Aldo-ketose-2-epimerase mutants and uses thereof

Abstract

The application provides a hexulos-2-epimerase mutant and application thereof, and belongs to the technical field of enzyme engineering. The hexulos-2-epimerase mutant comprises one or more than two mutations based on a reference sequence, the amino acid sequence of the reference sequence is shown as SEQ ID NO:1, the amino acid sequence of the mutant comprises amino acid mutations at at least one site in F22, F294, W177, K129, F130 and P132, the amino acid residues are numbered according to SEQ ID NO:1, and the hexulos-2-epimerase RsAE mutant can catalyze the conversion of D-glucose into D-mannose, and the specific enzyme activity is 18 U / mg enzyme.

Description

Technical Field

[0001] This application relates to the field of enzyme engineering technology, and in particular to a hexanoose-2-epimerase mutant and its uses. Background Technology

[0002] Functional sugars, as sucrose substitutes, have broad prospects in the food, pharmaceutical, and health care industries due to their unique physiological functions. D-mannose is one such functional sugar, possessing prebiotic properties and enhancing wound healing while preventing bacterial infections. D-mannose can directly bind to the fimbriae of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus, which cause urinary tract infections, causing them to detach from the urethral wall, thus effectively reducing the recurrence rate of urinary tract infections.

[0003] Currently, D-mannose is mainly prepared through chemical synthesis and enzymatic methods. Chemical synthesis has higher production costs, generates large amounts of acidic wastewater that pollute the environment, and the reaction byproducts make the separation and preparation of high-purity D-mannose difficult. In contrast, enzymatic methods offer milder reaction conditions, fewer byproducts, easier separation and purification, no environmental pollution, and lower costs.

[0004] Currently, the enzymes used for D-mannose preparation mainly include: mannose isomerase (using fructose as a substrate), aldose-ketoose isomerase (using glucose or fructose as a substrate), cellobiose 2-epimerase (using glucose as a substrate), and hexanoose-2-epimerase (specifically using glucose as a substrate). In enzymatic production processes, the degree of enzyme recovery and reuse is one of the important factors affecting the production cost and efficiency of D-mannose. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this application provides a hexanoose-2-epimerase mutant and the immobilization application of the mutant to achieve efficient, continuous, and low-cost conversion of D-glucose raw materials to produce D-mannose.

[0006] The specific technical solution of this application is as follows: This application provides a hexanoose-2-epimerase mutant, wherein the hexanoose-2-epimerase mutant comprises one or more mutations based on a reference sequence, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Preferably, for the aldose-2-epimerase mutant described above, the amino acid sequence of the mutant contains an amino acid mutation at at least one of the following sites: F22, F294, W177, K129, F130, and P132, with the amino acid residues numbered according to SEQ ID NO:1; preferably, it contains an amino acid mutation at the following sites: F22, F294, W177, K129, F130, and P132.

[0008] This application provides a hexanoose-2-epimerase mutant containing an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence as shown in SEQ ID NO:2.

[0009] Preferably, for any of the above-described aldose-2-epimerase mutants, the parent of the aldose-2-epimerase mutant is derived from Runella slithyformis.

[0010] Preferably, for any of the above-described aldose-2-epimerase mutants, the aldose is glucose or mannose.

[0011] This application provides a nucleic acid molecule that encodes a hexanoose-2-epimerase mutant as described in any of the preceding claims.

[0012] Preferably, for the nucleic acid molecule described above, the nucleic acid molecule contains the sequence shown in SEQ ID NO: 3 or the sequence shown in SEQ ID NO: 3.

[0013] This application provides an expression vector comprising the nucleic acid molecules described above.

[0014] Preferably, the expression vector described above is a plasmid, granule, bacteriophage, or viral vector, and is preferably a plasmid.

[0015] This application provides a host cell that includes the expression vector described above; Preferably, the host cell is a eukaryotic cell, a prokaryotic cell, or a bacterial cell, preferably a prokaryotic cell, and more preferably Escherichia coli.

[0016] This application provides an immobilized enzyme preparation comprising the aldose-2-epimerase mutant described in any of the preceding claims.

[0017] Preferably, the immobilized enzyme preparation described above is obtained by immobilizing the hexanoose-2-epimerase mutant on a resin.

[0018] Preferably, for the immobilized enzyme preparation described above, the resin is a reactive amino resin or an epoxy affinity resin.

[0019] This application provides the use of the aldose-2-epimerase mutant or the immobilized enzyme preparation described in any of the above claims in aldose conversion, preferably when the aldose is glucose, for converting glucose into mannose.

[0020] This application provides a method for converting aldose, comprising using the aldose-2-epimerase mutant or the immobilized enzyme preparation described in any of the above claims to catalyze the aldose reaction and convert the aldose; preferably, when the aldose is glucose, the glucose is converted.

[0021] Preferably, in the method described above, the reaction temperature is 20-55°C, more preferably 30-45°C; and / or The reaction is carried out at a pH of 6.5-10, preferably 7.5-8.5; and / or The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0022] Beneficial effects The hexanoose-2-epimerase RsAE mutant provided in this application can catalyze the conversion of D-glucose to D-mannose, with a specific enzyme activity of 18 U / mg enzyme, which is about 38.5% higher than the RsAE mutant (specific enzyme activity of 13 U / mg enzyme) described in Chinese Patent CN 118109446 A.

[0023] The RsAE mutant (free state, 0.15 mg / ml) of this application, when reacted at 37℃ for 60 min, achieved a conversion rate of 24.05% for D-glucose (10 g / L) to D-mannose; after 4 hours of reaction, the conversion rate was 25.02%, close to the equilibrium point. Immobilized RsAE mutant (0.5 mg enzyme / 300 μl resin) catalyzed the conversion of D-glucose (10 g / L) to D-mannose after 4 hours, achieving a conversion rate of 21.23%. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the reaction process curve of the mutant (0.03 mg / ml) described in Example 1 converting D-glucose into D-mannose.

[0025] Figure 2 This is a schematic diagram of the reaction process curve of the mutant (0.15 mg / ml) described in Example 1 converting D-glucose into D-mannose.

[0026] Figure 3 This is a schematic diagram of the mutant immobilization reaction described in Example 1.

[0027] Figure 4 This is a schematic diagram of the enzyme reaction curve for the conversion of D-glucose to D-mannose by an immobilized mutant. Detailed Implementation

[0028] The present application will now be described in detail with reference to 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 to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0029] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0030] definition As used herein, "epomerase" refers to an enzyme that catalyzes a configurational change in one of the asymmetric carbon atoms in a monosaccharide molecule (containing two or more asymmetric carbon atoms). In this application, the epimerase refers to an enzyme that isomerizes aldose. In this application, the aldose is glucose or mannose. The epimerase described in this application can convert glucose into mannose or mannose into glucose, preferably converting glucose into mannose.

[0031] As used herein, “homology,” “identity,” and “similarity” refer to the sequence similarity between two nucleic acid molecules. “Homology,” “identity,” or “similarity” can be determined by comparing positions in each sequence, and the sequences can be aligned for comparison purposes. When equivalent positions in the compared sequences are occupied by the same bases, the molecules are identical at that position; when equivalent sites are occupied by the same or similar amino acid residues (e.g., similar in spatial or electrical properties), the molecules can be considered homologous (similar) at that position. The expression of homology / similarity or identity percentage refers to the number of identical or similar amino acids at shared positions in the compared sequences. “Irrelevant” or “non-homologous” sequences share less than 40% identity with the sequences of this application, preferably less than 25%. The absence or presence of extra residues (amino acids or nucleic acids) also reduces identity and homology / similarity when comparing two sequences. In specific implementations, for two or more sequences or subsequences, determined by 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, for example, by the National Center for Biotechnology Information (NCBI), if their sequences exhibit approximately 60% identity in the specified region, or approximately 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, when compared and aligned for maximum correspondence within a comparison window or specified region, they can be considered substantially or significantly homologous, similar, or identical. This definition also relates to or can be used to test sequence complements. Therefore, to the extent permitted by the context of this paper, for example, if a nucleotide sequence can be predicted to be naturally present in a DNA duplex, or can be naturally present as one or both of the complementary strands, then a nucleotide sequence complementary to the specified target sequence or a variant thereof is itself considered "similar" to the target sequence, and when "similar" nucleic acid sequences are involved, this includes single-stranded sequences, their complementary sequences, double-stranded strand complexes, sequences capable of encoding the same or similar polypeptide products, and any permissible variants of any of the foregoing. Similarity must be limited to analyses of single nucleic acid strand sequences, which may 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 the implementation scheme, identity or similarity may be in regions of at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, 24, 25 or more nucleotides, or in regions of more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more nucleotides.

[0032] As used herein, the term "nucleotide" refers to naturally occurring nucleotides, as well as synthetic nucleotide analogs that can be recognized by cellular enzymes.

[0033] As used herein, the term "expression vector" refers to any naturally occurring or artificially constructed expression vector containing a nucleic acid molecule that can be catalyzed by cellular transcriptases and / or translatases. Exemplary expression vectors include plasmids, viruses (including bacteriophages), granules, artificial chromosomes, or transposons. In some embodiments, the expression vector is a plasmid.

[0034] As used herein, the term "host cell" refers to any biological cell that can be cultured in a culture medium and used to express recombinant genes. These host cells can be eukaryotic or prokaryotic cells, microbial cells such as bacterial cells, or cells derived from a cell line (such as an immortalized mammalian cell line), etc. In some embodiments, the host cell is a prokaryotic cell, such as Escherichia coli.

[0035] As used in this article, the term "recombinant" refers to nucleic acid molecules or polypeptides that are produced through artificial intervention in a non-natural environment.

[0036] hexanoyl-2-epimerase mutant This application provides a hexanoose-2-epimerase RsAE mutant, wherein the hexanoose-2-epimerase mutant contains one or more mutations based on a reference sequence, the amino acid sequence of which is shown in SEQ ID NO:1.

[0037] The amino acid sequence of SEQ ID NO:1 is as follows: MDRFARENLTEWKRILAYWEKFSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASKFKPALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPWNKSQNTHLHIIEAFTNLY RVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEFDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFQEVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK The term "corresponds" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds" means the position in one sequence that corresponds to a specified position in another sequence after two sequences have been aligned for homology or sequence identity.

[0038] This application does not impose any limitations on the methods for preparing mutants. Mutation can be carried out according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or the construction of synthetic oligonucleotides. The mutated DNA sequence can then be expressed in host cells to obtain mutants with amino acid substitutions, insertions, and / or deletions. In this application, site-directed mutagenesis is used based on the 3D structure and function analysis of the enzyme.

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

[0040] In some embodiments, the amino acid sequence of the mutant contains an amino acid mutation at at least one of the sites F22, F294, W177, K129, F130, and P132, the amino acid residues being numbered according to SEQ ID NO:1; preferably, it contains an amino acid mutation at the sites F22, F294, W177, K129, F130, and P132.

[0041] In this application, the sites mentioned above are counted starting from the N-terminus. For example, F22 refers to the 22nd amino acid mutation starting from the N-terminus of SEQ ID NO:1.

[0042] In this application, the phenylalanine F at position 22 can be mutated to tyrosine Y, or the phenylalanine F at position 294 can be mutated to tyrosine Y, or the tryptophan W at position 177 can be mutated to tyrosine Y, or the lysine K at position 129 can be mutated to glycine G, or the phenylalanine F at position 130 can be mutated to lysine K, or the proline P at position 132 can be mutated to glutamic acid E; preferably, the phenylalanine F at position 22 can be mutated to tyrosine Y, the phenylalanine F at position 294 can be mutated to tyrosine Y, the tryptophan W at position 177 can be mutated to tyrosine Y, the lysine K at position 129 can be mutated to glycine G, the phenylalanine F at position 130 can be mutated to lysine K, and the proline P at position 132 can be mutated to glutamic acid E.

[0043] This application provides a hexanoose-2-epimerase mutant containing an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence as shown in SEQ ID NO:2.

[0044] The amino acid sequence of SEQ ID NO:2 is as follows: MDRFARENLTEWKRILAYWEKYSPDYQRGGFHGQVNYDNQPVLDASRSIILISRILWTFSLAYRHFHRRRYLVLADRAYHYLYNHFRDTKNGGVYWSVTAAGVPLETRKQLYGHAFAIYGLSEYYAASGKKEALDFAQELFQTVDKHGYDAEKGGYFEAFGPSWETVDDLILSKMPYNKSQNTHLHIIEAFTNLY RVWPDALLKKRVVHLTDAFMEKLVSPETYRLRLFFDRDWQPKDETISYGHDIEASWLLWETAEVLNDHERSQKVKQLCIKMAEAACTGLGEDGALDYEYDPAANHRNHERSWWVLAEQMVGFYNAYELTGESHYKDKSLKSWEFIKKYVMDTQKGDWFQEVKPDLTPVRNAKVSFWKCPYHNSRACYEIVRRLEK 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 encompass sequences that contain one, two, or three or more amino acid mutations compared to the sequence shown in SEQ ID NO:2, but are substantially the same as its substantial function, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to the sequence shown in SEQ ID NO:2.

[0045] In some embodiments, the parent of the hexanoose-2-episomerase mutant is derived from... Runella slithyformis In some embodiments, the aldose is glucose or mannose.

[0046] Nucleic acid molecules, expression vectors, and host cells This application provides a nucleic acid molecule comprising encoding an aldose-2-epimerase mutant as described in any of the preceding embodiments. In some embodiments, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3 or a sequence as shown in SEQ ID NO: 3.

[0047] The nucleic acid sequence of SEQ ID NO:3 is as follows: In some embodiments, the nucleotide sequence encoding the aforementioned aldose-2-epimerase mutant is codon-optimized. Typically, codon optimization involves balancing the percentage of selected codons with the abundance of publicly available human transfer RNA, ensuring that neither 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. Differences in codon usage between the transfected gene and the host cell can affect protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are selected to balance with human usage frequencies. Typically, amino acid codon redundancy ensures that different codons encode one amino acid. In some embodiments, when selecting codons for substitution, it may be necessary to obtain a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.

[0048] This application provides an expression vector comprising the nucleic acid molecules described above. In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid.

[0049] For example, the nucleic acid molecule encoding the aforementioned aldose-2-epimerase mutant can be cloned into a suitable expression vector. The expression vector can be any suitable recombinant expression vector and can be used for transformation or transfection of any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses. In some embodiments, the expression vector is a plasmid.

[0050] In this application, the expression vector may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific to the type of host to which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operatively linked to a nucleotide sequence encoding a TCR. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in long terminal repeat sequences of mouse stem cell viruses, as well as other promoters known to those skilled in the art.

[0051] This application provides a host cell that includes the expression vector described above.

[0052] In this application, an expression vector is transformed into a host cell for further expression or cloning within the host cell. In some embodiments, a method for preparing an aldose-2-epimerase mutant is provided, the method comprising culturing a host cell containing nucleic acid encoding the aldose-2-epimerase mutant as described above under conditions suitable for expression of the aldose-2-epimerase mutant, and optionally recovering the aldose-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, and more preferably *Escherichia coli*.

[0053] The host cell refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0054] Immobilized enzyme preparations This application provides an immobilized enzyme preparation comprising the aforementioned aldose-2-epimerase mutant. In some embodiments, the aldose-2-epimerase mutant is obtained by immobilizing the aldose-2-epimerase mutant on a resin. In some embodiments, the resin is a reactive amino resin or an epoxy affinity resin.

[0055] In this application, no restrictions are placed on the preparation method of the immobilized enzyme preparation. Those skilled in the art can use conventional methods in the art to immobilize the mutant and thus fix the mutant on the resin to obtain the immobilized enzyme preparation.

[0056] In this application, the resin may be a resin conventionally used in the art, such as a reactive amino resin or an epoxy affinity resin.

[0057] Applications of hexanoose-2-epimerase mutants and methods for converting hexanoose. This application provides the use of the aldose-2-epimerase mutant or the immobilized enzyme preparation described above in aldose conversion.

[0058] In this application, the conversion refers to the conversion between aldoses, for example, when the aldose is glucose, it can be converted into mannose, and when the aldose is mannose, it can be converted into glucose. It is preferable to convert glucose into mannose.

[0059] This application provides a method for converting aldose, comprising using the aldose-2-epimerase mutant described in any one of the above claims or the immobilized enzyme preparation described in the above claims to catalyze the aldose reaction, thereby converting the aldose, preferably when the aldose is glucose, to convert glucose. In some embodiments, the reaction temperature is 20-55°C, preferably 30-45°C; and / or The reaction is carried out at a pH of 6.5-10, preferably 7.5-8.5; and / or The reaction time is 0.5-10 hours, preferably 0.5-6 hours.

[0060] For example, the reaction temperatures are 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, etc. The pH value of the reaction can be 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc.; 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.

[0061] In some embodiments, the concentration of hexanoose is 10-100 g / L.

[0062] For example, the concentration of hexalose can be 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L, etc.

[0063] The aldose-2-epimerase mutant described in this application has high specificity and can convert glucose into mannose. Compared with the RsAE mutant (with a specific enzyme activity of 13 U / mg enzyme) described in Chinese patent application CN 118109446 A, it is about 38.5% more specific.

[0064] Example This 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, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0065] Example 1: Gene cloning and expression preparation of RsAE mutant RsAE is a protein with a full-length 390 amino acid sequence encoded by the R. slithyformis (DSM 19594) gene fragment Runsl_4586. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the RsAE mutant gene fragment (G354Q+T355E+F22Y+F294Y+W177Y+K129G+F130K+P132E) and insert it between the restriction enzyme sites NdeI and NotI in the pET22b plasmid. Finally, the expression plasmid was transformed into E. coli BL21.

[0066] Recombinant Escherichia 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 culture was transferred at a 10% inoculation rate to a 500 mL shake flask containing 90 mL of fresh LB liquid medium and cultured on a shaker at 37°C and 150 rpm. When the bacterial cell concentration OD600 reached 0.6-0.8, the shaker temperature was adjusted to 28°C, and IPTG was added to a final concentration of 0.5 mM. Enzyme production was induced for 5 hours at 28°C and 150 rpm.

[0067] The collected bacterial cells were reconstituted using a phosphate buffer (pH 7.8) and then autoclaved. The supernatant was collected by centrifugation and finally purified by Ni column affinity adsorption.

[0068] Experimental Example 1: Enzyme activity assay of the reaction between RsAE mutant and D-glucose to produce D-mannose A 1 ml reaction system (50 mM phosphate buffer (pH 7.8)) with a glucose substrate concentration of 10 g / L was used to add 0.03 mg of the RsAE mutant prepared in Example 1. The reaction was carried out at 37°C for 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr, respectively. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 min. The catalytic solution after the reaction was terminated was centrifuged at 13000 rpm for 1 min, and the supernatant was used to prepare the HPLC test sample. The amount of mannose generated in the reaction system was determined by high-performance liquid chromatography. Chromatographic conditions: Agilent 1260 high-performance liquid chromatograph, Phenomen MARS Mca 5u 300X 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.

[0069] The reaction curve of the RsAE mutant converting D-glucose to D-mannose is shown below. Figure 1 As shown, enzyme activity is defined as the amount of enzyme required to produce 1 μmol of mannose in 1 min at 37°C, which is 1 U.

[0070] Calculations show that 1 mg of the RsAE mutant contains approximately 18 enzyme activity units, or 18 U / mg of enzyme. Furthermore, the Kcat for the conversion of D-glucose to D-mannose by the RsAE mutant is approximately 14 s. -1 .

[0071] Using a 1 ml reaction system (50 mM phosphate buffer (pH 7.8)), a glucose substrate concentration of 10 g / L, and 0.15 mg of the RsAE mutant prepared in Example 1, the reaction was carried out at 37°C for 1 hr, 2 hr, 3 hr, and 4 hr, respectively. The reaction was terminated by boiling in a water bath for 10 min after the reaction was completed. The sample preparation and detection methods were the same as above, and the results are as follows. Figure 2 As shown.

[0072] from Figure 2 It can be seen that when 0.15 mg of RsAE mutant was added and the reaction was carried out for 1 hour, the conversion rate of D-glucose was 24.05%; when the reaction was carried out for 4 hours, the conversion rate was 25.02%, which is close to the equilibrium point of the reaction.

[0073] Experimental Example 2: Immobilization of RsAE mutant Take 300 μl of reactive amino resin LXTE-703 (produced by Xi'an Lanxiao Technology New Material Co., Ltd.) and clean it thoroughly. Prepare 1 ml of 2% (v / v) glutaraldehyde solution using immobilization buffer (0.5 M phosphate buffer (pH 7.8). Add 1 ml of glutaraldehyde solution to the test tube containing the resin and shake in a 24°C environment for 80 min. After the reaction, wash repeatedly with deionized water 5-10 times, and finally wash once with 100 mM phosphate buffer (pH 7.8).

[0074] Add a solution containing 0.5 mg of RsAE mutant to a test tube containing the activated resin described above, and incubate at 24°C and 100 rpm for 20 hours. Wash the enzyme-fixed resin 2-3 times with 100 mM phosphate buffer (pH 7.8). The resulting fixed RsAE mutant is shown below. Figure 3 As shown.

[0075] Experimental Example 3: Determination of the reaction of immobilized RsAE mutants in converting D-glucose to D-mannose Take 300 μl of the RsAE mutant immobilized resin from Experiment 2 (approximately 0.5 mg of immobilized enzyme), add 1 ml of reaction solution (50 mM phosphate buffer (pH 7.8), 10 g / L substrate glucose), and react at 37℃ for 0.5 hr, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, and 6 hr, respectively. Terminate the reaction by boiling in a water bath for 10 min after the reaction is complete. The sample preparation and high-performance liquid chromatography conditions were the same as above for HPLC testing. The reaction progress curve of the immobilized RsAE mutant (G354Q+T355E+F22Y+F294Y+W177Y+K129G+F130K+P132E) converting D-glucose to D-mannose is shown below. Figure 4 As shown.

[0076] from Figure 4 It can be seen that the conversion rate of glucose substrate gradually increases with the extension of reaction time, and the conversion rate can reach 21.23% after 4 hours of reaction.

[0077] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A hexanoose-2-epimerase mutant, wherein the hexanoose-2-epimerase mutant has the amino acid sequence shown in SEQ ID NO:

2.

2. The aldose-2-epimerase mutant according to claim 1, wherein the parent of the aldose-2-epimerase mutant is derived from... Runella slithyformis .

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

4. A nucleic acid molecule encoding a hexanoose-2-epimerase mutant according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, wherein, The nucleic acid molecule is the sequence shown in SEQ ID NO:

3.

6. An expression vector comprising the nucleic acid molecule of claim 4 or 5, wherein the expression vector is a plasmid.

7. An immobilized enzyme preparation comprising the aldose-2-epimerase mutant according to any one of claims 1-3.

8. Use of the hexalose-2-epimerase mutant according to any one of claims 1-3 or the immobilized enzyme preparation according to claim 7 in hexalose conversion; The aldose is glucose.

9. The use according to claim 8, for converting glucose into mannose.

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