Aldohexose-2-epimerase mutant and its use

Abstract

The present application provides an aldohexose-2-epimerase mutant and its use. The aldohexose-2-epimerase mutant comprises one or more mutations based on a reference sequence, the amino acid sequence of the reference sequence is shown in SEQ ID NO: 1, and can convert aldohexose, for example, can convert D-glucose into D-mannose or convert D-mannose into D-glucose, and has a relatively high specific enzyme activity. Compared with the wild type, the specific enzyme activity of converting D-glucose into D-mannose is increased by about 2.2 times.

Description

Technical Field

[0001] The present application relates to the field of enzyme engineering technology, and in particular to an aldohexose-2-epimerase mutant and a use thereof. Background Art

[0002] As a sucrose substitute, functional sugar has broad prospects in the fields of food, pharmaceuticals and health care due to its unique physiological functions. D-mannose is one of the functional sugars, which has prebiotic function and can also enhance wound healing and avoid bacterial infection. D-mannose can directly bind to the pili of Escherichia coli, Enterococcus faecalis and Staphylococcus aureus that cause urinary tract infection, causing them to fall off the urethral wall, thereby effectively reducing the recurrence probability of urinary tract infection.

[0003] At present, the preparation of D-mannose mainly includes chemical synthesis and bioenzyme method. The production cost of chemical synthesis method is high, and a large amount of acidic waste liquid is easily generated during the production process to pollute the environment. In addition, the reaction byproducts make it difficult to separate and prepare high-purity D-mannose. The bioenzyme method has mild reaction conditions, few byproducts, easy separation and purification, no pollution to the environment and low cost. Therefore, the preparation of D-mannose by bioenzyme method has become a future research trend.

[0004] Specifically for the bioenzymatic method, mannose isomerase is currently used to act on fructose, aldose-ketose isomerase is used to act on glucose or fructose, and cellobiose 2-epimerase is used to act on glucose through a side reaction to prepare mannose. However, fructose, whether as a substrate residue or a byproduct, will have an adverse effect on the subsequent separation and purification of mannose. It can be seen that the low-cost and efficient bioenzymatic method for preparing D-mannose needs to be further developed. Summary of the invention

[0005] In response to the technical problems existing in the prior art, the present application provides an aldohexose-2-epimerase mutant and its use. The aldohexose-2-epimerase mutant provided by the present application can convert aldohexose, for example, it can convert D-glucose into D-mannose or convert D-mannose into D-glucose.

[0006] The specific technical solutions of this application are as follows:

[0007] 1. A mutant of aldohexose-2-epimerase, wherein the mutant of aldohexose-2-epimerase comprises one or more mutations based on a reference sequence, and the amino acid sequence of the reference sequence is shown in SEQ ID NO: 1.

[0008] 2. The aldohexose-2-epimerase mutant according to item 1, wherein the amino acid sequence of the mutant comprises an amino acid mutation corresponding to at least one of G354 and T355 of SEQ ID NO: 1, preferably comprises an amino acid mutation corresponding to G354 and T355 of SEQ ID NO: 1.

[0009] 3. An aldohexose-2-epimerase mutant comprising the amino acid sequence of any one of SEQ ID NOs: 2-4 or the amino acid sequence of any one of SEQ ID NOs: 2-4.

[0010] 4. The aldohexose-2-epimerase mutant according to any one of items 1 to 3, wherein the aldohexose is glucose or mannose.

[0011] 5. A nucleic acid molecule encoding the aldohexose-2-epimerase mutant according to any one of items 1 to 4.

[0012] 6. The nucleic acid molecule according to item 5, wherein the nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NOs: 5-7 or a sequence as shown in any one of SEQ ID NOs: 5-7.

[0013] 7. An expression vector comprising the nucleic acid molecule described in item 5 or 6.

[0014] 8. The expression vector according to item 7, wherein the expression vector is a plasmid, a cosmid, a phage or a viral vector, preferably a plasmid.

[0015] 9. A host cell comprising the expression vector according to item 7 or 8;

[0016] Preferably, the host cell is a eukaryotic cell, a prokaryotic cell or a bacterial cell, preferably a prokaryotic cell, and more preferably Escherichia coli.

[0017] 10. Use of the aldohexose-2-epimerase mutant according to any one of items 1 to 4 in aldohexose conversion.

[0018] 11. A method for converting aldohexose, comprising using the aldohexose-2-epimerase mutant according to any one of items 1 to 4 to catalyze an aldohexose reaction to convert the aldohexose.

[0019] 12. The method according to item 11, wherein the reaction temperature is 20-55°C, preferably 30-45°C; and / or

[0020] The pH value of the reaction is 6.5-10, preferably 7.5-8.5; and / or

[0021] The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0022] Effects of the Invention

[0023] The aldohexose-2-epimerase mutant described in the present application can convert aldohexose, for example, it can convert D-glucose into D-mannose or convert D-mannose into D-glucose. Due to its high specificity and good stability, it can convert the relatively cheap raw material D-glucose into D-mannose, significantly reducing the production cost of D-mannose while achieving green production of "reducing emissions and reducing consumption". Compared with the wild type, the mutant has an enzyme activity of about 2.2 times higher in converting D-glucose into D-mannose, and under the same conditions, the conversion rate of D-glucose into D-mannose is increased by about 3.6 times to 22.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the conversion rate of glucose when glucose is converted to mannose using wild-type and mutant aldohexose-2-epimerase.

[0025] Figure 2 Schematic diagram showing the change in glucose conversion rate as a function of reaction time when glucose is converted to mannose using an aldohexose-2-epimerase mutant. DETAILED DESCRIPTION

[0026] The present application is described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully communicated to those skilled in the art.

[0027] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0028] definition

[0029] As used herein, the "dimerase" refers to an enzyme that catalyzes the configurational change of an asymmetric carbon atom in a monosaccharide molecule (containing more than two asymmetric carbon atoms). In the present application, the dimerase refers to an enzyme that isomerizes aldohexose. In the present application, the aldohexose is glucose or mannose. The dimerase described in the present application can convert glucose into mannose or convert mannose into glucose.

[0030] As used herein, the "homology", "identity" and "similarity" refer to the sequence similarity between two nucleic acid molecules. "Homology", "identity" or "similarity" can be determined by comparing the positions in each sequence, and the sequences can be aligned for comparison purposes. When the equivalent position in the compared sequence is occupied by the same base, the molecules are identical at that position; when the equivalent site is occupied by the same or similar amino acid (e.g., similar in spatial properties or charged properties) residue, the molecules can be referred to as being homologous (similar) at that position. The expression of homology / similarity or identity percentage refers to a function of the number of identical or similar amino acids at the position shared by the compared sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, preferably less than 25% identity with the sequences of the present application. When comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of extra residues also reduces identity and homology / similarity. In a specific embodiment, two or more sequences or subsequences are considered to be substantially or significantly homologous, similar or identical if their sequences are about 60%, or about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical over a specified region, as determined using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below or by manual alignment and visual inspection provided online by, for example, the National Center for Biotechnology Information (NCBI). This definition also relates to or can be used to test the complement of a sequence. Thus, to the extent permitted by the context of the present invention, for example, if the nucleotide sequence can be predicted to occur naturally in a DNA duplex, or can occur naturally in the form of one or both of the complementary strands, then the nucleotide sequence that is complementary to a specified target sequence or a variant thereof is itself considered to be "similar" to the target sequence, and when referring to "similar" nucleic acid sequences, single-stranded sequences, their complementary sequences, double-stranded strand complexes, sequences that can encode the same or similar polypeptide products, and any allowed variants of any of the above. Circumstances in which similarity must be limited to the analysis of a single nucleic acid strand sequence can include, for example, the detection and quantification of the expression of a specific RNA sequence or coding sequence in a cell. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions.In embodiments, the identity or similarity can be over a region that is at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, 24, 25, or more nucleotides in length, or over a region that is more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than about 100 nucleotides in length.

[0031] As used herein, the term "nucleotide" refers to naturally occurring nucleotides, as well as synthetic nucleotide analogs that are recognized by cellular enzymes.

[0032] As used herein, the term "expression vector" refers to any natural or artificially constructed expression vector containing a nucleic acid molecule, wherein the nucleic acid molecule can be catalyzed by a cellular transcriptase and / or translation enzyme. Exemplary expression vectors include: plasmids, viruses (including bacteriophages), cosmids, artificial chromosomes or transposons, etc. In some embodiments, the expression vector is a plasmid.

[0033] As used herein, the term "host cell" refers to any biological cell that can be cultured in a culture medium and used to express a recombinant gene. These host cells can be eukaryotic or prokaryotic cells, or microorganisms such as bacterial cells, or cells derived from a cell line (such as a mammalian immortal cell line), etc. In some embodiments, the host cell is a prokaryotic cell, such as Escherichia coli.

[0034] As used herein, the term "recombinant" refers to a nucleic acid molecule or polypeptide that is in a non-naturally occurring environment and is produced by human intervention.

[0035] Aldohexose-2-epimerase mutant

[0036] The present application provides an aldohexose-2-epimerase mutant, wherein the aldohexose-2-epimerase mutant comprises one or more mutations based on a reference sequence, and the amino acid sequence of the reference sequence is shown in SEQ ID NO: 1.

[0037] The sequence of SEQ ID NO: 1 is as follows:

[0038] MDRFARENLTEWKRILAYWEKFSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASKFKPALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPWNKSQNTHLHIIEAFTNLY RVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEFDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFGTVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK

[0039] The sequence is a protein encoded by the Runella slithyformis-derived gene Runsl_4586, named RsAE (Runella slithyformis aldohexose 2-epimerase), which is classified as the N-acylglucosamine 2-epimerase (AGE) protein family in the database.

[0040] In some embodiments, the amino acid sequence of the mutant comprises an amino acid mutation corresponding to at least one of G354 and T355 of SEQ ID NO:1, preferably comprises an amino acid mutation corresponding to G354 and T355 of SEQ ID NO:1.

[0041] In the present application, the above-mentioned sites are counted from the N-terminus, for example, G354 refers to the amino acid mutation at the 354th amino acid from the N-terminus of SEQ ID NO: 1.

[0042] The term "corresponding to" has the meaning generally understood by those skilled in the art. Specifically, "corresponding to" means a position in one sequence that corresponds to a specified position in another sequence after the two sequences are aligned for homology or sequence identity.

[0043] In the present application, the glycine G at position 354 can be mutated to glutamine Q or the threonine T at position 355 can be mutated to glutamic acid E, or the glycine G at position 354 can be mutated to glutamine Q and the threonine T at position 355 can be mutated to glutamic acid E; preferably, the glycine G at position 354 can be mutated to glutamine Q and the threonine T at position 355 can be mutated to glutamic acid E.

[0044] The amino acid sequence in which glycine G at position 354 is mutated to glutamine Q is shown in SEQ ID NO: 2;

[0045] The amino acid sequence in which the threonine T at position 355 is mutated to glutamic acid E is shown in SEQ ID NO: 3;

[0046] The amino acid sequence in which the glycine G at position 354 is mutated to glutamine Q and the threonine T at position 355 is mutated to glutamic acid E is shown in SEQ ID NO:4.

[0047] The sequence of SEQ ID NO:2 is as follows:

[0048] MDRFARENLTEWKRILAYWEKFSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASKFKPALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPWNKSQNTHLHIIEAFTNLY RVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEFDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFQTVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK

[0049] The sequence of SEQ ID NO:3 is as follows:

[0050] MDRFARENLTEWKRILAYWEKFSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASKFKPALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPWNKSQNTHLHIIEAFTNLYRVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEFDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFGEVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK

[0051] The sequence of SEQ ID NO:4 is as follows:

[0052] MDRFARENLTEWKRILAYWEKFSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASKFKPALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPWNKSQNTHLHIIEAFTNLYRVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEFDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFQEVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK

[0053] Those skilled in the art will also understand that the mutant is not limited to the specific sequences listed above. The sequence of the mutant should include a sequence that contains one, two or three or more nucleotide mutations compared to the sequence shown in any one of SEQ ID NOs: 2-4, but is still substantially functionally the same as the sequence, and also includes a sequence that has 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 2-4.

[0054] The present application does not impose any restrictions on the method for preparing mutants, which can be mutated according to conventional methods in the art, such as using directed mutagenesis, random mutagenesis or the construction of synthetic oligonucleotides, and then expressing the mutated DNA sequence in a host cell to obtain a mutant with substitution, insertion and / or deletion of the amino acid sequence. In the present application, based on the 3D structure and function analysis of the enzyme, the mutation is carried out by using the method of site-directed mutagenesis.

[0055] The present application provides an aldohexose-2-epimerase mutant, which comprises an amino acid sequence as described in any one of SEQ ID NOs: 2 to 4 or an amino acid sequence as described in any one of SEQ ID NOs: 2 to 4. In some embodiments, the aldohexose is glucose or mannose.

[0056] Nucleic acid molecules, expression vectors and host cells

[0057] The present application provides a nucleic acid molecule comprising a nucleic acid encoding any of the above-mentioned aldohexose-2-epimerase mutants. In some embodiments, the nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NOs: 5-7 or a sequence as shown in any one of SEQ ID NOs: 5-7.

[0058] In some embodiments, the nucleotide sequence encoding the above-mentioned aldohexose-2-epimerase mutant is codon optimized. Generally, codon optimization involves balancing the percentage of selected codons with the abundance of disclosed human transfer RNAs so that none of them is overloaded or limited. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Codon usage differences between transfected genes and host cells may affect protein expression and immunogenicity of nucleic acid constructs. Generally, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Generally, the redundancy of amino acid codons makes different codons encode a kind of amino acid. In some embodiments, when selecting a codon for replacement, it may be necessary that the resulting mutation is a silent mutation so that the codon changes do not affect the amino acid sequence. Generally, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence.

[0059] The sequence of SEQ ID NO: 5 is as follows:

[0060]

[0061] The sequence of SEQ ID NO: 6 is as follows:

[0062]

[0063] The sequence of SEQ ID NO: 7 is as follows:

[0064]

[0065] The present application provides an expression vector comprising the nucleic acid molecule described above. In some embodiments, the expression vector is a plasmid, a cosmid, a phage or a viral vector, preferably a plasmid.

[0066] For example, the nucleic acid molecules encoding the above-mentioned aldohexose-2-epimerase mutants can be cloned into suitable expression vectors, which can be any suitable recombinant expression vectors and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. In some embodiments, the expression vector is a plasmid.

[0067] In the present application, the expression vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, taking into account whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter, and a promoter found in the long terminal repeat sequence of a mouse stem cell virus, and other promoters known to the skilled person are also contemplated.

[0068] The present application provides a host cell, which comprises the expression vector described above.

[0069] In the present application, the expression vector is transformed into a host cell to further express or clone in the host cell. In some embodiments, a method for preparing an aldohexose-2-epimerase mutant is provided, the method comprising culturing a host cell comprising a nucleic acid encoding an aldohexose-2-epimerase mutant as provided above under conditions suitable for expressing the aldohexose-2-epimerase mutant, and optionally recovering the aldohexose-2-epimerase mutant from the host cell (or host cell culture medium). In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell or a bacterial cell, preferably a prokaryotic cell, more preferably an Escherichia coli.

[0070] The host cell refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to parent cells in nucleic acid content, but may contain mutations.

[0071] Use of aldohexose-2-epimerase mutant and method for converting aldohexose

[0072] The present application provides use of any of the above-mentioned aldohexose-2-epimerase mutants in aldohexose conversion.

[0073] In the present application, the conversion refers to the conversion between aldohexoses, for example, when the aldohexose is D-glucose, it can be converted into D-mannose, and when the aldohexose is D-mannose, it can be converted into D-glucose.

[0074] The present application provides a method for converting aldohexose, comprising using any of the above-described aldohexose-2-epimerase mutants to catalyze an aldohexose reaction to convert aldohexose. In some embodiments, the reaction temperature is 20-55°C, preferably 30-45°C; and / or

[0075] The pH value of the reaction is 6.5-10, preferably 7.5-8.5; and / or

[0076] The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0077] For example, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, etc.;

[0078] The pH value of the reaction can be 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc.;

[0079] The reaction time can be 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, etc.

[0080] In some embodiments, the concentration of aldohexose is 10-100 g / L.

[0081] For example, the concentration of aldohexose can be 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, etc.

[0082] The aldohexose-2-epimerase mutant described in the present application has high specificity. When the aldohexose is glucose, it can convert glucose into mannose, and when the aldohexose is mannose, it can convert mannose into glucose. The enzyme activity of the aldohexose-2-epimerase mutant is high. According to calculation and analysis, 1 mg of the aldohexose-2-epimerase mutant (G354Q+T355E) contains about 13 enzyme activity units, which is about 2.2 times higher than that of the wild type. The mutant can increase the conversion rate of D-glucose to D-mannose, for example, the mutant (G354Q+T355E) can increase the conversion rate by about 3.6 times.

[0083] Example

[0084] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0085] Example 1 Preparation of gene cloning and expression of wild-type and mutant RsAE (Runs1_4586)

[0086] The gene fragment Runs1_4586 of R. slithyformis (DSM 19594) is numbered as AEI50906.1 in GenBank, and it encodes a protein with a full-length 390 amino acid sequence. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment of Runs1_4586 (wild type) and the gene fragment of the mutant, and inserted into the restriction enzyme cutting sites NdeI and NotI of the pET22b plasmid, and finally the expression plasmid was transformed into Escherichia coli BL21.

[0087] The recombinant E. coli was inoculated into a test tube containing 5 mL of LB liquid medium and cultured overnight at 25°C and 150 rpm. The next day, the seed solution was transferred to a 500 mL shake flask containing 90 mL of fresh LB liquid medium at a 10% inoculation rate, and cultured on a shaker at 37°C and 150 rpm. When the bacterial concentration OD600 reached 0.6-0.8, the shaker temperature was adjusted to 28°C, IPTG was added at a final concentration of 0.5 mM, and expression and enzyme production were induced at 28°C and 150 rpm for 5 hours.

[0088] The collected bacteria were re-dissolved and disrupted by high pressure using phosphate buffer (pH 7.8), and then the supernatant was collected by centrifugation. Finally, the RsAE enzyme (wild type and mutant) was purified by Ni column affinity adsorption.

[0089] Example 2 Identification of Enzyme Activity of RsAE Wild Type and Mutants

[0090] 1ml reaction system (50mM phosphate buffer (pH 7.8)), substrate glucose concentration is 10g / L, 0.15mg enzyme prepared in Example 1 (wild type (WT), mutant (W198H), mutant (G354Q) and mutant (G354Q+T355E) of RsAE) are added respectively, and the reaction is carried out at 37°C for 120min. After the reaction is completed, the reaction is terminated by boiling water bath for 10min. The catalytic solution after the termination reaction is centrifuged at 13000rpm for 1min, and the supernatant is taken to prepare a high-pressure liquid chromatography test sample, and the amount of mannose generated in the reaction system is determined, wherein the chromatographic conditions of the high-pressure liquid chromatography are: Agilent Agilent1260 high-performance liquid chromatograph, Pheromon MARS Mca 5u 300X 7.8mm chromatographic column, detector temperature is 35°C, column temperature is 80°C, mobile phase is pure water; flow rate is 0.5ml / min, injection volume is 10μl, and the results are as follows Figure 1 shown.

[0091] from Figure 1 It can be seen that under the above reaction conditions, the conversion rates of the wild type (WT), mutant (W198H), mutant (G354Q) and mutant (G354Q+T355E) of RsAE from D-glucose substrate to D-mannose were 6.3%, 4.8%, 19.5% and 22.6%, respectively, indicating that the introduction of the mutation W198H reduced the enzyme activity of RsAE in converting D-glucose substrate to D-mannose, while the introduction of the mutations G354Q and T355E significantly increased the enzyme activity.

[0092] Example 3 Determination of enzyme activity of the reaction of converting D-glucose to D-mannose by RsAE mutant (G354Q+T355E)

[0093] 1ml reaction system (50mM phosphate buffer (pH 7.8)), substrate glucose concentration of 10g / L, 0.03mg RsAE mutant prepared in Example 1 was added, and the reaction was carried out at 37°C for 0.5hr, 1hr, 2hr, 3hr, 4hr, 5hr and 6hr respectively. After the reaction, the reaction was terminated by boiling water bath for 10min. HPLC test sample preparation and high pressure liquid chromatography conditions were the same as in Example 2. The reaction progress curve of RsAE mutant (G354Q+T355E) converting D-glucose to D-mannose is shown in Figure 2 The enzyme activity is defined as: 1U of enzyme is required to produce 1μmol of mannose in 1min at 37℃.

[0094] from Figure 2 And through computational analysis, 1 mg of RsAE mutant (G354Q+T355E) contains about 13 units of enzyme activity, i.e. 13U / mg enzyme.

[0095] Example 4 Enzyme kinetics of the reaction of converting D-glucose to D-mannose by RsAE mutant (G354Q+T355E)

[0096] 1ml reaction system (50mM phosphate buffer (pH 7.8)), substrate glucose concentrations of 10g / L, 20g / L, 40g / L, 60g / L, 80g / L and 100g / L, respectively, 0.03mg of the RsAE mutant (G354Q+T355E) prepared in Example 1 was added respectively, and the reaction was carried out at 37°C for 60min. After the reaction was completed, the reaction was terminated by boiling water bath for 10min. The HPLC test sample preparation and high pressure liquid chromatography conditions were the same as those in Example 2. The measured data were calculated and analyzed by Lineweaver-Burk equation (double reciprocal plotting method), and the Km of the reaction of converting D-glucose to D-mannose by RsAE mutant (G354Q+T355E) was about 440mM, and the Kcat was about 10s -1 .

[0097] The above is only the preferred embodiment of the present application, and does not limit the present application in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present application without departing from the technical solution of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A mutant of aldohexose-2-epimerase, the amino acid sequence of which is shown in SEQ ID NO: 2 or SEQ ID NO:

4. 2 . The aldohexose-2-epimerase mutant according to claim 1 , wherein the aldohexose is glucose.

3. A nucleic acid molecule encoding the aldohexose-2-epimerase mutant according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, wherein The nucleotide sequence thereof is shown in SEQ ID NO: 6 or 7. An expression vector comprising the nucleic acid molecule according to claim 3 or 4.

6. The expression vector according to claim 5, wherein The expression vector is a plasmid, a cosmid or a viral vector.

7. The expression vector according to claim 6, wherein The viral vector is a bacteriophage. The expression vector according to claim 5 , wherein the expression vector is a plasmid.

9. A host cell comprising the expression vector according to any one of claims 5 to 8.

10. The host cell according to claim 9, wherein the host cell is a eukaryotic cell or a prokaryotic cell. The host cell according to claim 9 , wherein the host cell is a prokaryotic cell.

12. The host cell of claim 11, wherein the prokaryotic cell is a bacterial cell.

13. The host cell according to claim 12, wherein the bacterial cell is Escherichia coli.

14. Use of the aldohexose-2-epimerase mutant according to any one of claims 1 to 2 in converting glucose into mannose.

15. A method for converting glucose into mannose, comprising using the aldohexose-2-epimerase mutant according to any one of claims 1 to 2 to catalyze a glucose reaction to convert glucose into mannose.

16. The method according to claim 15, wherein the reaction temperature is 30-45°C; and / or The pH of the reaction is 7.5-8.5; and / or The reaction time is 0.5-6 hours.

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