRPPDVTTNPPRQWGVNAPPVYTPDPDVLVIDRSFRDLLYGNAALHRVWDKGTWLEGPAWSSQGRYLLFSDVVASRQYRYLWETGEVTVFRPESYHANGNTFDYQGRQITCEHFLRRVIRWEHDGSAHVVADKFEGHSLNSPNDVIAHPDGSIWFTDPGYGDNVAEGHADMPGGEANPDGHIRWKIGEELIGTLGGSRRQADHTFRVDATGHLDVVLTQAQLPNPNGLCFSPDFSRLYVISSGKQPGGHGHDGDQAVHVFDLHKEELPLSNPRLFTNMRFEGVQMGPDGMKADVHGNIWCGVSGPVGLCGVFVFNPEGHLIGRIRLPQSVSNLTFGGPKRDWLFMCAGDSLYRLQVNTQGAAPA SEQ ID NO:4(Acidobacterium capsulatum) MHPLARVAAASALLLFLYPTNSRAQAPVPGAAVKSSMRVLRFSPALDQLIAPGAKPEV VHGGYVFTEGPMWREGRLWFSDEEGDHVHALTASGQDAVLVDFQHGKYALPDGHKT GPNAMAPAKDGSVVMCEQYGRAVVRLEGPASHLRVVPFFDSYHGKRLNSPNDLVFLP DGSFFFTDPPYGLKGGDHDPAKQLKFDGVFHYKNGKLTPIIENLTLPNGIALSPDNKTL YVNNSGPAMRVIAYPLHADGSVGEPRDVIDFTSKDGPGVPDGMKIDSHGNIWTTAPGG IRIVTPQGKVLGQILLPEVAANLAWGGDGTTLYITARTHVYRLQTRVKGYLPAFRR SEQ ID NO:5(Bryobacteraceae bacterium) MKLPAIFLLLLASTVLTAQDARPPRRFELKPLSPRFHDLIAPDARLTRVAGGFGFTEGPV WDPKGFVCVSDEVRNRIFRVFPDGRTETLLRIIDSDGSTFDRDHHFITTASALRALIDVS PDGNYKVIADRYEGRRFNSPNDVVLGPGGALYFTDPTLDLPKGEKQELDFQGVFRLGP DGSVRLLIRDLEQPNGLAFSPGGKRLYVDDTSRKTIHVYDVTPDGGVANGRIFGREEGP PGSGSPDGMRVDSKGNLYVTGPYGIWIWDPAGNHLGTIALPETAANLCWGDADSRTLY ITATTSLYRIRAKVRGYIR SEQ ID NO:6(Leptospirillum sp.) MGKNVNRKWKASAKMISLGLGASLVLGAGMQAQAVTVSTIAGSFHERGAIDGAGTN ARFEFPQGILAAPDGTIYIADTGNDMIRKMNPSTKSVENVAGSDHRARYRDGVGANAR FNNPEGMAISPDGKTLYIADSRNNMIRKIDLATKTVSTIAGHSFPSSGDGVGKEAGFETP RGLAISPDGKTLYVADSGNNAIRKIDLATNTVTTLAGAGKLMSGSADGVGVQATFHEP RSLAISGDGQVLYIADTRNNLIRKMVLATNSVSTLAGHPGFPGTLNGPGPDAYFYHPVS VTIDGNKLYVADGANADIRMVDLSTGVVSTVAGATLNGGVPIAGREDGSGVEGHFQFP GALTVYHGVLFVADTPADTIREVSF SEQ ID NO:7(Aspergillus niger) MKSPTILSCALPVLLAALIGGSYAQTASIPASYQYLLPPLFHGNLSEDFVSGTESAQLSL NSALKEAQRAAFISYDKEFDEVIGQEPSAQIVSRGTYGYAYEEAGIWVHDLNQVGATGEI LGGRSVANLPQMWFTSAISSGPTYLSVLDLKTNEVTQPNLVKPHGSESFVLSNPNGGFY YDGYVYITMLGDATHVAGLVKINPRTLETSTVLNSYYGLGLPSVDDVSIMRDKETKQT HIFFSTLDLVPFGPPISAHASNVTIQLPNAFWSFTPDTETLRPVIPRSDVLGPNGVSIDGK NSYLYVTDTPDHIHPTGTGFPAIFRYTLSSENGFIPSNKQLFGPPRHGVADGLKVDAMG RVWTGEAEGVVVRNMAGKVIGIFNKEVFMGRQTPMTEMANFALAGDKLILALDAII VVQLAESIGVAHDLVL SEQ ID NO:8 (Acetobacter senegalensis) MTHSLSRRLFGLSGMAGAVTVALPAYAQDDTAKAPDPADPESRERTYCYVGGYTQHG PPGGNGEGITVFEMDPKTGVLTNLMTFTDIASPSYIALSPDKRFLYAVSEINDFDTAHSG SVTAFSIDHATGALTKLNTVASGGADPAHLSVHPSGRYVMVANYTGGTVGVLHVLAD GSLGRMTELLHNTGPRMPDRAADNPPGNFAISDHSSAHPHMVKADPTGKYVLVMDA GLDRVYVWTINLDTGKLTPAAVPFTTMTPGSAPRHFEFGSDGGHKLYLLCEQDSKVVVA DFDPQTGKIAPQQTVSSVTAHFRGSTLAAGILLSPDEHFLYVSNRLGDSLAVFQVAKDG GLTLVDEIWTHADYGRSLMFDPSGSYLYVANQRSDSITSFRVDKTTGKISFTWDFTPIGS PTCFEFLRVPAS SEQ ID NO:9(Acidovorax soli) MLGHFTRSGSAWRGLGLALLWCWGATNAAAAVPCNASELAYLGTDGVGIRAVRFDA CAGTLALLGQPADTRKSRWLLKHPRLPVLYAAAEGAEGDGRVLAYTIDGRTGGLSLLG EGTAGGAGTTHLWLDAPSMTLLAANFAAGTTVSSIALLPDGKPGALVSTIKATGSGPHR RQTSPHAHGVAIDPSGRHALVADMGADRVFVYGFERSTRVLAPDDAAAPRSFQAPAGS GPRRAVFSTTGRMVYVLSELTAEISVLRWDASSGRLAPVQTLALSSEEFAGTRSASELA LGPDGRFLYLGNRGESQLLVYRVDAGSGELTPVQRLPSGGEAPWAFDIHPSGQWMLVA NYRSNRVSLMRIDAASGRLSDTGVALESPAPVSVVFVN SEQ ID NO:10 (Luteitalea pratensis) MRPAASKTLTTRTLTRRSFVTAATGLASVAPALARGATSQASTSSGGLFAYVGTYSSPPV DAPPGKVDLPPGNGRGIHIFRVDRTTGTLTAAAGVTDHYTSPSALVFDATGTHLYSTNAT DLVDNGASGTVSAFTVDRTSGQLQLLNSVASGGTGPTYAASVHRGGRHLLVANYAGGSV AVLPILPDGRLGPATDVKKTTGVVGPKRATHAPPGSFAISGHDTPHAHMIGTDPAGRFVI VPDLGLDRIFVWAFDANAGTLSPADPPSVALPPDGPRHFHRDGRWLYAIQEEGSTI VLFDYDAERGRLSARQTISSLPAGYAGSNFCSGILISQDGRFVYAGNRLHDSVGIFAVGR DGTLTFVDDIWTRGDYPRSMTIDPSGRFMYVCNQRADHVTAFAIDGATGRLAFTGQYT PVGNPSAIAFLDTGRG SEQ ID NO:11(Gluconobacter oxydans) MSHPVSRRNFAVGLVAGVAAAGTGVAGAADPEKAVPTPPPPPKPVAKTICFVGGYTKH GPPGGGTGNGQGISVFDMDRDTGVLTPITTFTDIASPSFLAISKDQRFLYALSEIDDFNK DGDGSVTAFAIDPKTGSLRKLNVVSSKGAVPAHLSIHHSGRYVLVANYVGGCVAVLPIR SDGSLGEASDVVHNTGPRQPERASDNPQGNFAVSDHSGSHPHMIHSDPSGKFVLADDA GLDRVYVWTLNIDTGKLIPAKTPYYDMEPGSAPRHFQFNHSGRILYNLCEQDSKVVVS NFDPATGAISDIQTVSTVTSHFRGSTLAAEILISASGKFVYVSNRLGDSLAVFAIGADGTL TLQDEVWMHADYGRALMFDPSGAFLFCANQRSDAVTSFKVDKKTGEIAFTNNFTPVG SPTTFAFMNTQV SEQ ID NO:12(Sulfobacillus thermosulfidooxidans) MHRPFFRVVFTVAVSFALSACGFNPSTASSKHSAKAPASPKTSHRQSPQSKSLVPWGAT AASSILPGDILIADSHNNRILLVTPQKKIVWQYPKPGQPSLLHDDDDVFFGPHFDEIITNQ EGYNVISIINFKTRQVVWNYGHAGVPGSLPGYLNTPDDAFLYAKPKGDIITVADIRNQRI LFISRKTHQILKQYGQTGVMAVNPPATYSAPNGDFPAPHGGMLVTQINGNDAMLLNRH NQVRYTVHFPSFLSYPSDANFTPHGNIIVAFYTNPGAVVEMSPTGKVLWLYRPLSGPGR LDRPSLAVKLPNGLVLLNDDYNDRVIVINPKTNQIVWQYGHTGIPGTAPGYLNIPDGLD LLPPGIVPGGHNPIGHHLWSYPHNGY SEQ ID NO:13(Leptospirillum ferrooxidans) MNKSKHPKGRGIRSIKTAAVAVSLGIGMTLMAGQAFQAQAVSLKTIAGSFHERGDNDG NGASARFEFPQGIVAAPDGSIYVADTGNDMIRKITMSSGTGTVETIAGVNHHARFRDGA GAAARFNNPEGLAISPDGKTLYVADSRNNRIRKIDLATKTVSTLAGHAFASGNDGIGDH AGFAQPRGLAISPDGKTLYIGDSGNNMIRKMDLTTLQVTTLAGQGALVPGHADGVGTQ ATFLEPRGLAISPDGQILYIADTRNHLIRKLVLATNSVSTLAGHPGFPGTLNGPGPDAYF YDPMMLAIDGNKLYVCDAANADLRLIDLNSDTVSTVAGATINGGVPIKGREDGDGTL GHFEFPGAVTIYHGVLYVTDTPADTIRSVSF SEQ ID NO:14(Thiobacillus sp) MNAKKLVLALLCLGCGTFAMAAPIAYIPLGSANQVIAVDAANDKIIRSYSGVENPHGLVATPDGDYLVAGSLKETPVPAGAPSDTPNSKLYVIHPEHGHVMAEIPVSGWTHHEAVTPDGKYVISTHPTRGGVSVVDVINNKVYKTIKTGPSPNYTLVTKDGKR AYVSNSGNGTLSEIDLSKWEVTRSLEAGPTPEHLVFSPDEKTIYVSNDRAGTVSVVSVEQGKVEKTYKIGKRVHGLDLGDDGKTLFISSRSDDKLVALDTQTGEQRVLTLSPEPYHLDKIPGTGKVYVSSSKAPKIWVVDQKTFTVTDTIQLPAGEGHQIAVIR It should be noted that, although the technical solutions of the present disclosure are introduced with specific examples, those skilled in the art will appreciate that the present disclosure should not be limited thereto. The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing L-ascorbic acid, characterized in that: Using 2-keto-L-gulonic acid and / or its metal salt as substrate, reacting it with an enzyme catalyst under acidic conditions to generate L-ascorbic acid; Optionally, the acidic condition is pH ≤ 6.

0.

2. The method according to claim 1, characterized in that The enzyme catalyst comprises one or more hydrolases with an EC number of EC 3; Optionally, the hydrolase comprises a carboxylate hydrolase with an EC number of EC 3.1.

1. Preferably, the carboxylate hydrolase is a lactonase that can hydrolyze lactone.

3. The method according to claim 2, characterized in that The lactonase is selected from L-arabinonolactonase with an EC number of EC 3.1.1.15, At least one of the following: gluconolactonase of EC 3.1.1.17, 1,4-lactonase of EC number EC 3.1.1.25, D-arabinolactonase of EC number EC 3.1.1.30, 6-phosphogluconolactonase of EC number EC 3.1.1.31 and xylono-1,4-lactonase of EC number EC 3.1.1.

68.

4. The method according to claim 2 or 3, characterized in that: The lactonase is derived from Tannerella forsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceaebacterium, Leptospirillum sp., Aspergillus niger, Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis, Gluconobacter oxydans, Sulfobacillusthermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp.

5. The method according to any one of claims 2 to 4, characterized in that: The lactonase is selected from any one of the following (a1) to (a3): (a1) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a2) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2).

6. The method according to any one of claims 1 to 5, characterized in that: L-ascorbic acid is generated under acidic conditions, wherein the acidic conditions are pH ≤ 5.5, preferably pH 2.0 to 5.

5.

7. The method according to any one of claims 1 to 6, characterized in that: The 2-keto-L-gulonic acid metal salt is a soluble salt of 2-keto-L-gulonic acid; preferably, the soluble salt is selected from potassium salt, sodium salt or calcium salt.

8. The method according to any one of claims 1 to 7, characterized in that: The 2-keto-L-gulonic acid and / or its metal salt comprises 2-keto-L-gulonic acid, 2-keto-L-gulonic acid hydrate, 2-keto-L-gulonic acid sodium, 2-keto-L-gulonic acid potassium and / or 2-keto-L-gulonic acid calcium.

9. The method according to any one of claims 1 to 8, characterized in that: The enzyme catalyst is in the form of pure enzyme, crude enzyme, cell culture, cell lysate or whole cell; optionally, the pure enzyme or crude enzyme is in free or immobilized form, and the whole cell is in free or immobilized form.

10. The method according to any one of claims 1 to 9, characterized in that: In the step of synthesizing L-ascorbic acid, the reaction system contains calcium ions and reacts under anaerobic conditions at 0 to 100° C.; Optionally, the calcium ions are provided in the form of nitrate, sulfate or chloride.

11. At least one of the following uses of lactonase: (A) Use in the preparation of L-ascorbic acid; (B) Use as a catalyst for preparing L-ascorbic acid; Alternatively, the lactonase is derived from Tannerella forsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium, Leptospirillum sp., Aspergillus niger, Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis, Gluconobacter oxydans, Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp; Preferably, the lactonase is selected from any one of the following (a1) to (a3): (a1) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a2) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2).

12. A catalyst for preparing L-ascorbic acid, comprising a lactonase derived from Tannerellaforsythia, Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceaebacterium, Leptospirillum sp., Aspergillus niger, Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis, Gluconobacter oxydans, Sulfobacillusthermosulfidooxidans, Leptospirillum ferrooxidans and / or Thiobacillus sp.; Optionally, the lactonase is selected from any one of the following (a1) to (a3): (a1) A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a2) A polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1, 3 to 14, in which one or more amino acids are substituted, deleted or added, and having the activity of lactonizing 2-keto-L-gulonic acid and / or its metal salt to L-ascorbic acid in one step; (a3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (a1) or (a2).

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