Method for preparing D-erythorbic acid

Abstract

The invention discloses a method for preparing D-erythorbic acid, and belongs to the field of erythorbic acid production. According to the method for preparing the D-erythorbic acid, provided by the invention, 2-keto-D-gluconic acid and / or a metal salt thereof are / is taken as a substrate and react with an enzyme catalyst under an acidic condition, so that the 2-keto-D-gluconic acid and / or a derivative thereof are / is converted into the D-erythorbic acid and / or the derivative thereof in one step; the method does not need a large amount of acid-base and organic solvent methanol for lactonization cyclization, the process is simple and green, the substrate conversion rate is high, the production efficiency is improved, and the cost is reduced. Besides, the reaction system is acidic and is consistent with the acidic condition required by later concentration and crystallization, so that concentration and crystallization can be carried out without an acidification reaction step after the enzyme catalyst is removed after the reaction is finished, separation and purification of later products are simplified, and the production cost is further reduced.

Description

Technical Field

[0001] The present invention belongs to the field of isoascorbic acid production and relates to a method for preparing D-isoascorbic acid, and in particular to a method for converting 2-keto D-gluconic acid and / or its derivatives into D-isoascorbic acid and / or its derivatives by in vitro enzyme catalysis. Background Art

[0002] Isovitamin C, also known as D-Isoascorbic acid and Erythorbic acid, is an optically active isomer of D-isoascorbic acid. Isovitamin C and its metal salts have strong reducing properties and are safe and effective food additives. They are widely used in various foods, such as food antioxidants and preservatives and colorants for meat products. It can also be used as an auxiliary material for drugs, medicines or sanitary products; as a stabilizer for chemical raw materials; as an important component of deoxidizing, anti-corrosion and descaling solvents; as a chemical additive for acidification of oil field formations; as an electrolyte in electrolysis and electroplating, etc.

[0003] At present, the production method of D-isoascorbic acid in industry is mainly indirect fermentation (or semi-synthesis), including fermentation, extraction, and esterification conversion. First, glucose in the fermentation medium is converted into 2-keto-D-gluconic acid (2-keto-D-gluconic acid, 2KGA) by bacteria, and then the fermentation broth is purified and concentrated, and then the D-isoascorbic acid product is obtained through esterification, conversion (including intramolecular ester exchange and tautomerization reaction), acidification and refining. Usually, the esterification conversion process of D-isoascorbic acid includes the following steps: esterification reaction step, 2-keto-D-gluconic acid is catalyzed by concentrated sulfuric acid in methanol to generate 2-keto-D-gluconic acid methyl ester; conversion reaction step, 2-keto-D-gluconic acid methyl ester is converted into D-isoascorbic acid sodium salt with NaHCO3; acidification reaction step, H2SO4 is added to D-isoascorbic acid sodium salt to acidify it into D-isoascorbic acid. In this process, a large amount of acid, alkali and organic solvent methanol are needed. The treatment of acid, alkali and organic solvent increases the difficulty and cost of subsequent separation and purification of the product.

[0004] With the continuous expansion of the market demand for D-isoascorbic acid and the increasingly fierce market competition, it is very important to explore new production methods for D-isoascorbic acid. Therefore, it is urgent to develop a green and low-cost preparation method for D-isoascorbic acid. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] Currently, most methods for producing D-isoascorbic acid involve chemical methods, which have complex reaction steps and require the use of large amounts of acid, alkali and organic solvent methanol, which is not conducive to the separation and purification of the product, making production difficult and costly.

[0007] Solutions for solving problems

[0008] [1]. A method for preparing D-isoascorbic acid, wherein an enzyme catalyst is contacted with 2-keto-D-gluconic acid and / or a metal salt thereof in a system containing 2-keto-D-gluconic acid and / or a metal salt thereof to generate D-isoascorbic acid under acidic conditions.

[0009] [2] The method according to [1], wherein the 2-keto-D-gluconic acid metal salt is a soluble salt of 2-keto-D-gluconic acid; optionally, the soluble salt is selected from a potassium salt of 2-keto-D-gluconic acid, a sodium salt of 2-keto-D-gluconic acid or a calcium salt of 2-keto-D-gluconic acid.

[0010] [3] The method according to [1] or [2], wherein the 2-keto-D-gluconic acid and / or its metal salt is selected from at least one of 2-keto-D-gluconic acid, 2-keto-D-gluconic acid hydrate, sodium 2-keto-D-glucose and 2-keto-D-gluconic acid hemi-calcium salt.

[0011] [4] The method according to any one of [1] to [3], wherein the acidic condition is pH ≤ 6.0, preferably pH ≤ 5.5.

[0012] [5] The method according to any one of [1] to [4], wherein the enzyme catalyst comprises at least one hydrolase having an EC number of EC 3;

[0013] 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; optionally, the lactonase is selected from at least one of the lactonases with EC numbers of EC 3.1.1.15, EC3.1.1.17, EC 3.1.1.25, EC 3.1.1.30, EC 3.1.1.31 and EC 3.1.1.68.

[0014] [6] The method according to [5], wherein the lactonase is selected from at least one of (a1) to (a5):

[0015] (a1) L-arabinonolactonase from Tannerella forsythia;

[0016] (a2) one or more of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0017] (a3) 1,4-lactonase from Aspergillus niger;

[0018] (a4) one or more of 6-phosphogluconolactonase from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0019] (a5) One or more lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.

[0020] [7] The method according to [5] or [6], wherein the lactonase is selected from any one of the following (b1) to (b3):

[0021] (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0022] (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0023] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0024] [8] The method according to any one of [1] to [7], wherein the enzyme catalyst is in the form of pure enzyme, crude enzyme, cell lysate, cell culture or whole cells; optionally, the pure enzyme, crude enzyme or whole cell is in free or immobilized form.

[0025] [9] A method according to any one of [1] to [8], wherein:

[0026] In the step of synthesizing D-isoascorbic acid, the reaction system contains calcium ions, and optionally, the calcium ions are provided by a soluble salt of calcium;

[0027] In the step of synthesizing D-isoascorbic acid, the reaction is carried out under anaerobic conditions at 0 to 100°C.

[0028]

[10] . Use of a lactonase in the preparation of D-isoascorbic acid and / or in a catalyst for the preparation of D-isoascorbic acid, wherein the lactonase is selected from at least one of (a1) to (a5):

[0029] (a1) L-arabinonolactonase from Tannerella forsythia;

[0030] (a2) one or more of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0031] (a3) 1,4-lactonase from Aspergillus niger;

[0032] (a4) one or more of 6-phosphogluconolactonase from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0033] (a5) one or more lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.;

[0034] Optionally, the lactonase is selected from any one of the following (b1) to (b3):

[0035] (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0036] (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0037] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0038]

[11] . A catalyst for preparing D-isoascorbic acid, the catalyst comprising a lactonase as shown in at least one of (a1) to (a5):

[0039] (a1) L-arabinonolactonase from Tannerella forsythia;

[0040] (a2) at least one of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0041] (a3) 1,4-lactonase from Aspergillus niger;

[0042] (a4) at least one of 6-phosphogluconolactonase derived from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0043] (a5) at least one lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.;

[0044] Optionally, the lactonase is selected from any one of the following (b1) to (b3):

[0045] (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0046] (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0047] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0048] Effects of the Invention

[0049] The present disclosure provides a method for preparing D-isoascorbic acid, using 2-keto-D-gluconic acid and / or its metal salt as a substrate, and the substrate is directly converted into D-isoascorbic acid and / or its derivatives in one step through the catalytic action of an enzyme catalyst, and there is no need to add a large amount of acid, alkali and organic solvent methanol in the reaction system for lactonization and ring formation, which is green and environmentally friendly; and the substrate conversion rate is high, the production efficiency is improved, and it is beneficial to reduce costs. In addition, the entire preparation process is carried out under acidic conditions, which is consistent with the acidic conditions required for later concentration and crystallization. Therefore, after the reaction is completed, the enzyme catalyst is removed without going through the acidification reaction step, and the concentration and crystallization can be carried out, which simplifies the separation and purification of later products and further reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The pathway for the one-step conversion of 2-keto-D-gluconic acid to D-isoascorbic acid is shown.

[0051] Figure 2 The HPLC detection chart of the one-step conversion of 2-keto-D-gluconic acid into D-isoascorbic acid catalyzed by lactonase is shown. DETAILED DESCRIPTION

[0052] 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.

[0053] 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.

[0054] 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.

[0055] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0056] 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.

[0057] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In this document, "or its combination" means "or any combination thereof", and "any one", "any one" means "any one", "any one" or "any one".

[0065] 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.

[0066] 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.

[0067] 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.

[0068] As used herein, the term "suitable reaction conditions" refers to those conditions under which an enzyme catalyst catalyzes the lactonization of 2-keto-D-gluconolacton to produce D-isoascorbic 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] <Method for preparing D-isoascorbic acid>

[0077] The present disclosure provides a method for preparing D-isoascorbic acid, wherein an enzyme catalyst is added to a system containing 2-keto-D-gluconic acid and / or its metal salt, the enzyme catalyst is brought into contact with the 2-keto-D-gluconic acid and / or its metal salt, and D-isoascorbic acid is generated under acidic conditions.

[0078] In some embodiments, the method for preparing D-isoascorbic acid is carried out under anaerobic conditions.

[0079] (2-Keto-D-gluconic acid and / or its metal salt)

[0080] According to some embodiments of the present disclosure, the 2-keto-D-gluconic acid metal salt is a soluble salt of 2-keto-D-gluconic acid, and the soluble salt is selected from a potassium salt, a sodium salt or a calcium salt.

[0081] In some specific embodiments, the 2-keto-D-gluconic acid and / or its metal salt is selected from 2-keto-D-gluconic acid, 2-keto-D-gluconic acid hydrate, sodium 2-keto-D-gluconic acid and / or 2-keto-D-gluconic acid hemi-calcium salt.

[0082] (Acidic conditions)

[0083] 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.

[0084] 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).

[0085] 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.

[0086] (Enzyme Catalyst)

[0087] In the present disclosure, the enzyme catalyst has the function of lactonization of 2-keto-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step, and has the ability to produce D-isoascorbic acid in the absence of other cofactors. All proteins of organisms such as microorganisms, plants, animals, etc. that have the function of lactonization of 2-keto-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step are also included. Even newly discovered enzymes are included as long as they have the function of lactonization of 2-keto-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step.

[0088] 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.

[0089] According to some embodiments of the present disclosure, the enzyme catalyst includes one or more of the hydrolases with EC number EC 3; optionally, the hydrolases include carboxylic acid ester hydrolases with EC number EC 3.1.1, in particular, the carboxylic acid hydrolases are lactonase, which catalyzes a reversible reaction, specifically hydrolyzing lactones (cyclic esters formed by intramolecular reactions between carboxyl and hydroxyl groups) and reforming lactones. In some optional embodiments, the lactonase includes L-arabinon lactonase (EC 3.1.1.15), gluconolactonase (EC 3.1.1.17), 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 (EC3.1.1.68) or other enzymes with no enzyme number but lactonase activity.

[0090] The method disclosed herein can also be implemented by using functional derivatives of the lactonase. These functional derivatives are different from the lactonase itself by adding, inserting, deleting and / or replacing one or more amino acid residues in the normal sequence of the lactonase (unmodified), but these derivatives still have lactonase activity using experimental determinations known in the art. These functional derivatives can be prepared by peptide chemical synthesis methods known in the art or chemical modification methods of proteins, or by recombinant methods known in the art based on the DNA sequence disclosed herein. Amino acid substitution can refer to the replacement of an amino acid at a certain position in the amino acid sequence by other amino acids, as long as the altered sequence retains the activity of the original amino acid sequence in whole or in part. Amino acid substitution can be a conservative amino acid substitution, which means that compared with the original amino acid sequence, several amino acids are replaced by amino acids of similar or similar properties 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.

[0091] 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.

[0092] Exemplarily, the lactonase is selected from at least one of (a1) to (a5):

[0093] (a1) L-arabinonolactonase from Tannerella forsythia (EC 3.1.1.15);

[0094] (a2) one or more of gluconolactonase (EC 3.1.1.17) from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0095] (a3) 1,4-lactonase from Aspergillus niger (EC 3.1.1.25);

[0096] (a4) one or more of 6-phosphogluconolactonase (EC 3.1.1.31) from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0097] (a5) One or more lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.

[0098] 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).

[0099] 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).

[0100] 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).

[0101] 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).

[0102] 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).

[0103] [Enzyme form]

[0104] 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.

[0105] 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.

[0106] (Metal Ions)

[0107] According to some embodiments of the present disclosure, the reaction system contains metal ions, preferably calcium ions, and the calcium ions are provided by soluble salts of calcium; optionally, provided by calcium nitrate, calcium sulfate or calcium chloride.

[0108] (Buffer)

[0109] According to some embodiments of the present disclosure, the reaction system further contains a buffer to ensure 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-D-gluconic acid and / or its metal salt.

[0110] In some embodiments of the present disclosure, any buffer suitable for the pH value or pH range under the above acidic conditions, such as acetate buffer, citrate buffer, can be used in the method for preparing D-isoascorbic acid. In some embodiments of the present disclosure, the concentration of the buffer can be determined according to the concentration of the substrate used, and is preferably in the range of 1mM to 200mM, such as 50mM to 200mM, 100mM to 200mM.

[0111] (Reaction temperature)

[0112] In some embodiments of the present disclosure, the temperature of the reaction depends on factors such as the nature and amount of the enzyme catalyst and the amount of the substrate 2-keto-D-gluconic acid and / or its metal salt. The method for preparing D-isoascorbic acid described in the present disclosure is carried out at a reaction temperature of 0°C to 100°C. In some preferred embodiments of the present disclosure, the method for preparing D-isoascorbic acid is carried out at a reaction temperature of 25°C to 80°C. In some more preferred embodiments of the present disclosure, the method for preparing D-isoascorbic acid is carried out at a reaction temperature of 37°C to 75°C, exemplarily, the reaction temperature is 30°C, 35°C, 37°C, 40°C, 45°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 72°C, 75°C.

[0113] (Reaction time)

[0114] According to some embodiments of the present disclosure, the reaction time of the method for preparing D-isoascorbic acid is not less than 20 h. Preferably, the reaction time is not less than 24 h, 30 h, 40 h, 50 h, 60 h or 70 h. Exemplarily, the reaction time can be 24 h, 30 h, 35 h, 40 h, 45 h, 50 h, 60 h, 65 h, 70 h, 75 h, 80 h, 85 h or 90 h.

[0115] <Application of lactonase>

[0116] According to some embodiments of the present disclosure, the present disclosure provides the use of a lactonase in the preparation of D-isoascorbic acid and / or in a catalyst for the preparation of D-isoascorbic acid, wherein the lactonase is 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, Bryobacteraceaebacterium, Leptospirillum sp.; a 1,4-lactonase (EC 3.1.1.25) derived from Aspergillus niger; a 1,4-lactonase (EC 3.1.1.25) derived from Acetobacter senegalensis, Acidovoraxsoli, Luteitalea pratensis, Gluconobacter oxydans 6-phosphogluconolactonase (EC 3.1.1.31); xylono-1,4-lactonase (EC 3.1.1.68), or lactonase from Sulfobacillusthermosulfidooxidans, Leptospirillum ferrooxidans, Thiobacillus sp.

[0117] In some embodiments, the lactonase is selected from at least one of (a1) to (a5):

[0118] (a1) L-arabinonolactonase from Tannerella forsythia (EC 3.1.1.15);

[0119] (a2) one or more of gluconolactonase (EC 3.1.1.17) from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0120] (a3) 1,4-lactonase from Aspergillus niger (EC 3.1.1.25);

[0121] (a4) one or more of the 6-phosphogluconolactonase (EC 3.1.1.31) from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0122] (a5) One or more lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.

[0123] Optionally, the lactonase is selected from any one of the following (b1) to (b3):

[0124] (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0125] (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0126] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0127] <Catalyst for preparing D-isoascorbic acid>

[0128] According to some embodiments of the present disclosure, a catalyst for preparing D-isoascorbic acid is provided, wherein the catalyst comprises a lactonase that can directly convert 2-keto-D-gluconic acid and / or its metal salt into D-isoascorbic acid in one step, wherein the EC number of the lactonase is EC 3.1.1.x, in particular, a lactonase with the EC number of 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 / or EC 3.1.1.68.

[0129] In some embodiments, the lactonase is selected from at least one of (a1) to (a5):

[0130] (a1) L-arabinonolactonase from Tannerella forsythia (EC 3.1.1.15);

[0131] (a2) one or more of gluconolactonase (EC 3.1.1.17) from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.;

[0132] (a3) 1,4-lactonase from Aspergillus niger (EC 3.1.1.25);

[0133] (a4) one or more of the 6-phosphogluconolactonase (EC 3.1.1.31) from Acetobacter senegalensis, Acidovorax soli, Luteitaleapratensis and Gluconobacter oxydans;

[0134] (a5) one or more of lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillumferrooxidans and Thiobacillus sp.

[0135] Optionally, the lactonase is selected from any one of the following (b1) to (b3):

[0136] (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0137] (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step;

[0138] (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

[0139] Example

[0140] The embodiments of the present disclosure will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0141] 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.

[0142] Materials and Methods

[0143] Drugs and materials

[0144] 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.

[0145] Preparation and purification of plasmids and recombinant proteins

[0146] 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).

[0147] 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.

[0148] 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:

[0149] SEQ ID NO: 1 shows the amino acid sequence of L-arabinonolactonase from Tannerella forsythia (Uniprot ID: A0A1D3UGI3);

[0150] SEQ ID NO: 2 shows the amino acid sequence of gluconolactonase from Zymomonas mobilis (Uniprot ID: Q01578);

[0151] SEQ ID NO: 3 shows the amino acid sequence of gluconolactonase from Acetobacter pasteurianus (UniprotID: S6CYH2);

[0152] SEQ ID NO: 4 shows the amino acid sequence of gluconolactonase from Acidobacterium capsulatum (Uniprot ID: C1F8R6);

[0153] SEQ ID NO: 5 shows the amino acid sequence of gluconolactonase from Bryobacteraceae bacterium (Uniprot ID: A0A949VV58);

[0154] SEQ ID NO: 6 shows the amino acid sequence of gluconolactonase from Leptospirillum sp. (Uniprot ID: B6AQ12);

[0155] SEQ ID NO: 7 shows the amino acid sequence of 1,4-lactonase from Aspergillus niger (Uniprot ID: A2R8C2);

[0156] SEQ ID NO: 8 shows the amino acid sequence of 6-phosphogluconolactonase from Acetobacter senegalensis (Uniprot ID: A0A0U5F503);

[0157] SEQ ID NO: 9 shows the amino acid sequence of 6-phosphogluconolactonase from Acidovorax soli (UniprotID: A0A7X0PKY7);

[0158] SEQ ID NO: 10 shows the amino acid sequence of 6-phosphogluconolactonase from Luteitalea pratensis (Uniprot ID: A0A143PK71).

[0159] SEQ ID NO: 11 shows the amino acid sequence of 6-phosphogluconolactonase from Gluconobacter oxydans (Uniprot ID: A0A067Z318).

[0160] SEQ ID NO: 12 shows the amino acid sequence of lactonase derived from Sulfobacillus thermosulfidooxidans (Uniprot ID: A0A1W1WCN5).

[0161] SEQ ID NO: 13 shows the amino acid sequence of lactonase from Leptospirillum ferrooxidans (Uniprot ID: 10INF7).

[0162] SEQ ID NO: 14 shows the amino acid sequence of lactonase from Thiobacillus sp. (Uniprot ID: A0A9D5J2S7).

[0163] Exemplarily, the preparation method of the pET plasmid carrying the corresponding protein encoding gene is as follows:

[0164] 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.

[0165] Table 1

[0166]

[0167] 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 picked and 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 activity determination.

[0168] Enzyme activity assay used

[0169] Principle of determination: In the presence of hydrochloric acid, silicate and molybdate react to generate silicomolybdate yellow, and D-isoascorbic acid reduces silicomolybdate yellow to generate silicomolybdate blue. The concentration of D-isoascorbic 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 D-isoascorbic acid generated in the reaction system can be determined.

[0170] Reaction system: In a 1mL reaction system, 200mM acetic acid buffer (pH 5.0), 200mM 2-keto-D-gluconic acid hydrate, 1mM CaCl2 and 0.1-1g / L enzyme catalyst were included. Anaerobic reaction was carried out at 37°C for 16h, 120μL of the sample was taken, 10μL of 4M HCl was added to terminate the reaction, the sample was allowed to stand for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid generated in the supernatant was determined.

[0171] Definition of enzyme activity: The amount of enzyme required to generate 1 μM D-isoascorbic acid within 1 minute is one unit of enzyme activity.

[0172] 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 D-isoascorbic acid generated according to the standard curve.

[0173] The experimental results are as follows Figure 2 As shown, the experimental results show that the enzyme catalysts in Table 1 are all active.

[0174] HPLC detection conditions:

[0175] The chromatographic column was ZORBAX SB-C18; the mobile phase was methanol / phosphate buffer (v:v, 2:98), wherein the phosphate buffer was 10 mM potassium dihydrogen phosphate (pH 2.5); the flow rate was 0.6 mL / min; the column temperature was 30°C; the detector was an ultraviolet detector (detection wavelength was 254 nm); and the injection volume was 10 μL.

[0176] Example 1

[0177] In a 1mL reaction system, 200mM acetic acid buffer (pH 5.0), 200mM 2-keto-D-gluconic acid hemi-calcium salt and 0.14g / L ZmGL were contained. Anaerobic reaction was carried out at 37℃ for 40h, 120μL of the sample was taken, 10μL of 4M HCl was added to terminate the reaction, the sample was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results are shown in Figure 2 The HPLC test results showed that the amount of D-isoascorbic acid produced was 4.2 mM.

[0178] Example 2

[0179] In a 1mL reaction system, 200mM acetic acid buffer (pH 4.0), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 2g / L APGL were included. Anaerobic reaction was carried out at 30°C for 60h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 24.3mM.

[0180] Example 3

[0181] In a 1mL reaction system, 500mM 2-keto-D-gluconate sodium, 2mM CaCl2 and 5g / LAcGL were included, 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 placed for 10min, centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 48.4mM.

[0182] Example 4

[0183] In a 1mL reaction system, 100mM acetic acid buffer (pH 5.0), 100mM 2-keto-D-gluconic acid hemi-calcium salt and 0.2g / L BbGL were included. Anaerobic reaction was carried out at 37°C for 60h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 5.4mM.

[0184] Example 5

[0185] In a 1mL reaction system, 500mM 2-keto-D-gluconic acid hemi-calcium salt and 6g / L LsGL were included, and the pH was adjusted to 4.0. Anaerobic reaction was carried out at 45°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, and the mixture was placed for 10min, centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 53.4mM.

[0186] Example 6

[0187] In a 1mL reaction system, 200mM acetic acid buffer (pH 5.0), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 0.5g / L TfAL were included. Anaerobic reaction was carried out at 30°C for 24h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 5.5mM.

[0188] Example 7

[0189] In a 1mL reaction system, 200mM acetic acid buffer (pH 5.0), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 1g / L AnL were included. Anaerobic reaction was carried out at 37°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 16.4mM.

[0190] Example 8

[0191] In a 1mL reaction system, 200mM acetic acid buffer (pH 3.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 2g / L Abs6PGL were included. Anaerobic reaction was carried out at 37°C for 60h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 20.7mM.

[0192] Example 9

[0193] In a 1mL reaction system, 200mM acetic acid buffer (pH 3.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 2g / L As6PGL were contained. Anaerobic reaction was carried out at 30°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 19.4mM.

[0194] Example 10

[0195] In a 1mL reaction system, 200mM acetic acid buffer (pH 4.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 2g / L Lp6PGL were included. Anaerobic reaction was carried out at 50°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 21.3mM.

[0196] Embodiment 11

[0197] In a 1mL reaction system, 200mM acetic acid buffer (pH 3.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 1g / L Go6PGL were included. Anaerobic reaction was carried out at 40°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 19.3mM.

[0198] Example 12

[0199] In a 1mL reaction system, 200mM acetic acid buffer (pH 4.0), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 1g / L StLac were included. Anaerobic reaction was carried out at 60℃ for 60h, 120μL of sample was taken, 10μL 4M HCl was added, and the mixture was placed for 10min. The mixture was centrifuged at 12000rpm for 10min, and the supernatant was taken. The amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 25.8mM.

[0200] Example 13

[0201] In a 1mL reaction system, 200mM acetic acid buffer (pH 3.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 2g / L LfLac were included. Anaerobic reaction was carried out at 50°C for 48h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 18.7mM.

[0202] Embodiment 14

[0203] In a 1mL reaction system, 200mM acetic acid buffer (pH 3.5), 200mM sodium 2-keto-D-gluconate, 1mM CaCl2 and 1g / L ThsLac were included. Anaerobic reaction was carried out at 30°C for 24h, 120μL of the sample was taken, 10μL of 4M HCl was added, the mixture was placed for 10min, centrifuged at 12000rpm for 10min, the supernatant was taken, and the amount of D-isoascorbic acid produced was detected by high performance liquid chromatography (HPLC). The HPLC test results showed that the amount of D-isoascorbic acid produced was 15.3mM.

[0204] SEQ ID NO:1 (Tannerella forsythia)

[0205] MRGFGKIGIILLCAVMLTGMAVSCSQVEHINAEVVYQTEAQLGEGAIWHPDRQSLFWVDIESKTLYEFLPRKKKCRSWKFDRKVTTVVPENEETMVVALENGIIRLNLKSKAKEEIAPIETRGGSLRCNDGKCSPNGWLWVGTMSNDGEKGKATLYCVRPDGRV DAMVKGVSISNGIVWSRNRKYIYYNDTPTGKIKRYRYDARSGDIIANGIAVNIPQGSGMPDGMAIDRNDNLWVAQWGGFGVYCYNAYTGELITKVDVPAPNVTSCTFGGRNMDTLYITTAREGLTDEELAAYPLSGCIFACKPGAVGLRPNYFGQELKKENNED

[0206] SEQ ID NO:2(Zymomonas mobilis subsp.mobilis)

[0207] MTTGRMSRRECLSAAVMVPIAAMTATATITGSAQAAKNNMNGSTIGKITKFSPRLDAILDVSTPIEVIASDIQWSEGPVWVKNGNFLLFSDPANIMRKWTPDAGVSIFLKPSGHAEPIPAGQFREPGSNGMKVGPDGKIWVADSGTRAIMKVDPVTRQRSVVVDNYKGKRFNSPNDL FFSKSGAVYFTDPPYGLTNLDESDIKEMNYNGVFRLSPDGRLDLIEAGLSRPNGLALSPDETKLYVSNSDRASPNIWVYSLDSNGLPTSRTLLRNFRKEYFDQGLAGLPDGMNIDKQGNLFASAPGGIYIFAPDGECLGLISGNPGQPLSNCCFGEKGQTLFISASHNVVRVRTKTFG

[0208] SEQ ID NO:3 (Acetobacter pasteurianus)

[0209] MAPPRSRLARRAMLKGITALSATVAAPVLARGADEVLPDTVRPPDVTTNPPRQWGVNAPPVYTPPDVLVIDRSFRDLYGNAALHRVWDKGTWLEGPAWSSQGRYLLFSDVVASRQYRYLWETGEVTVFRPESYHANGNTFDYQGRQITCEHFLRRVIRWEHDGSAHVVADKFEGHSLNSPNDVIAHPDGSIWFTDPGYGD NVAEGHADMPGGEANPDGHIRWKIGEELIGTLGGSRRQADHTFRVDATGHLDVVLTQAQLPNPNGLCFSPDFSRLYVISSGKQPGGHGHDGDQAVHVFDLHKEELPLSNPRLFTNMRFEGVQMGPDGMKADVHGNIWCGVSGPVGLCGVFVFNPEGHLIGRILPRQSVSNLTFGGPKRDWLFMCAGDSLYRLQVNTQGAAPA

[0210] SEQ ID NO:4(Acidobacterium capsulatum)

[0211] MHPLARVAAASALLLFLYPTNSRAQAPVPGAAVKSSMRVLRFSPALDQLIAPGAKPEV

[0212] VHGGYVFTEGPMWREGRLWFSDEEGDHVHALTASGQDAVLVDFQHGKYALPDGHKT

[0213] GPNAMAPAKDGSVVMCEQYGRAVVRLEGPASHLRVVPFFDSYHGKRLNSPNDLVFLP

[0214] DGSFFFTDPPYGLKGGDHDPAKQLKFDGVFHYKNGKLTPIIENLTLPNGIALSPDNKTL

[0215] YVNNSGPAMRVIAYPLHADGSVGEPRDVIDFTSKDGPGVPDGMKIDSHGNIWTTAPGG

[0216] IRIVTPQGKVLGQILLPEVAANLAWGGDGTTLYITARTHVYRLQTRVKGYLPAFRR

[0217] SEQ ID NO:5(Bryobacteraceae bacterium)

[0218] MKLPAIFLLLLASTVLTAQDARPPRRFELKPLSPRFHDLIAPDARLTRVAGGFGFTEGPV

[0219] WDPKGFVCVSDEVRNRIFRVFPDGRTETLLRIIDSDGSTFDRDHHFITTASALRALIDVS

[0220] PDGNYKVIADRYEGRRFNSPNDVVLGPGGALYFTDPTLDLPKGEKQELDFQGVFRLGP

[0221] DGSVRLLIRDLEQPNGLAFSPGGKRLYVDDTSRKTIHVYDVTPDGGVANGRIFGREEGP

[0222] PGSGSPDGMRVDSKGNLYVTGPYGIWIWDPAGNHLGTIALPETAANLCWGDADSRTLY

[0223] ITATTSLYRIRAKVRGYIR

[0224] SEQ ID NO:6(Leptospirillum sp.)

[0225] MGKNVNRKWKASAKMISLGLGASLVLGAGMQAQAVTVSTIAGSFHERGAIDGAGTN

[0226] ARFEFPQGILAAPDGTIYIADTGNDMIRKMNPSTKSVENVAGSDHRARYRDGVGANAR

[0227] FNNPEGMAISPDGKTLYIADSRNNMIRKIDLATKTVSTIAGHSFPSSGDGVGKEAGFETP

[0228] RGLAISPDGKTLYVADSGNNAIRKIDLATNTVTTLAGAGKLMSGSADGVGVQATFHEP

[0229] RSLAISGDGQVLYIADTRNNLIRKMVLATNSVSTLAGHPGFPGTLNGPGPDAYFYHPVS

[0230] VTIDGNKLYVADGANADIRMVDLSTGVVSTVAGATLNGGVPIAGREDGSGVEGHFQFP

[0231] GALTVYHGVLFVADTPADTIREVSF

[0232] SEQ ID NO:7(Aspergillus niger)

[0233] MKSPTILSCALPVLLAALIGGSYAQTASIPASYQYLLPPLFHGNLSEDFVSGTESAQLSL

[0234] NSALKEAQRAAFISYDKEFDEVIGQEPSAQIVSRGTYGYAYEAGIWVHDLNQVGATGEI

[0235] LGGRSVANLPQMWFTSAISSGPTYLSVLDLKTNEVTQPNLVKPHGSESFVLSNPNGGFY

[0236] YDGYVYITMLGDATHVAGLVKINPRTLETSTVLNSYYGLGLPSVDDVSIMRDKETKQT

[0237] HIFFSTLDLVPFGPPISAHASNVTIQLPNAFWSFTPDTETLRPVIPRSDVLGPNGVSIDGK

[0238] NSYLYVTDTPDHIHPTGTGFPAIFRYTLSSENGFIPSNKQLFGPPRHGVADGLKVDAMG

[0239] RVWTGEAEGVVVRNMAGKVIGIFNKEVFMGRQTPMTEMANFALAGDKLILALDAII

[0240] VVQLAESIGVAHDLVL

[0241] SEQ ID NO:8 (Acetobacter senegalensis)

[0242] MTHSLSRRLFGLSGMAGAVTVALPAYAQDDTAKAPDPADPESRERTYCYVGGYTQHG

[0243] PPGGNGEGITVFEMDPKTGVLTNLMTFTDIASPSYIALSPDKRFLYAVSEINDFDTAHSG

[0244] SVTAFSIDHATGALTKLNTVASGGADPAHLSVHPSGRYVMVANYTGGTVGVLHVLAD

[0245] GSLGRMTELLHNTGPRMPDRAADNPPGNFAISDHSSAHPHMVKADPTGKYVLVMDA

[0246] GLDRVYVWTINLDTGKLTPAAVPFTTMTPGSAPRHFEFGSDGGHKLYLLCEQDSKVVVA

[0247] DFDPQTGKIAPQQTVSSVTAHFRGSTLAAGILLSPDEHFLYVSNRLGDSLAVFQVAKDG

[0248] GLTLVDEIWTHADYGRSLMFDPSGSYLYVANQRSDSITSFRVDKTTGKISFTWDFTPIGS

[0249] PTCFEFLRVPAS

[0250] SEQ ID NO:9(Acidovorax soli)

[0251] MLGHFTRSGSAWRGLGLALLWCWGATNAAAAVPCNASELAYLGTDGVGIRAVRFDA

[0252] CAGTLALLGQPADTRKSRWLLKHPRLPVLYAAAEGAEGDGRVLAYTIDGRTGGLSLLG

[0253] EGTAGGAGTTHLWLDAPSMTLLAANFAAGTTVSSIALLPDGKPGALVSTIKATGSGPHR

[0254] RQTSPHAHGVAIDPSGRHALVADMGADRVFVYGFERSTRVLAPDDAAAPRSFQAPAGS

[0255] GPRRAVFSTTGRMVYVLSELTAEISVLRWDASSGRLAPVQTLALSSEEFAGTRSASELA

[0256] LGPDGRFLYLGNRGESQLLVYRVDAGSGELTPVQRLPSGGEAPWAFDIHPSGQWMLVA

[0257] NYRSNRVSLMRIDAASGRLSDTGVALESPAPVSVVFVN

[0258] SEQ ID NO:10 (Luteitalea pratensis)

[0259] MRPAASKTLTTRTLTRRSFVTAATGLASVAPALARGATSQASTSSGGLFAYVGTYSSPPV

[0260] DAPPGKVDLPPGNGRGIHIFRVDRTTGTLTAAAGVTDHYTSPSALVFDATGTHLYSTNAT

[0261] DLVDNGASGTVSAFTVDRTSGQLQLLNSVASGGTGPTYAASVHRGGRHLLVANYAGGSV

[0262] AVLPILPDGRLGPATDVKKTTGVVGPKRATHAPPGSFAISGHDTPHAHMIGTDPAGRFVI

[0263] VPDLGLDRIFVWAFDANAGTLSPADPPSVALPPDGPRHFAHRDGRWLYAIQEEGSTI

[0264] VLFDYDAERGRLSARQTISSLPAGYAGSNFCSGILISQDGRFVYAGNRLHDSVGIFAVGR

[0265] DGTLTFVDDIWTRGDYPRSMTIDPSGRFMYVCNQRADHVTAFAIDGATGRLAFTGQYT

[0266] PVGNPSAIAFLDTGRG

[0267] SEQ ID NO:11 (Gluconobacter oxydans)

[0268] MSHPVSRRNFAVGLVAGVAAAGTGVAGAADPEKAVPTPPPPPKPVAKTICFVGGYTKH

[0269] GPPGGGTGNGQGISVFDMDRDTGVLTPITTFTDIASPSFLAISKDQRFLYALSEIDDFNK

[0270] DGDGSVTAFAIDPKTGSLRKLNVVSSKGAVPAHLSIHHSGRYVLVANYVGGCVAVLPIR

[0271] SDGSLGEASDVVHNTGPRQPERASDNPQGNFAVSDHSGSHPHMIHSDPSGKFVLADDA

[0272] GLDRVYVWTLNIDTGKLIPAKTPYYDMEPGSAPRHFQFNHSGRILYNLCEQDSKVVVS

[0273] NFDPATGAISDIQTVSTVTSHFRGSTLAAEILISASGKFVYVSNRLGDSLAVFAIGADGTL

[0274] TLQDEVWMHADYGRALMFDPSGAFLFCANQRSDAVTSFKVDKKTGEIAFTNNFTPVG

[0275] SPTTFAFMNTQV

[0276] SEQ ID NO:12(Sulfobacillus thermosulfidooxidans)

[0277] MHRPFFRVVFTVAVSFALSACGFNPSTASSKHSAKAPASPKTSHRQSPQSKSLVPWGAT

[0278] AASSILPGDILIADSHNNRILLVTPQKKIVWQYPKPGQPSLLHDDDDVFFGPHFDEIITNQ

[0279] EGYNVISIINFKTRQVVWNYGHAGVPGSLPGYLNTPDDAFLYAKPKGDIITVADIRNQRI

[0280] LFISRKTHQILKQYGQTGVMAVNPPATYSAPNGDFPAPHGGMLVTQINGNDAMLLNRH

[0281] NQVRYTVHFPSFLSYPSDANFTPHGNIIVAFYTNPGAVVEMSPTGKVLWLYRPLSGPGR

[0282] LDRPSLAVKLPNGLVLLNDDYNDRVIVINPKTNQIVWQYGHTGIPGTAPGYLNIPDGLD

[0283] LLPPGIVPGGHNPIGHHLWSYPHNGY

[0284] SEQ ID NO:13(Leptospirillum ferrooxidans)

[0285] MNKSKHPKGRGIRSIKTAAVAVSLGIGMTLMAGQAFQAQAVSLKTIAGSFHERGDNDG

[0286] NGASARFEFPQGIVAAPDGSIYVADTGNDMIRKITMSSGTGTVETIAGVNHHARFRDGA

[0287] GAAARFNNPEGLAISPDGKTLYVADSRNNRIRKIDLATKTVSTLAGHAFASGNDGIGDH

[0288] AGFAQPRGLAISPDGKTLYIGDSGNNMIRKMDLTTLQVTTLAGQGALVPGHADGVGTQ

[0289] ATFLEPRGLAISPDGQILYIADTRNHLIRKLVLATNSVSTLAGHPGFPGTLNGPGPDAYF

[0290] YDPMMLAIDGNKLYVCDAANADLRLIDLNSDTVSTVAGATINGGVPIKGREDGDGTL

[0291] GHFEFPGAVTIYHGVLYVTDTPADTIRSVSF

[0292] SEQ ID NO:14 (Thiobacillus sp)

[0293] MNAKKLVLALLCLGCGTFAMAAPIAYIPLGSANQVIAVDAANDKIIRSYSGVENPHGLV

[0294] ATPDGDYLVAGSLKETPVPAGAPSDTPNSKLYVIHPEGHVMAEIPVSGWTHHEAVTPD

[0295] GKYVISTHPTRGGVSVVDVINNKVYKTIKTGPSPNYTLVTKDGKRAYVSNSGNGTLSEI

[0296] DLSKWEVTRSLEAGPTPEHLVFSPDEKTIYVSNDRAGTVSVVSVEQGKVEKTYKIGKR

[0297] VHGLDLGDDGKTLFISSRSDDKLVALDTQTGEQRVLTLSPEPYHLDKIPGTGKVYVSSS

[0298] KAPKIWVVDQKTFTVTDTIQLPAGEGHQIAVIR

[0299] 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.

[0300] 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 D-isoascorbic acid, characterized in that: In a system containing 2-keto-D-gluconic acid and / or its metal salt, an enzyme catalyst is contacted with 2-keto-D-gluconic acid and / or its metal salt to generate D-isoascorbic acid under acidic conditions.

2. The method according to claim 1, characterized in that The 2-keto-D-gluconic acid metal salt is a soluble salt of 2-keto-D-gluconic acid; optionally, the soluble salt is selected from the potassium salt of 2-keto-D-gluconic acid, the sodium salt of 2-keto-D-gluconic acid or the calcium salt of 2-keto-D-gluconic acid.

3. The method according to claim 1 or 2, characterized in that: The 2-keto-D-gluconic acid and / or its metal salt is selected from at least one of 2-keto-D-gluconic acid, 2-keto-D-gluconic acid hydrate, sodium 2-keto-D-gluconic acid and 2-keto-D-gluconic acid hemi-calcium salt.

4. The method according to any one of claims 1 to 3, characterized in that: The acidic condition is pH ≤ 6.0, preferably pH ≤ 5.

5.

5. The method according to any one of claims 1 to 4, characterized in that: The enzyme catalyst comprises at least one of the hydrolases with EC number EC3; 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; optionally, the lactonase is selected from at least one of the lactonases with EC numbers of 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.

6. The method according to claim 5, characterized in that The lactonase is selected from at least one of (a1) to (a5): (a1) L-arabinonolactonase from Tannerella forsythia; (a2) one or more of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.; (a3) 1,4-lactonase from Aspergillus niger; (a4) one or more of 6-phosphogluconolactonase from Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis and Gluconobacter oxydans; (a5) one or more of the lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and Thiobacillus sp.

7. The method according to claim 5 or 6, characterized in that: The lactonase is selected from any one of the following (b1) to (b3): (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

8. The method according to any one of claims 1 to 7, characterized in that: The enzyme catalyst is in the form of pure enzyme, crude enzyme, cell lysate, cell culture or whole cell; optionally, the pure enzyme, crude enzyme or whole cell is in free or immobilized form.

9. The method according to any one of claims 1 to 8, characterized in that: In the step of synthesizing D-isoascorbic acid, the reaction system contains calcium ions, and optionally, the calcium ions are provided by a soluble salt of calcium; In the step of synthesizing D-isoascorbic acid, the reaction is carried out under anaerobic conditions at 0 to 100°C.

10. Use of lactonase in the preparation of D-isoascorbic acid and / or in a catalyst for the preparation of D-isoascorbic acid, characterized in that: The lactonase is selected from at least one of (a1) to (a5): (a1) L-arabinonolactonase from Tannerella forsythia; (a2) one or more of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.; (a3) 1,4-lactonase from Aspergillus niger; (a4) one or more of 6-phosphogluconolactonase from Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis and Gluconobacter oxydans; (a5) one or more lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and Thiobacillus sp.; Optionally, the lactonase is selected from any one of the following (b1) to (b3): (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

11. A catalyst for preparing D-isoascorbic acid, wherein the catalyst comprises a lactonase as shown in at least one of (a1) to (a5): (a1) L-arabinonolactonase from Tannerella forsythia; (a2) at least one of gluconolactonase derived from Acetobacter pasteurianus, Acidobacterium capsulatum, Bryobacteraceae bacterium and Leptospirillum sp.; (a3) 1,4-lactonase from Aspergillus niger; (a4) at least one of 6-phosphogluconolactonase derived from Acetobacter senegalensis, Acidovorax soli, Luteitalea pratensis and Gluconobacter oxydans; (a5) at least one lactonase derived from Sulfobacillus thermosulfidooxidans, Leptospirillum ferrooxidans and Thiobacillus sp.; Optionally, the lactonase is selected from any one of the following (b1) to (b3): (b1) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b2) 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-D-gluconic acid and / or its metal salt to D-isoascorbic acid in one step; (b3) A polypeptide encoded by a polynucleotide encoding the polypeptide shown in (b1) or (b2).

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