Method for preparing L-guluronic acid

Abstract

The invention discloses a method for preparing L-guluronic acid, and belongs to the technical field of bioengineering and enzyme engineering. According to the present invention, galactose oxidase (GOA) is adopted to catalyze gluconic acid (Glucic acid) so as to generate the L-guluronic acid (L-guluronic acid), and the L-guluronic acid (L-guluronic acid) can be used to catalyze the L-guluronic acid (L-guluronic acid), such that the L-guluronic acid (L-guluronic acid) can be obtained; furthermore, according to the invention, the galactose oxidase from fusarium graminearum is subjected to enzyme engineering modification, the galactose oxidase mutant is provided, and the activity of the galactose oxidase mutant for catalyzing oxidation of gluconic acid to generate L-guluronic acid is obviously improved. Compared with the existing production technology, the method for producing the L-guluronic acid by using the enzyme catalysis method provided by the invention has the advantages of simple production process, environmental friendliness, high yield, easiness in large-scale preparation and the like.

Description

Technical Field

[0001] The present invention relates to a method for preparing L-guluronic acid, in particular to a method for preparing L-guluronic acid by using galactose oxidase to catalyze gluconic acid, and belongs to the technical field of bioengineering and enzyme engineering. Background Art

[0002] L-guluronic acid is mainly derived from brown algae and is one of the comonomers of alginic acid. L-guluronic acid (G2013) is a new patented drug with immunomodulatory and anti-inflammatory properties (WO2018011419). It belongs to the family of nonsteroidal anti-inflammatory drugs (NSAIDs) and can treat diseases by reducing the levels of inflammatory mediators. Currently, G2013 is being used in clinical trials for ankylosing spondylitis, rheumatoid arthritis, and multiple sclerosis, and its efficacy and safety are better than conventional treatments (Khadem Azarian S, Akhlaghi M, Mahmoudi M, et al. A randomized clinical trial for the assessment of the efficacy and safety of guluronic acid (G2013) in patients with rheumatoid arthritis [J]. Immunopharmacology and immunotoxicology, 2019, 41(1): 95-101.). G2013 can also treat non-alcoholic steatohepatitis and reduce the expression of related inflammatory factors (Tahmasebi S, Neishaboori H, Jafari D, et al. The effects of guluronic acid (G2013), a new emerging treatment, on inflammatory factors in nonalcoholic steatohepatitis patients under in vitro conditions [J]. Immunopharmacology and Immunotoxicology, 2021, 43 (5): 562-570.).In addition, G2013 can also reduce the expression of factors related to prostate cancer and tumor metastasis, thereby inhibiting the proliferation of cancer cells. Currently, clinical trials are applied to the first and second stages of breast cancer (Bagherian Z, Mirshafiey A, Mohsenzadegan M, et al. Evaluation of G2013 (α-L-guluronic acid) efficacy on PC-3 cells through inhibiting the expression of inflammatory factors [J]. Clinical and Experimental Pharmacology and Physiology, 2022, 49 (2): 254-263.).

[0003] At present, the production methods of L-guluronic acid mainly include chemical catalysis and hydrolysis. The chemical catalysis method uses Pt / C to catalyze gluconic acid to produce L-guluronic acid, but this method has poor selectivity (generating a lot of byproduct D-2-keto-gluconic acid), low yield and easy over-oxidation to produce glucaric acid (Dirkx JMH, Van Der Baan HS, Van DenBroek J M. The preparation of D-glucaric acid by the oxidation of D-gluconicacid catalysed by platinum on carbon [J]. Carbohydrate Research, 1977, 59 (1): 63-72.). The hydrolysis method includes acid hydrolysis or enzymatic hydrolysis of alginic acid to produce L-guluronic acid, but the supply of alginic acid raw materials is limited, and the hydrolysis process is difficult to control, the yield is low, the generated product is impure, and the subsequent product separation steps are complicated, resulting in a high production cost of L-guluronic acid (JP2007230902A).

[0004] Therefore, it is urgent to develop a new method for preparing L-guluronic acid with simple production process, environmental friendliness, high yield and easy scalability. Summary of the Invention

[0005] Problems to be solved by the invention

[0006] The current methods for preparing L-guluronic acid result in impure products, low yields, low production efficiency and high production costs, which are not conducive to the large-scale production of L-guluronic acid.

[0007] Solutions for solving problems

[0008] Based on the existing problems, the present invention utilizes galactose oxidase to prepare L-guluronic acid. The galactose oxidase (GOase) used in the present invention is derived from Fusarium graminearum and is a single copper metalloenzyme whose active center is composed of a cross-linked tyrosine-cysteine free radical and a copper ion, which can specifically oxidize the alcoholic hydroxyl group at position 6 of galactose to generate an aldehyde group. The present invention catalyzes gluconic acid to generate L-guluronic acid by galactose oxidase in one step. The method has the advantages of cheap raw materials, low production cost, environmental friendliness, safety to the human body, and easy scale. In addition, the present invention also obtains a mutant with significantly improved activity in catalyzing the oxidation of gluconic acid to generate L-guluronic acid by enzyme engineering of galactose oxidase.

[0009] The technical solutions of the present invention are as follows:

[0010] [1] A galactose oxidase mutant, wherein the mutant is selected from any one of the following groups (I) to (V):

[0011] (I) the galactose oxidase mutant comprises a mutation at at least one of positions 290, 329, 330, 406, 495, and 496 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2;

[0012] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0013] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0014] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0015] (b) the full-length complementary polynucleotide of (a);

[0016] (IV) a fragment of the mutant shown in any one of (I), (II) or (III), wherein the fragment still has galactose oxidase activity;

[0017] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0018] [2] The galactose oxidase mutant according to [1], wherein the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has a mutated amino acid at at least one of the following positions:

[0019] F290Y, Y329H, Y329S, Y329R, Y329P, Y329F, K330L, K330V, K330S, K330T, K330R, T406R, T406K, Y495T, Y495S.

[0020] [3] The galactose oxidase mutant according to [1] or [2], wherein the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m2) 15 ) is a mutation shown in any one of:

[0021] (m1)K330L;

[0022] (m2)K330V;

[0023] (m3)K330S;

[0024] (m4)K330T;

[0025] (m5)T406R;

[0026] (m6)T406K;

[0027] (m7)Y495S;

[0028] (m8)Y495T;

[0029] (m9)T406R, F290Y;

[0030] (m 10 )T406R, Y329H;

[0031] (m 11 )T406R, Y329S;

[0032] (m 12 )T406R, Y329R;

[0033] (m 13 )T406R, Y329P;

[0034] (m 14 )T406R, K330R;

[0035] (m 15 )T406R, K330R, Y329F;

[0036] Preferably, the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0037] (m5)T406R;

[0038] (m 12 )T406R, Y329R;

[0039] (m 14 )T406R, K330R;

[0040] (m 15 )T406R, K330R, Y329F.

[0041] [4]. An isolated polynucleotide, wherein the polynucleotide encodes the galactose oxidase mutant according to any one of [1] to [3].

[0042] [5]. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide described in [4].

[0043] [6] A recombinant host cell, wherein the recombinant host cell comprises the galactose oxidase mutant according to any one of [1] to [3], the isolated polynucleotide according to [4], or the recombinant expression vector according to [5];

[0044] Optionally, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus or Corynebacterium;

[0045] Preferably, the recombinant host cell is derived from Escherichia coli.

[0046] [7]. A cell culture comprising the recombinant host cell according to [6].

[0047] [8]. A product comprising the galactose oxidase mutant according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6], or the cell culture according to [7].

[0048] [9] A method for preparing L-guluronic acid and / or its metal salt, comprising the step of using the galactose oxidase mutant according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6], the cell culture according to [7], the product according to [8], or galactose oxidase;

[0049] Optionally, the galactose oxidase source includes but is not limited to Fusarium graminearum, Fusarium acuminatum, Fusarium thapsinum, Paenibacillus sp.;

[0050] Preferably, the galactose oxidase comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0051] Optionally, the method uses gluconic acid and / or its metal salt as a substrate;

[0052] Optionally, the substrate gluconic acid and / or its metal salt includes but is not limited to sodium gluconate, potassium gluconate and / or calcium gluconate;

[0053] Optionally, the concentration of the substrate is 20 to 200 mM, preferably 50 to 100 mM;

[0054] In the step of preparing L-guluronic acid and / or its metal salt, the reaction temperature is 20-40° C., the pH is 5.5-8.5, and the reaction time is not less than 2 hours.

[0055]

[10] Use of the galactose oxidase mutant according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6] or the cell culture according to [7] in the preparation of L-guluronic acid and / or its metal salts;

[0056] Optionally, the method uses gluconic acid and / or its metal salt as a substrate.

[0057] Effects of the Invention

[0058] 1. The present invention uses galactose oxidase to catalyze the conversion of gluconic acid to L-guluronic acid, and develops a new method for preparing L-guluronic acid that is simple, green, friendly, has high yield, uses cheap raw materials, and is easy to scale up.

[0059] 2. The present invention can achieve efficient production of L-guluronic acid by constructing a series of galactose oxidase mutants with enhanced activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the catalytic pathway for the production of L-guluronic acid from gluconic acid.

[0061] Figure 2 The specific enzymatic activities of the galactose oxidase mutants catalyzing gluconic acid oxidation are shown.

[0062] Figure 3 This is the HPLC spectrum of the product L-guluronic acid produced by the galactose oxidase mutant catalyzing gluconic acid.

[0063] Figure 4 This is a diagram for galactose oxidase saturation mutation screening. DETAILED DESCRIPTION

[0064] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0065] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0066] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0067] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0068] In this specification, 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 be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0069] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0070] 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.

[0071] As used herein, "galactose oxidase" (GOase; EC 1.1.3.9) enzymes are copper-dependent enzymes that catalyze the oxidation of primary alcohols to their corresponding aldehydes in the presence of dimolecular oxygen. They act selectively in both a regiospecific and enantiospecific manner, enabling synthetic methods requiring little or no functional group protection and producing the desired stereoisomers. The oxidation is mild and controlled, so that the activity does not lead to excessive oxidation of the alcohol to its corresponding carboxylic acid.

[0072] In this specification, "horseradish peroxidase" (HRP; EC 1.11.1.7) enzymes are iron-dependent enzymes that activate and maintain GOase catalytic activity by oxidizing the inactive redox state of the active site that occurs during the normal GOase catalytic cycle. In the examples included herein, type I HRP is specifically used in a catalytic manner, however, it is not intended to be exclusive in this role, as there are other isoforms of this enzyme class and chemical reagents that can achieve this role.

[0073] As used herein, "catalase" refers to an iron-dependent enzyme (EC 1.11.1.6) that acts on hydrogen peroxide, a byproduct of GOase oxidation, which renders GOase inactive above a specific level of hydrogen peroxide. In the examples herein, catalase is specifically used as a catalytic maintenance enzyme, but in some embodiments, it can be replaced by other methods, such as electrochemical decomposition of hydrogen peroxide.

[0074] Throughout this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0075] As used herein, the term "wild-type" refers to an object found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used herein, "naturally occurring" and "wild-type" are synonyms.

[0076] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0077] As used herein, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In the present invention, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to a "deletion."

[0078] In some embodiments, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can maintain the normal function of the protein. A representative example of a conservative mutation is a conservative substitution.

[0079] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0080] As used herein, the terms "sequence identity" or "percent identity" in the context of comparing two nucleic acids or polypeptides means that they are identical or have a specified percentage of identical sequences when compared and aligned for maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or as measured by visual inspection. In other words, nucleotide or amino acid sequence identity can be defined as the ratio of the number of identical nucleotides or amino acids to the total number of nucleotides or amino acids in the aligned portions when two or more nucleotide or amino acid sequences are aligned to maximize the number of identical nucleotides or amino acids, adding gaps as needed.

[0081] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0082] 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.

[0083] As used herein, the term "expression vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences for expressing a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. A recombinant expression vector may include, for example, i) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcriptional and translational initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, the expression vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK or pQlink series, etc., which can be replicated and expressed in prokaryotic cells.

[0084] In this specification, the term "host cell" means any cell type that is easily transformed, transfected, transduced, etc. with a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector comprising the present invention. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the polynucleotide or recombinant expression vector encoding the mutant polypeptide is introduced, and the recombinant host cell is specifically achieved by transformation. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell refers to a prokaryotic cell, and specifically, the host cell is derived from a microorganism of the genus Escherichia, the genus Bacillus, or the genus Corynebacterium. In some preferred embodiments, the host cell is derived from the genus Escherichia, more preferably Escherichia coli, including Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Trans T1, Escherichia coli MC1061, Escherichia coli BL21, etc.

[0085] In this specification, the term "cell culture" refers to a combination of cells and a cell culture medium, wherein the cells are cultured in the cell culture medium outside a living organism.

[0086] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0087] In this specification, "conversion" may also refer to the enzymatic conversion (or bioconversion) of substrate(s) into corresponding product(s). "Percent conversion" refers to the percentage of substrate converted to product over a period of time under specified conditions. Thus, the "enzyme activity" or "activity" of a galactose oxidase polypeptide can be expressed as the "percent conversion" of substrate to product over a specific period of time.

[0088] In this specification, "cultivation" refers to growing a microbial cell population under any suitable conditions (for example, using a liquid, gel or solid culture medium), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0089] In this specification, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0090] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] The technical solution of the present invention is described in detail below:

[0092] <First Aspect>

[0093] In a first aspect of the present invention, a galactose oxidase mutant is provided, wherein the mutant is selected from any one of the following groups consisting of (I) to (IV):

[0094] (I) the galactose oxidase mutant comprises a mutation at at least one of positions 290, 329, 330, 406, 495, and 496 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2;

[0095] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0096] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0097] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0098] (b) the full-length complementary polynucleotide of (a);

[0099] (IV) a fragment of the mutant shown in any one of (I), (II) or (III), wherein the fragment still has galactose oxidase activity;

[0100] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0101] In some specific embodiments, the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions:

[0102] F290Y, Y329H, Y329S, Y329R, Y329P, Y329F, K330L, K330V, K330S, K330T, K330R, T406R, T406K, Y495T, Y495S.

[0103] Furthermore, the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m 15 ) is a mutation shown in any one of:

[0104] (m1) K330L (R1-1 in the embodiment of the present invention);

[0105] (m2) K330V (R1-2 in the embodiment of the present invention);

[0106] (m3) K330S (R1-3 in the embodiment of the present invention);

[0107] (m4) K330T (R1-4 in the embodiment of the present invention);

[0108] (m5) T406R (R1-5 in the embodiment of the present invention);

[0109] (m6) T406K (R1-6 in the embodiment of the present invention);

[0110] (m7) Y495S (R1-7 in the embodiments of the present invention);

[0111] (m8) Y495T (R1-8 in the embodiments of the present invention);

[0112] (m9) T406R, F290Y (R1-9 in the embodiments of the present invention);

[0113] (m 10 ) T406R, Y329H (R1-10 in the embodiment of the present invention);

[0114] (m 11 ) T406R, Y329S (R1-11 in the embodiment of the present invention);

[0115] (m 12 ) T406R, Y329R (R1-12 in the embodiment of the present invention);

[0116] (m 13 ) T406R, Y329P (R1-13 in the embodiment of the present invention);

[0117] (m 14 ) T406R, K330R (R1-14 in the embodiment of the present invention);

[0118] (m 15 )T406R, K330R, Y329F (R1-15 in the embodiments of the present invention).

[0119] In some preferred embodiments, the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0120] (m5)T406R;

[0121] (m 12 )T406R, Y329R;

[0122] (m 14 )T406R, K330R;

[0123] (m 15 )T406R, K330R, Y329F.

[0124] In some specific embodiments, the galactose oxidase mutant is prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. In some exemplary embodiments, the galactose oxidase mutant is prepared as a lyophilized powder, in powder form (e.g., acetone powder), or as an enzyme solution. In some preferred embodiments, the galactose oxidase mutant is in the form of a substantially pure preparation.

[0125] <Second Aspect>

[0126] In a second aspect of the present disclosure, an isolated polynucleotide is provided, wherein the polynucleotide encodes the galactose oxidase mutant as described in the first aspect of the present disclosure.

[0127] The polynucleotides disclosed herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0128] The polynucleotide encoding the mutant of the present disclosure includes: a coding sequence encoding only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and non-coding sequences.

[0129] <Third Aspect>

[0130] In the third aspect of the present disclosure, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the polynucleotide described in the second aspect of the present disclosure.

[0131] In some embodiments, the polynucleotide of the second aspect is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide.

[0132] <Fourth Aspect>

[0133] In the fourth aspect of the present disclosure, a recombinant host cell is provided, wherein the recombinant host cell comprises the galactose oxidase mutant described in the first aspect of the present disclosure, the isolated polynucleotide described in the second aspect of the present disclosure, or the recombinant expression vector described in the third aspect of the present disclosure.

[0134] In some embodiments, an expression vector containing a heterologous polynucleotide encoding a galactose oxidase mutant polypeptide is introduced into an appropriate host cell to express the corresponding galactose oxidase mutant polypeptide.

[0135] In some optional embodiments, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus or Corynebacterium;

[0136] In some preferred embodiments, the recombinant host cell is derived from Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis;

[0137] In some more preferred embodiments, the recombinant host cell is derived from Escherichia coli.

[0138] <Fifth Aspect>

[0139] In a fifth aspect of the present disclosure, a cell culture comprising the recombinant host cell according to the fourth aspect of the present disclosure is provided.

[0140] <Sixth Aspect>

[0141] In the sixth aspect of the present invention, a product is provided, which comprises the galactose oxidase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, and the cell culture described in the fifth aspect.

[0142] In some optional embodiments, the product may include enzyme preparations, bacterial preparations, kits, etc.

[0143] <Seventh Aspect>

[0144] In the seventh aspect of the present invention, a method for preparing L-guluronic acid and / or its metal salt is provided, comprising the steps of utilizing the galactose oxidase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, the product described in the sixth aspect or the galactose oxidase.

[0145] In some optional embodiments, the source of the galactose oxidase includes but is not limited to Fusarium graminearum, Fusarium acuminatum, Fusarium thapsinum, Paenibacillus sp., and the like.

[0146] In some preferred embodiments, the galactose oxidase comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0147] In some exemplary embodiments, the L-guluronic acid and / or its metal salt can be L-guluronic acid sodium salt, L-guluronic acid potassium salt, L-guluronic acid calcium salt, etc.

[0148] In some optional embodiments, the method uses gluconic acid and / or its metal salt as a substrate.

[0149] In some optional embodiments, the substrate gluconic acid and / or its metal salts include but are not limited to sodium gluconate, potassium gluconate and / or calcium gluconate.

[0150] In some optional embodiments, the concentration of the substrate is 20-200 mM, preferably 50-100 mM.

[0151] In the step of preparing L-guluronic acid and / or its metal salt, catalase is also included.

[0152] In the step of preparing L-guluronic acid and / or its metal salt, the reaction temperature is 15-50°C, preferably 20-35°C, more preferably 25-30°C; the pH is 5.5-8.5, preferably 6.5-8.0, more preferably 7.0-7.5.

[0153] In the step of preparing L-guluronic acid and / or its metal salt, the reaction time is not less than 2 hours, for example, it can be 2 hours, 3 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc., preferably not less than 4 hours, more preferably not less than 6 hours. The reaction time is based on the concentration of the product L-guluronic acid in the reaction system no longer changes.

[0154] <Eighth Aspect>

[0155] In the seventh aspect of the present invention, provided is the use of the galactose oxidase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, the product described in the sixth aspect, or the galactose oxidase in the preparation of L-guluronic acid and / or its metal salts.

[0156] In some optional embodiments, the source of the galactose oxidase includes but is not limited to Fusarium graminearum, Fusarium acuminatum, Fusarium thapsinum, Paenibacillus sp., and the like.

[0157] In some preferred embodiments, the galactose oxidase comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0158] In some exemplary embodiments, the L-guluronic acid and / or its metal salt can be L-guluronic acid sodium salt, L-guluronic acid potassium salt, L-guluronic acid calcium salt, etc.

[0159] In some optional embodiments, the method uses gluconic acid and / or its metal salt as a substrate.

[0160] In some optional embodiments, the substrate gluconic acid and / or its metal salts include but are not limited to sodium gluconate, potassium gluconate and / or calcium gluconate.

[0161] Example

[0162] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0163] The materials and instruments used in the examples of the present invention are as follows:

[0164] Horseradish peroxidase (HRP), Shanghai Merrill, product number: M86891;

[0165] 2,2-Azobis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS), Aladdin product number: A109612;

[0166] Catalase, Maclean, product number: C804187;

[0167] Gluconic acid, Beijing Dehang Wuzhou Technology, product number: 8206010100;

[0168] L-guluronic acid, Shanghai Yuanye Biotechnology, product number: B25952;

[0169] Microplate reader, model: Infinite 200PRO9M Nano;

[0170] High performance liquid chromatograph, model: LC-20ADXR.

[0171] Vector construction method for galactose oxidase mutants: Mutation primers were designed using homologous recombination. PCR amplification was performed using the pET22b vector containing the GOase-encoding gene (encoding the amino acids shown in SEQ ID NO: 1) as a template. The PCR reaction system consisted of 15 μL of 2× PrimerStar mixture, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 0.5 μL of template DNA, and 12.5 μL of double-distilled water. PCR amplification conditions were: pre-denaturation at 98°C for 2 minutes, followed by 25 cycles of 98°C for 40 seconds, 53°C for 20 seconds, and 72°C for 2 minutes, followed by a final incubation at 72°C for 10 minutes. The PCR product was digested with Dpn I at 37°C for 1 hour and then purified using a PCR product purification kit. The purified PCR product was ligated for homologous recombination using the ClonExpress II OneStep Cloning Kit (Vazyme). 7 μL of PCR product was mixed with 2 μL of CE II buffer and 1 μL of recombinase Exnase II and incubated at 37°C for 30 minutes. The ligation product was directly transformed into competent Escherichia coli BL21 cells, plated onto antibiotic-containing LB solid medium plates, and incubated inverted at 37°C overnight.

[0172] Expression and screening of galactose oxidase mutants: Single colonies from the mutant library were inoculated into 1 mL of LB liquid medium containing antibiotics in a 96-well plate and cultured overnight in a high-throughput shaker at 37°C, 800 rpm. The overnight culture was transferred to 1 mL of TB liquid medium containing antibiotics in a 96-well plate at a 1% inoculum volume and cultured in a high-throughput shaker at 30°C, 800 rpm until the OD 600 nm =0.6-0.8, 0.2 mM IPTG and 0.5 mM CuSO4 were added, and the cells were induced at 25°C, 600 rpm, and cultured for 18 hours. The cells were harvested using a high-speed centrifuge, resuspended in buffer (100 mM PBS, pH 7.0) plus 0.5 mg / mL lysozyme, and shaken in a high-throughput shaker at 37°C, 800 rpm for 2 hours to lyse the cells. The supernatant was collected by high-speed centrifugation, and the crude enzyme solution was then used for screening using the HRP-ABTS colorimetric assay (the reaction generates hydrogen peroxide, and HRP catalyzes both hydrogen peroxide and ABTS to generate green ABTS free radicals).

[0173] Expression and purification of galactose oxidase mutants after buffer optimization: The excellent mutants finally screened were expanded and cultured, and the bacteria were collected and resuspended in optimized buffer (100mM Tris-HCl, pH 7.4, 150mM NaCl, 0.5mMCuSO4) plus 0.5mg / mL lysozyme. The bacterial cells were lysed using a high-pressure homogenizer, and the supernatant was collected by centrifugation and purified by nickel column. The GOase mutant protein was obtained after buffer (100mM PBS, pH 7.0) replacement.

[0174] Galactose oxidase mutant activity assay: High-throughput screening: 20 μL of crude enzyme solution was added to a 200 μL reaction solution of 100 mM PBS, pH 7.0, containing 50 mM gluconic acid, 0.02 mg / mL HRP, and 1 mM ABTS at room temperature. The reaction solution was observed for color change and absorbance was measured at 420 nm. Mutants that changed color faster and had a higher absorbance than the wild-type were screened for further rescreening. Pure enzyme was added to a 200 μL reaction solution of 100 mM PBS, pH 7.0, containing 100 mM gluconic acid, 0.05 mg / mL HRP, and 2 mM ABTS at 30°C. The increase in ABTS radical absorbance was immediately monitored at 420 nm. The molar absorptivity coefficient ζ = 36 mmol / L / cm.

[0175] Detection method for the product of gluconic acid catalyzed by the galactose oxidase mutant: HPLC detection using an Agilent MetaCarb 87H, 300×7.8 mm column and 5 mM H2SO4 as the mobile phase at 35°C, 0.5 mL / min, and detection at UV 210 nm.

[0176] Example 1: Wild-type galactose oxidase catalyzes the production of L-guluronic acid from gluconic acid

[0177] 5 μg of pure GOase (SEQ ID NO: 1) was added to 200 μL of 100 mM PBS, pH 7.0 reaction solution (the reaction solution contained 100 mM gluconic acid, 0.05 mg / mL HRP, and 2 mM ABTS). The increase in the absorbance of ABTS free radicals was immediately monitored at 420 nm at 30°C, and the specific enzyme activity of GOase catalyzing gluconic acid was measured to be 0.05 U / mg.

[0178] 20 mg of pure GOase was added to 1 mL of 100 mM PBS, pH 7.0 (containing 10 g / L gluconic acid and 1 mg / mL catalase) and reacted at room temperature. The product, L-guluronic acid, was detected by high-performance liquid chromatography (HPLC). The column used was an Agilent MetaCarb 87H, 300 × 7.8 mm, with a mobile phase of 5 mM H₂SO₄, a flow rate of 0.5 mL / min, a column oven temperature of 35°C, and a collection time of 20 minutes. L-guluronic acid production was detectable after 24 hours of reaction.

[0179] Example 2: Rational design and modification of galactose oxidase

[0180] Using the above-mentioned GOase as the starting protein, mutant R1 (GOase-W290F-R330K-Q406T, shown in SEQ ID NO: 2) was obtained through literature review and previous combined mutagenesis. R1 and gluconic acid were molecularly docked using Autodock computing software. After analysis, three sites 330, 406, and 495 were selected for site-directed mutagenesis. Using the vector pET22b containing the R1 encoding gene as shown in SEQ ID NO: 2 as a template, PCR amplification was performed to construct an expression vector for the R1 mutant. After screening, mutants that were superior to R1 were obtained. After protein expression, the mutant proteins were obtained, and the specific enzyme activity of the mutants in oxidizing gluconic acid was measured. After determination, the optimal mutant obtained in this round was R1-T406R.

[0181] Table 1

[0182] name mutation site Amino acid before mutation Amino acid after mutation Specific enzyme activity (U / mg) Amino acid sequence R1 none - - 0.15 SEQ ID NO:2 R1-1 330 K L 0.16 SEQ ID NO:3 R1-2 330 K V 0.18 SEQ ID NO:4 R1-3 330 K S 0.19 SEQ ID NO:5 R1-4 330 K T 0.17 SEQ ID NO:6 R1-5 406 T R 0.70 SEQ ID NO:7 R1-6 406 T K 0.45 SEQ ID NO:8 R1-7 495 Y S 0.20 SEQ ID NO:9 R1-8 495 Y T 0.19 SEQ ID NO: 10

[0183] Example 3: Site-directed saturation mutagenesis of galactose oxidase

[0184] The mutant R1-5 was used as the starting protein to analyze the protein structure. The Autodock molecule was used to dock R1-5 and gluconic acid, and gluconic acid was selected to be located in the active center. Amino acids within the range (a total of 11 amino acids) were subjected to site-directed saturation mutagenesis, such as Figure 4 After screening, mutants with superior performance to R1-5 were obtained. These mutant proteins were expressed and their specific enzymatic activity in gluconic acid oxidation was determined. The superior mutants obtained in this round of screening were R1-T406R-Y329R and R1-T406R-K330R.

[0185] Table 2

[0186] name mutation site Amino acid before mutation Amino acid after mutation Specific enzyme activity (U / mg) Amino acid sequence R1-5 none - - 0.70 SEQ ID NO:7 R1-9 290 F Y 1.24 SEQ ID NO:11 R1-10 329 Y H 1.56 SEQ ID NO:12 R1-11 329 Y S 1.69 SEQ ID NO:13 R1-12 329 Y R 2.04 SEQ ID NO:14 R1-13 329 Y P 1.76 SEQ ID NO:15 R1-14 330 K R 1.90 SEQ ID NO:16

[0187] Example 4: Iterative saturation mutagenesis of galactose oxidase

[0188] In the previous round of screening, two better mutants R1-12 and R1-14 were screened, and the 329 site had the most mutants. Therefore, in this round, R1-14 was used as the starting protein to perform site-directed saturation mutagenesis on sites 290 and 329. Figure 4 . After screening, a mutant superior to R1-14 was obtained, and the mutant protein was obtained by protein expression, and the specific enzyme activity of the mutant in oxidizing gluconic acid was determined. The specific enzyme activity of the mutant R1-15 (R1-T406R-K330R-Y329F) with improved catalytic gluconic acid activity was 3.82U / mg. R1, R1-5 (R1-T406R), R1-14 (R1-T406R-K330R) and R1-15 (R1-T406R-K330R-Y329F) were expressed and purified according to the expression and purification steps of the galactose oxidase mutants after the above-mentioned buffer optimization, and the enzyme activity was determined. Finally, the optimal specific activity was determined to be 7.75U / mg, which was 0.7 times higher than that of mutant R1-14, 2.2 times higher than that of mutant R1-5, and 11.6 times higher than that of R1 ( Figure 2 The amino acid sequence of the mutant R1-T406R-K330R-Y329F is shown in SEQ ID NO: 17.

[0189] Example 5: Galactose oxidase mutant R1-5 oxidizes gluconic acid to produce L-guluronic acid

[0190] Pure enzyme was incubated in 1 mL of 100 mM PBS, pH 7.0, containing 10 g / L gluconic acid, 1 mg / mL catalase, and 15 mg of enzyme. The reaction was carried out at room temperature in a metal bath at 600 rpm. Samples were taken at 0, 2, 4, 6, 8, 10, and 12 hours. High-performance liquid chromatography (HPLC) was used to determine the amount of L-guluronic acid produced. The column used was an Agilent MetaCarb 87H, 300 × 7.8 mm, with a mobile phase of 5 mM H₂SO₄, a flow rate of 0.5 mL / min, a column oven temperature of 35°C, and a collection time of 20 minutes. Complete conversion of gluconic acid to L-guluronic acid occurred within 8 hours of the reaction.

[0191] Example 6: Galactose oxidase mutant R1-14 oxidizes gluconic acid to produce L-guluronic acid

[0192] Pure enzyme was incubated in 1 mL of 100 mM PBS, pH 7.0, containing 10 g / L gluconic acid, 1 mg / mL catalase, and 10 mg of enzyme. The reaction was carried out at room temperature in a metal bath at 600 rpm. Samples were taken at 0, 2, 4, 6, 8, 10, and 12 hours. The amount of L-guluronic acid produced was determined by high-performance liquid chromatography (HPLC). The column used was an Agilent MetaCarb 87H, 300 × 7.8 mm, with a mobile phase of 5 mM H₂SO₄, a flow rate of 0.5 mL / min, a column oven temperature of 35°C, and a collection time of 20 minutes. Complete conversion of gluconic acid to L-guluronic acid occurred within 6 hours of the reaction.

[0193] Example 7: Galactose oxidase mutant R1-15 oxidizes gluconic acid to produce L-guluronic acid

[0194] The pure enzyme was added to 1 mL of 100 mM PBS, pH 7.0 reaction solution, containing 10 g / L gluconic acid, 1 mg / mL catalase, and 5 mg of enzyme. The reaction was carried out at room temperature and a metal bath at 600 rpm. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. The amount of L-guluronic acid produced was detected by high performance liquid chromatography. The chromatographic column used was Agilent MetaCarb 87H, 300 × 7.8 mm, the mobile phase was 5 mM H2SO4, the flow rate was 0.5 ml / min, the column oven temperature was 35 ° C, and the acquisition time was 20 min. The reaction completely converted gluconic acid to L-guluronic acid within 6 h ( Figure 3 ).

[0195] SEQ ID NO: 1

[0196] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSWSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0197] SEQ ID NO:2

[0198] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0199] SEQ ID NO:3

[0200] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYLSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0201] SEQ ID NO:4

[0202] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYVSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0203] SEQ ID NO:5

[0204] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYSSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0205] SEQ ID NO:6

[0206] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYTSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0207] SEQ ID NO:7

[0208] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0209] SEQ ID NO:8

[0210] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYKDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0211] SEQ ID NO:9

[0212] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRASHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0213] SEQ ID NO:10

[0214] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYTDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRATHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0215] SEQ ID NO:11

[0216] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSYSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0217] SEQ ID NO:12

[0218] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLHKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0219] SEQ ID NO:13

[0220] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLSKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0221] SEQ ID NO:14

[0222] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLRKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0223] SEQ ID NO:15

[0224] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLPKSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0225] SEQ ID NO:16

[0226] ASAPIGSAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTYTIDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVPAAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPSTGIVSDRTVTVTKHDMFCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGRVFTIGGSFSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKGKILTFGGSPDYRDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPDGSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVKVGGRITISTDSSISKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGVPSVASTIRVTQ*

[0227] SEQ ID NO:17

[0228] *

[0229] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0230] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A galactose oxidase mutant, wherein: The mutant is selected from any one of the following groups (I) to (V): (I) the galactose oxidase mutant comprises a mutation at at least one of positions 290, 329, 330, 406, 495, and 496 corresponding to the sequence of SEQ ID NO: 2, compared to the sequence of SEQ ID NO: 2; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2; (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions: (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I); (b) the full-length complementary polynucleotide of (a); (IV) a fragment of the mutant shown in any one of (I), (II) or (III), wherein the fragment still has galactose oxidase activity; (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

2. The galactose oxidase mutant according to claim 1, wherein The galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions: F290Y, Y329H, Y329S, Y329R, Y329P, Y329F, K330L, K330V, K330S, K330T, K330R, T406R, T406K, Y495T, Y495S.

3. The galactose oxidase mutant according to claim 1 or 2, wherein The galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m2) 15 ) is a mutation shown in any one of: (m1)K330L; (m2)K330V; (m3)K330S; (m4)K330T; (m5)T406R; (m6)T406K; (m7)Y495S; (m8)Y495T; (m9)T406R, F290Y; (m 10 )T406R、Y329H; (m 11 )T406R、Y329S; (m 12 )T406R、Y329R; (m 13 )T406R、Y329P; (m 14 )T406R、K330R; (m 15 )T406R、K330R、Y329F; Preferably, the galactose oxidase mutant corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations: (m5)T406R; (m 12 )T406R、Y329R; (m 14 )T406R、K330R; (m 15 )T406R、K330R、Y329F。 4. An isolated polynucleotide, wherein The polynucleotide encodes the galactose oxidase mutant according to any one of claims 1 to 3.

5. A recombinant expression vector, wherein: The recombinant expression vector comprises the polynucleotide according to claim 4.

6. A recombinant host cell, wherein The recombinant host cell comprises the galactose oxidase mutant according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, or the recombinant expression vector according to claim 5; Optionally, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus or Corynebacterium; Preferably, the recombinant host cell is derived from Escherichia coli.

7. A cell culture comprising the recombinant host cell of claim 6.

8. A product comprising the galactose oxidase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, or the cell culture according to claim 7.

9. A method for preparing L-guluronic acid and / or its metal salt, wherein: The method comprises the steps of using the galactose oxidase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, the cell culture according to claim 7, the product according to claim 8, or the galactose oxidase; Optionally, the galactose oxidase source includes but is not limited to Fusarium graminearum, Fusarium acuminatum, Fusarium thapsinum, Paenibacillus sp.; Preferably, the galactose oxidase comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; Optionally, the method uses gluconic acid and / or its metal salt as a substrate; Optionally, the substrate gluconic acid and / or its metal salt includes but is not limited to sodium gluconate, potassium gluconate and / or calcium gluconate; Optionally, the concentration of the substrate is 20 to 200 mM, preferably 50 to 100 mM; In the step of preparing L-guluronic acid and / or its metal salt, the reaction temperature is 20-40° C., the pH is 5.5-8.5, and the reaction time is not less than 2 hours.

10. Use of the galactose oxidase mutant according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, or the cell culture according to claim 7 in the preparation of L-guluronic acid and / or its metal salts; Optionally, the method uses gluconic acid and / or its metal salt as a substrate.

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