Aldohexose-2-epimerase or mutant thereof and use thereof

Abstract

Provided are an aldohexose-2-epimerase or a mutant thereof and a use thereof. The aldohexose-2-epimerase comprises a sequence of any one of SEQ ID NOs: 1-5 or a sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 1-5, or the aldohexose-2-epimerase mutant comprises one or more mutations based on a reference sequence, wherein the amino acid sequence of the reference sequence is as shown in SEQ ID NO: 5. The aldohexose-2-epimerase or the mutant thereof can convert aldohexose, for example, converting D-glucose into D-mannose or D-mannose into D-glucose. Due to high specificity and good stability, the aldohexose-2-epimerase or the mutant thereof can convert the relatively inexpensive raw material D-glucose into D-mannose, significantly reducing the production cost of D-mannose while achieving green production with "emission reduction and energy savings".

Description

Aldohexose-2-epimerase or its mutant, and use thereof Technical Field

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

[0002] Functional sugars, as sucrose substitutes, hold broad promise in the food, pharmaceutical, and healthcare sectors due to their unique physiological properties. D-mannose, a functional sugar, possesses prebiotic properties and can enhance wound healing and prevent bacterial infections. D-mannose can directly bind to the pili of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus, causing them to detach from the urethral wall, effectively reducing the recurrence of UTIs.

[0003] Currently, the preparation of D-mannose mainly includes chemical synthesis and bioenzymatic methods.

[0004] Chemical synthesis methods are costly, and the process easily generates large amounts of acidic wastewater, polluting the environment. Furthermore, reaction byproducts make the isolation and preparation of high-purity D-mannose difficult. Currently, mannose can be produced by isomerizing glucose under alkaline conditions, but this method suffers from poor yields and a high refining burden due to the decomposition of the sugar. Separately, the production of mannose by isomerizing fructose under alkaline conditions not only faces similar yield and refining burden issues, but also suffers from the problem of fructose remaining in the reaction system, making it difficult to separate from the mannose.

[0005] The bioenzymatic method has mild reaction conditions, few by-products, easy separation and purification, no pollution to the environment, and low cost. Therefore, the use of bioenzymatic methods to prepare D-mannose has become a future research trend.

[0006] Enzymatic methods currently typically utilize mannose isomerase acting on fructose, aldose-ketose isomerase acting on glucose or fructose, and cellobiose 2-epimerase acting on glucose via a side reaction to produce mannose. However, fructose, whether as a substrate residue or a byproduct, can adversely affect the subsequent separation and purification of mannose. Therefore, further development of low-cost, efficient enzymatic methods for the production of D-mannose is needed. Summary of the Invention

[0007] In response to the technical problems existing in the prior art, the present application provides an aldohexose-2-epimerase or a mutant thereof and its use. The aldohexose-2-epimerase or a mutant thereof provided in the present application can convert aldohexose, for example, it can convert D-glucose into D-mannose or convert D-mannose into D-glucose.

[0008] The specific technical solutions of this application are as follows:

[0009] 1. An aldohexose-2-epimerase or a mutant thereof, wherein the aldohexose-2-epimerase comprises a sequence of any one of SEQ ID NOs: 1-5 or a sequence having at least 90% identity to a sequence of any one of SEQ ID NOs: 1-5; or

[0010] 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:5.

[0011] 2. The aldohexose-2-epimerase or a mutant thereof 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: 5, preferably comprises an amino acid mutation corresponding to G354 and T355 of SEQ ID NO: 5.

[0012] 3. An aldohexose-2-epimerase mutant comprising an amino acid sequence as described in any one of SEQ ID NOs: 6 to 8 or an amino acid sequence having at least 90% identity to an amino acid sequence as described in any one of SEQ ID NOs: 6 to 8.

[0013] 4. The aldohexose-2-epimerase or a mutant thereof according to any one of items 1 to 3, wherein the aldohexose is glucose or mannose.

[0014] 5. A nucleic acid molecule encoding the aldohexose-2-epimerase or a mutant thereof according to any one of items 1 to 4.

[0015] 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: 9-16 or a sequence having at least 90% identity to a sequence as shown in any one of SEQ ID NOs: 9-16.

[0016] 7. An expression vector comprising the nucleic acid molecule according to item 5 or 6.

[0017] 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.

[0018] 9. A host cell comprising the expression vector according to item 7 or 8;

[0019] Preferably, the host cell is a eukaryotic cell, a prokaryotic cell or a bacterial cell, preferably a prokaryotic cell, and more preferably Escherichia coli.

[0020] 10. Use of the aldohexose-2-epimerase or a mutant thereof according to any one of items 1 to 4 in aldohexose conversion.

[0021] 11. A method for converting aldohexose, comprising using the aldohexose-2-epimerase or a mutant thereof according to any one of items 1 to 4 to catalyze an aldohexose reaction to convert the aldohexose.

[0022] 12. The method according to item 11, wherein the reaction temperature is 20-55°C, preferably 30-45°C; and / or

[0023] The pH value of the reaction is 6.5-10, preferably 7.5-8.5; and / or

[0024] The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0025] Effects of the Invention

[0026] The aldohexose-2-epimerase or its 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 "reduced emissions and reduced consumption".

[0027] The aldohexose-2-epimerase described in the present application is used for the conversion between monosaccharides (aldohexose), that is, converting D-glucose into D-mannose or converting D-mannose into D-glucose, and the aldohexose-2-epimerase has a relatively high activity in converting aldohexose. Among them, the enzyme activity of using the enzyme to convert D-glucose into D-mannose is about 6 U / mg, which can greatly improve the efficiency of converting D-glucose to produce D-mannose.

[0028] Compared with the wild type (aldohexose-2-epimerase), the mutant has an approximately 2.2-fold increase in the specific enzymatic activity of converting D-glucose into D-mannose, and under the same conditions, the conversion rate of D-glucose into D-mannose is increased by approximately 3.6-fold to 22.6%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a graph showing changes in glucose conversion rate over time when glucose is converted into mannose using aldohexose-2-epimerase.

[0030] FIG2 is a graph showing how the conversion rate of glucose changes with reaction temperature when glucose is converted into mannose using aldohexose-2-epimerase.

[0031] FIG3 is a graph showing how the conversion rate of glucose changes with the reaction pH when glucose is converted into mannose using aldohexose-2-epimerase.

[0032] FIG4 is a graph showing the conversion rate of glucose when glucose is converted to mannose using wild-type and mutant aldohexose-2-epimerase.

[0033] FIG5 is a graph showing changes in glucose conversion rate over reaction time when glucose is converted into mannose using an aldohexose-2-epimerase mutant. DETAILED DESCRIPTION

[0034] 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 fully convey the scope of the present application to those skilled in the art.

[0035] 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 difference in the functions of the 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 this application shall be as defined by the attached claims.

[0036] definition

[0037] As used herein, the "epimerase" refers to an enzyme that catalyzes the configurational change of an asymmetric carbon atom in a monosaccharide molecule (containing two or more asymmetric carbon atoms). In the present application, the epimerase refers to an enzyme that isomerizes aldohexose. In the present application, the aldohexose is glucose or mannose. The epimerase described in the present application can convert glucose into mannose or convert mannose into glucose.

[0038] 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 the purpose of comparison. When the equivalent position in the compared sequences 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 (for example, similar in spatial properties or charged properties) residue, the molecules can be said to be 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 when compared and aligned for maximum correspondence over a comparison window or designated 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, for example, as provided online by 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 herein, a nucleotide sequence that is complementary to a specified target sequence or a variant thereof is itself considered "similar" to the target sequence, for example, if the nucleotide sequence can be predicted to occur naturally in a DNA duplex, or can occur naturally as one or both of the complementary strands, and references to "similar" nucleic acid sequences include single-stranded sequences, their complementary sequences, double-stranded complexes, sequences that are capable of encoding the same or similar polypeptide products, and any permitted variants of any of the foregoing. Instances where similarity must be limited to analysis of a single nucleic acid strand sequence can include, for example, the detection and quantification of 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.

[0039] As used herein, the term "nucleotide" refers to naturally occurring nucleotides, as well as synthetic nucleotide analogs that are recognized by cellular enzymes.

[0040] 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 transcription and / or translation enzyme. Exemplary expression vectors include: plasmids, viruses (including bacteriophages), cosmids, artificial chromosomes, or transposons. In some embodiments, the expression vector is a plasmid.

[0041] 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 an immortalized mammalian cell line). In some embodiments, the host cell is a prokaryotic cell, such as Escherichia coli.

[0042] 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.

[0043] Aldohexose-2-epimerase

[0044] The present application provides an aldohexose-2-epimerase comprising a sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 1-5 or a sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 1-5.

[0045] The aldohexose-2-epimerase comprises a sequence that is at least 90% identical to the sequence of any one of SEQ ID NOs: 1-5, for example, can comprise a sequence that is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to the sequence of any one of SEQ ID NOs: 1-5.

[0046] In this application, the sequence of SEQ ID NO: 1 is as follows:

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

[0048] GPSWEXVDDLILSZMPWNKSQN, where X is T, P, or L, and Z is K or R

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

[0050] The sequence of SEQ ID NO:4 is as follows:

[0051] KGDWBGTVKPDLTPVRNAKOSFWKCPYHNSRACYE, wherein B is F or Y, and O is V or I.

[0052] The sequence of SEQ ID NO:5 is as follows:

[0053] Those skilled in the art will also understand that the aldohexose-2-epimerase is not limited to the specific sequences listed above. The sequences of the mutants should include sequences that contain one, two, or three or more mutations compared to the sequences shown in any one of SEQ ID NOs: 1-5, but are still substantially functionally identical thereto, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to the sequences shown in any one of SEQ ID NOs: 1-5.

[0054] In some embodiments, the aldohexose-2-epimerase comprises a sequence as set forth in any one of SEQ ID NOs: 1 to 5 or a sequence as set forth in any one of SEQ ID NOs: 1 to 5. In some embodiments, the aldohexose is glucose or mannose, preferably D-glucose or D-mannose.

[0055] Aldohexose-2-epimerase mutant

[0056] 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: 5.

[0057] The sequence is a protein encoded by the Runella slithyformis-derived gene Runs1_4586, named RsAE (Runella slithyformis aldohexose 2-epimerase), which is classified in the database as a member of the N-acylglucosamine 2-epimerase (AGE) protein family.

[0058] 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: 5, preferably comprises an amino acid mutation corresponding to G354 and T355 of SEQ ID NO: 5.

[0059] 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: 5.

[0060] The term "corresponding" has the meaning generally understood by those skilled in the art. Specifically, "corresponding" refers to a position in one sequence that corresponds to a specified position in the other sequence after the two sequences are aligned for homology or sequence identity.

[0061] In the present application, the glycine G at position 354 in SEQ ID NO: 5 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.

[0062] The amino acid sequence in which glycine G at position 354 is mutated to glutamine Q is shown in SEQ ID NO: 6;

[0063] The amino acid sequence in which the threonine T at position 355 is mutated to the glutamic acid E is shown in SEQ ID NO: 7;

[0064] The amino acid sequence in which glycine G at position 354 is mutated to glutamine Q and threonine T at position 355 is mutated to glutamic acid E is shown in SEQ ID NO:8.

[0065] The sequence of SEQ ID NO:6 is as follows:

[0066] The sequence of SEQ ID NO:7 is as follows:

[0067] The sequence of SEQ ID NO:8 is as follows:

[0068] Those skilled in the art will also understand that the mutants are not limited to the specific sequences listed above. The sequences of the mutants should include sequences that contain one, two, or three or more nucleotide mutations compared to any one of SEQ ID NOs: 6-8, but are still substantially functionally identical thereto, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to any one of SEQ ID NOs: 6-8.

[0069] This application does not impose any restrictions on the method for preparing mutants. Mutations can be performed according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or the construction of synthetic oligonucleotides, and then the mutated DNA sequence is expressed in a host cell to obtain mutants with substitutions, insertions, and / or deletions in the amino acid sequence. In this application, mutations were performed using site-directed mutagenesis based on the 3D structure and function analysis of the enzyme.

[0070] The present application provides an aldohexose-2-epimerase mutant comprising an amino acid sequence as described in any one of SEQ ID NOs: 6 to 8 or an amino acid sequence as described in any one of SEQ ID NOs: 6 to 8. In some embodiments, the aldohexose is glucose or mannose.

[0071] Nucleic acid molecules, expression vectors and host cells

[0072] The present application provides a nucleic acid molecule encoding any of the above-described aldohexose-2-epimerases. In some embodiments, the nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NOs: 9-16 or a sequence having at least 90% identity to a sequence as shown in any one of SEQ ID NOs: 9-16.

[0073] In some embodiments, the nucleotide sequence encoding the above-mentioned aldohexose-2-epimerase or its 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 restricted. 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 change does not affect the amino acid sequence. Generally, the last nucleotide of a codon can remain unchanged and will not affect the amino acid sequence.

[0074] The sequence of SEQ ID NO: 9 is as follows:

[0075] The sequence of SEQ ID NO: 10 is as follows:

[0076] The sequence of SEQ ID NO: 11 is as follows:

[0077] The sequence of SEQ ID NO: 12 is as follows:

[0078] The sequence of SEQ ID NO: 13 is as follows:

[0079] The sequence of SEQ ID NO: 14 is as follows:

[0080] The sequence of SEQ ID NO: 15 is as follows:

[0081] The sequence of SEQ ID NO: 16 is as follows:

[0082] The present application provides an expression vector comprising the nucleic acid molecule described above. In some embodiments, the expression vector is a plasmid, cosmid, phage or viral vector, preferably a plasmid.

[0083] For example, the nucleic acid molecules encoding the above-mentioned aldohexose-2-epimerase or its mutants can be cloned into a suitable expression vector, which can be any suitable recombinant expression vector 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.

[0084] 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 into which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), 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, SV40 promoter, RSV promoter, and a promoter found in the long terminal repeat sequence of the mouse stem cell virus, and other promoters known to those skilled in the art are also contemplated.

[0085] The present application provides a host cell comprising the expression vector described above.

[0086] 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 aldohexose-2-epimerase or its mutant is provided, the method comprising culturing a host cell comprising a nucleic acid encoding aldohexose-2-epimerase or its mutant as provided above under conditions suitable for expressing aldohexose-2-epimerase or its mutant, and optionally recovering aldohexose-2-epimerase or its 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 Escherichia coli.

[0087] The term "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 the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.

[0088] Use of aldohexose-2-epimerase or its mutant and method for converting aldohexose

[0089] The present application provides use of any of the above-mentioned aldohexose-2-epimerase or a mutant thereof in aldohexose conversion.

[0090] 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.

[0091] The present application provides a method for converting aldohexose, comprising using any of the above-described aldohexose-2-epimerase or a mutant thereof to catalyze an aldohexose reaction to convert aldohexose. In some embodiments, the reaction temperature is 20-55°C, preferably 30-45°C; and / or

[0092] The pH value of the reaction is 6.5-10, preferably 7.5-8.5; and / or

[0093] The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0094] For example, the reaction temperature is 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, etc.;

[0095] The pH value of the reaction can be 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc.;

[0096] 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.

[0097] In some embodiments, the concentration of aldohexose is 10-100 g / L.

[0098] 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.

[0099] The aldohexose-2-epimerase or its mutant described in the present application has high specificity. When the aldohexose is D-glucose, it can convert D-glucose into D-mannose, and when the aldohexose is D-mannose, it can convert D-mannose into D-glucose. In addition, the aldohexose-2-epimerase has high enzymatic activity. According to computational analysis, the specific enzyme activity of the enzyme in catalyzing the isomerization of D-glucose to D-mannose is approximately 6 enzyme activity units / mg enzyme, which can greatly improve the efficiency of converting D-glucose to produce D-mannose.

[0100] In addition, the enzyme activity of the aldohexose-2-epimerase mutant is high. Through calculation and analysis, 1 mg of the aldohexose-2-epimerase mutant (G354Q+T355E) contains approximately 13 enzyme activity units, which is about 2.2 times higher than the wild type. The mutant can also 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.

[0101] Example

[0102] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0103] Example 1 Aldohexose-2-epimerase

[0104] Example 1 Gene cloning and expression preparation of RsAE (Runs1_4586)

[0105] The R. slithyformis (DSM 19594) gene fragment Runs1_4586, accession number AEI50906.1 in GenBank, encodes a protein with a full-length 390 amino acid sequence (SEQ ID NO: 5). Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the Runs1_4586 gene fragment (SEQ ID NO: 10) and insert it into the pET22b plasmid between the NdeI and NotI restriction enzyme sites. The expression plasmid was then transformed into Escherichia coli BL21 to generate recombinant E. coli.

[0106] 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 to obtain seed liquid. The next day, the seed liquid was transferred to a 500 mL shake flask containing 90 mL of fresh LB liquid medium at a 10% inoculum volume and cultured on a shaker at 37°C and 150 rpm until the bacterial concentration OD 600 When the pH value reached 0.6-0.8, the shaking temperature was adjusted to 28°C, IPTG was added at a final concentration of 0.5 mM, and the expression and enzyme production were induced at 28°C and 150 rpm for 5 hours.

[0107] The collected bacteria were re-dissolved in phosphate buffer (pH 7.8) and disrupted under high pressure, then the supernatant was collected by centrifugation and finally purified by Ni column affinity adsorption to obtain aldohexose-2-epimerase RsAE (SEQ ID NO: 5).

[0108] Example 2 Identification of Enzyme Activity of RsAE

[0109] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)), a test substrate (as shown in Table 1) at a concentration of 10 g / L was added 0.03 mg of the RsAE obtained in Example 1. The reaction was incubated at 37°C for 60 min. After completion of the reaction, the reaction was terminated in a boiling water bath for 10 min. The catalytic solution after the termination reaction was centrifuged at 13,000 rpm for 1 min, and the supernatant was used to prepare an HPLC test sample. The amount of mannose produced in the reaction system was determined using high-performance liquid chromatography, as shown in Table 1. The chromatographic conditions were: Agilent 1260 HPLC, Phenomenon MARS Mca 5u 300 x 7.8 mm column, detector temperature 35°C, column temperature 80°C, mobile phase pure water, flow rate 0.5 ml / min, injection volume 10 μl.

[0110] Table 1

[0111] As can be seen from Table 1, when the substrates catalyzed by RsAE are D-glucose and D-mannose, the reactions can generate specific products D-mannose and D-glucose, respectively, but cannot catalyze other substrates, indicating that the enzyme described in this application has specificity.

[0112] Example 3 Determination of enzyme activity of RsAE in the reaction of converting D-glucose to D-mannose

[0113] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) with a substrate glucose concentration of 10 g / L, 0.03 mg of RsAE prepared in Example 1 was added. The reaction was incubated at 37°C for 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr, respectively. The reaction was terminated by 10 min in a boiling water bath. HPLC test sample preparation and high-pressure liquid chromatography conditions were the same as in Example 2. The reaction progress curve for the conversion of D-glucose to D-mannose by RsAE is shown in Figure 1, where enzyme activity is defined as 1 U of enzyme required to produce 1 μmol of mannose in 1 min at 37°C.

[0114] From the relationship between glucose conversion rate and reaction time in FIG1 and through calculation and analysis, it was found that 1 mg of RsAE contained approximately 6 enzyme activity units, i.e., 6 U / mg of enzyme.

[0115] Example 4 Study on the performance of the enzyme RsAE in converting D-glucose to D-mannose

[0116] (1) Enzyme kinetics studies

[0117] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) with a substrate glucose concentration of 10 g / L to 100 g / L, 0.03 mg of the RsAE obtained in Example 1 was added, and the reaction was incubated at 37°C for 60 min. After completion of the reaction, the reaction was terminated by placing the mixture in a boiling water bath for 10 min. HPLC test sample preparation and high-pressure liquid chromatography conditions were the same as in Example 2. The measured data were calculated and analyzed using the Lineweaver-Burk equation (double reciprocal plotting method), and the Km for the conversion of D-glucose to D-mannose by RsAE was approximately 440 mM, and the Kcat was approximately 5 s -1 .

[0118] (2) Thermal stability of RsAE

[0119] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) containing 10 g / L glucose, 0.03 mg of the RsAE obtained in Example 1 was added. The reaction was carried out at 30°C, 40°C, 50°C, and 60°C for 120 min, followed by terminating in a boiling water bath for 10 min. HPLC test sample preparation and HPLC conditions were the same as in Example 2. The relationship between the calculated glucose substrate conversion and reaction temperature is shown in Figure 2, indicating that RsAE exhibits excellent stability below 40°C.

[0120] (3) Optimal pH value of RsAE

[0121] A 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) was prepared at pH values ​​of 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. The substrate glucose concentration was 10 g / L. 0.03 mg of RsAE was added, and the reaction was incubated at 37°C for 120 min. The reaction was terminated by a 10-min boiling water bath. HPLC test sample preparation and high-pressure liquid chromatography conditions were the same as in Example 2. The calculated relationship between glucose substrate conversion and reaction system pH is shown in Figure 3. The optimal pH for the conversion of D-glucose to D-mannose by RsAE is around 8.0.

[0122] Example 2 Mutants of Aldohexose-2-Epimerase

[0123] Example 5 Preparation of gene cloning and expression of wild type (SEQ ID NO: 5) and mutants of RsAE (Runs1_4586)

[0124] RsAE enzymes (wild type and mutants) were obtained in the same manner as in Example 1.

[0125] Example 6 Identification of Enzyme Activity of RsAE Wild Type and Mutants

[0126] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) with a substrate glucose concentration of 10 g / L, 0.15 mg of each of the enzymes prepared in Example 5 (wild type (WT), mutant (W198H), mutant (G354Q), and mutant (G354Q+T355E) of RsAE) were added. The reaction was incubated at 37° C. for 120 min, and then terminated in a boiling water bath for 10 min. The terminated catalytic solution was centrifuged at 13,000 rpm for 1 min, and the supernatant was used to prepare a high-pressure liquid chromatography (HPLC) test sample, and the amount of mannose produced in the reaction system was determined. The HPLC conditions were: an Agilent 1260 HPLC, a Pheromones MARS Mca 5u 300×7.8 mm column, a detector temperature of 35° C., a column temperature of 80° C., a mobile phase of pure water, a flow rate of 0.5 ml / min, and an injection volume of 10 μl. The results are shown in FIG4 .

[0127] As can be seen from Figure 4, 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 W198H mutation reduced the enzyme activity of RsAE in converting D-glucose substrate to D-mannose, while the introduction of the G354Q and T355E mutations significantly increased the enzyme activity.

[0128] Example 7 Determination of enzyme activity in the reaction of converting D-glucose to D-mannose by RsAE mutant (G354Q+T355E)

[0129] To a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) with a substrate glucose concentration of 10 g / L, 0.03 mg of the RsAE mutant prepared in Example 5 was added. The reaction was incubated at 37°C for 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr, respectively. The reaction was terminated by 10 min in a boiling water bath. HPLC analysis was performed using the same sample preparation and HPLC conditions as in Example 6. The reaction progress curve for the conversion of D-glucose to D-mannose by the RsAE mutant (G354Q+T355E) is shown in Figure 5. Enzyme activity is defined as 1 U of enzyme required to produce 1 μmol of mannose in 1 min at 37°C.

[0130] From Figure 5 and through computational analysis, 1 mg of the RsAE mutant (G354Q+T355E) contains approximately 13 units of enzyme activity, ie, 13 U / mg enzyme.

[0131] Example 8 Enzyme Kinetics Study on the Conversion of D-Glucose to D-Mannose by RsAE Mutant (G354Q+T355E)

[0132] 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 5 was added, and the reaction was incubated at 37°C for 60min. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10min. The HPLC test sample preparation and high pressure liquid chromatography conditions were the same as in Example 6. The measured data were calculated and analyzed by the Lineweaver-Burk equation (double reciprocal plotting method), and the Km of the reaction of converting D-glucose to D-mannose by the RsAE mutant (G354Q+T355E) was approximately 440mM, and the Kcat was approximately 10s -1 .

[0133] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. An aldohexose-2-epimerase or a mutant thereof, wherein the aldohexose-2-epimerase comprises the sequence described in any one of SEQ ID NO: 1-5 or a sequence having at least 90% identity with the sequence described in any one of SEQ ID NO: 1-5; or the aldohexose-2-epimerase mutant comprises one or more than two mutations based on a reference sequence, and the amino acid sequence of the reference sequence is as shown in SEQ ID NO:

5.

2. The aldohexose-2-epimerase or a mutant thereof according to claim 1, wherein the amino acid sequence of the mutant comprises an amino acid mutation at at least one site corresponding to G354 and T355 of SEQ ID NO: 5, preferably comprises amino acid mutations at the G354 and T355 sites corresponding to SEQ ID NO:

5.

3. An aldohexose-2-epimerase mutant, which comprises the amino acid sequence described in any one of SEQ ID NO: 6-8 or an amino acid sequence having at least 90% identity with the amino acid sequence described in any one of SEQ ID NO: 6-8.

4. The aldohexose-2-epimerase or mutant thereof according to any one of claims 1-3, wherein, The aldohexose is glucose or mannose.

5. A nucleic acid molecule, which encodes the aldohexose-2-epimerase or a mutant thereof according to any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, wherein, The nucleic acid molecule comprises the sequence shown in any one of SEQ ID NO: 9-16 or a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO: 9-16.

7. An expression vector, which comprises the nucleic acid molecule according to claim 5 or 6.

8. The expression vector according to claim 7, wherein, The expression vector is a plasmid, cosmid, phage or viral vector, preferably a plasmid.

9. A host cell, which comprises the expression vector according to claim 7 or 8; Preferably, the host cell is a eukaryotic cell, prokaryotic cell or bacterial cell, preferably a prokaryotic cell, and more preferably Escherichia coli.

10. Use of the aldohexose-2-epimerase or a mutant thereof according to any one of claims 1-4 in the conversion of aldohexose.

11. A method for converting aldohexose, which comprises using the aldohexose-2-epimerase or a mutant thereof according to any one of claims 1-4 to catalyze an aldohexose reaction to convert the aldohexose.

12. The method according to claim 11, wherein, The temperature of the reaction is 20-55 °C, preferably 30-45 °C; and / or The pH value of the reaction is 6.5-10, preferably 7.5-8.5; and / or The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

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