A novel mannose 6-phosphate phosphatase and uses thereof

Abstract

The application discloses a new mannose 6-phosphate phosphatase and application thereof, and belongs to the technical field of genetic engineering and biological catalysis.The application discloses a new mannose 6-phosphate phosphatase, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO.2.The mannose 6-phosphate phosphatase disclosed by the application can catalyze the generation of mannose from mannose 6-phosphate at high temperature through enzyme molecule modification and optimization.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biocatalysis, and more specifically to a novel mannose-6-phosphate phosphatase and its applications. Background Technology

[0002] Mannose is a monosaccharide, an isomer of glucose, with the hydroxyl group on the second carbon atom facing a different direction, belonging to the aldose family. It exists in nature in various forms, especially common in plants and some microorganisms. Mannose has a sweet and slightly bitter taste, with a sweetness approximately 60% that of sucrose, but less than glucose. It is readily soluble in water and slightly soluble in ethanol. It is found in certain fruits such as cranberries, peaches, and apples; some legumes, carrots, and other vegetables; and in tree gums such as guar gum and locust bean gum.

[0003] The synthesis and preparation methods of mannose are mainly divided into several categories: chemical synthesis, natural extraction, enzymatic conversion, and bio-fermentation. In the chemical synthesis method, glucose is mainly used as the target material for epimerization, which is catalyzed by the addition of calcium hydroxide and ammonium molybdate under alkaline conditions. The yield of the chemical method is about 20%-30%, with many byproducts and high purification costs. The natural extraction method obtains mannose from plants containing mannan through acid hydrolysis. This method is limited by the source of raw materials, has cumbersome preparation steps, complex purification, and high production costs. Patent CN113913481A discloses a method for preparing mannose by converting starch using a multi-enzyme system. It uses low-value starch as a substrate and combines isoamylase, dextran phosphorylase, glucose phosphate mutase, the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase, and mannose 6-phosphate phosphatase to prepare mannose. A catalytic concentration of 100 g / L starch produces 58 g / L of mannose. However, the mannose-6-phosphate phosphatase in this enzyme exhibits low activity and poor stability, affecting the overall conversion rate and production cost. Therefore, finding a novel mannose-6-phosphate phosphatase with high activity, high stability, good compatibility in multi-enzyme cascade systems, and the ability to efficiently participate in mannose synthesis is of great significance for overcoming existing technological bottlenecks and achieving efficient bioconversion from starch to mannose.

[0004] Therefore, providing a novel mannose-6-phosphate phosphatase and its applications is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a novel mannose-6-phosphate phosphatase and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A type of mannose-6-phosphate phosphatase (M6PP), derived from BacteriumIts amino acid sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the method for constructing the above-mentioned mannose-6-phosphate phosphatase includes: 1) Obtain double-stranded DNA sequences from genomic DNA amplification; 2) Chemically synthesize DNA sequences to obtain double-stranded DNA of polypeptides.

[0009] The biological material related to the above-mentioned mannose-6-phosphate phosphatase is any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the above-mentioned mannose-6-phosphate phosphatase; (a2) An expression cassette comprising the nucleic acid molecule described in (a1); (a3) A vector comprising the nucleic acid molecule described in (a1) or the expression cassette described in (a2); (a4) A host cell comprising the nucleic acid molecule of (a1), the expression cassette of (a2), or the vector of (a3).

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the mannose-6-phosphate phosphatase in the above-mentioned biological material is shown in SEQ ID NO.2.

[0011] Preferably, in the above-mentioned biological material, the host cell is a prokaryotic cell or a eukaryotic cell. The prokaryotic cell is preferably a bacterial cell; more preferably, it is *Escherichia coli*.

[0012] Furthermore, the application of the aforementioned mannose-6-phosphate phosphatase or biomaterials in the catalytic production of mannose.

[0013] Preferably, the mannose-6-phosphate phosphatase or biological material catalyzes the production of mannose from mannose-6-phosphate at high temperature.

[0014] Preferably, the enzyme catalytic system uses maltodextrin as a substrate to prepare mannose.

[0015] Preferably, when maltodextrin is used as a substrate to synthesize mannose, the reaction system is a reaction system of maltodextrin and multiple enzymes.

[0016] It should be noted that, in this invention, the polynucleotide sequence encoding the heat-stable mannose-6-phosphate phosphatase (M6PP) can be inserted into a vector to construct a vector containing the polynucleotide described in this invention. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Vectors applicable in this invention include, but are not limited to: expression vectors based on the T7 promoter in bacteria, such as the pET20b vector; pMSXND expression vectors in mammalian cells; and vectors derived from baculoviruses in insect cells. In short, any plasmid and vector can be used to construct recombinant expression vectors as long as they can replicate and remain stable in the host. An important characteristic of expression vectors is that they typically contain a replication origin, a promoter, a marker gene, and translational regulatory elements. Methods well-known to those skilled in the art can be used to construct expression vectors containing the M6PP polynucleotide sequence and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The polynucleotide sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of Escherichia coli; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0017] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as ampicillin resistance, tetracycline and kanamycin for Escherichia coli, or dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture.

[0018] Those skilled in the art are well aware of how to select appropriate vectors / transcriptional regulatory elements (such as promoters, enhancers, etc.) and selective marker genes.

[0019] Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant mannose-6-phosphate phosphatase (M6PP), generally involving the following steps: (1) transforming or transducing suitable host cells with the nucleotide encoding mannose-6-phosphate phosphatase (M6PP) of the present invention, or with a vector containing the polynucleotide; (2) culturing the host cells in a suitable culture medium; and (3) isolating and purifying the protein from the cells.

[0020] In step (2), the culture medium used in the culture can be selected from various conventional culture media, depending on the host cells used. The cells are cultured under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period of time.

[0021] "Complementary" or "complementary" refers to the natural binding of polynucleotides through base pairing under permissible salt concentration and temperature conditions. For example, the sequence "CTGA" can bind to the complementary sequence "GACT". Complementarity between two single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0022] "Identity percentage" refers to the percentage of identical or similar sequences among two or more amino acid or nucleic acid sequences. The identity percentage can be determined using software, such as the GeneDoc program.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a new mannose-6-phosphate phosphatase and its application. The mannose-6-phosphate phosphatase (M6PP), through enzyme molecule modification and optimization, has the characteristic of being able to catalyze the conversion of mannose-6-phosphate to mannose at high temperature. It can be converted into mannose-6-phosphate by using maltodextrin as a substrate and utilizing a multi-enzyme cascade catalysis such as α-glucan phosphorylase, and finally dephosphorylating the group to obtain the mannose product. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 This is a pathway diagram for the conversion of maltodextrin to mannose, involving the reaction catalyzed by M6PP to produce mannose from mannose-6-phosphate.

[0026] Figure 2 This is a map of the pET28a-M6PP plasmid.

[0027] Figure 3 The expression of M6PP is shown below; where M: protein marker; T: cell lysate, representing the total protein expression level; and S: supernatant, representing the soluble protein expression level.

[0028] Figure 4This is an HPLC pattern of mannose production via multi-enzyme catalysis using maltodextrin as a substrate. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise stated, all units used in this manual are international standard units.

[0031] The term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0032] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, glucose refers to D-glucose.

[0033] In this manual, “mannose-6-phosphate phosphatase”, “mannose-6-phosphate phosphatase”, “mannose-6-phosphate phosphatase”, and “M6PP” can be used interchangeably, as they can catalyze the synthesis of mannose from mannose-6-phosphate.

[0034] As used in this article, the term "carbon n position" refers to the carbon position Cn determined according to the carbon number specified in the IUPAC nomenclature, where n is an integer of 1 or greater than 1.

[0035] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0036] The following materials were used in the embodiments of this invention: maltodextrin, a product of Sigma-Aldrich; mannose-6-phosphate, a product of Sigma-Aldrich; mannose, a product of Sigma-Aldrich; pET28a vector, Novagen, Madison, WI; Escherichia coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA; all enzymes in this invention are available from Sigma-Aldrich, and all enzymes can be obtained by prokaryotic expression using genetic engineering methods.

[0037] The amino acid sequence of mannose-6-phosphate phosphatase is shown in SEQ ID NO.1.

[0038] MLRAVIFDMDGVIVDSEPIHLFAEKSLLAKFGISVTDEEIQSYMGKSLRFLLKNFIDRYHLNTSVEKLYAVQQKTLFELYDEKAELLPGLLDLLKILKDDHIDMAVASSSSRALISLVLKKFHLSPYLKVIVSGEEVKKPKPNPDIFLETVSKLGYHPSECVVIEDSTAGVQAAKSAGTACVGFRSPHSGNQDLKIADIIVDDLSSLNIQKLKALMYEIK; SEQ ID NO: 1.

[0039] The nucleotide sequence of mannose-6-phosphate phosphatase is shown as SEQ ID NO. 2.

[0040] atgctgcgtgcggttattttcgatatggacggtgtgatagtagactctgaaccgatccacctgtttgcggagaaaagcctgcttgcaaaattcggtatttctgttacggacgaagagattcagtcatacatgggtaaatcactgcgcttcttgctgaaaaactttatcgaccgttatcatctgaataccagtgtggagaaattgtatgccgtgcaacagaaaacgctttttgaactgtatgatgagaaagcagagcttttaccggggctgttagatctgctgaagattctgaaagatgatcatatcgatatggctgtagccagtagtagttcacgtgccctgattagcctggtgctcaagaaatttcatctgagcccgtatcttaaagtgattgtgtccggtgaagaagtgaaaaaacctaagccgaatccggatattttcttagaaaccgtctctaagctgggctatcatccgtcagaatgcgtcgttattgaagacagcacagcaggggttcaggcggctaaatccgcaggaacagcatgcgtcggatttcgctccccacacagcggtaaccaggatctcaaaattgcagatatcatcgtagatgacctgtcaagcctgaacatccagaaacttaaagccctgatgtatgaaatcaaa; SEQ ID NO: 2.

[0041] The amino acid sequence of isoamylase is shown in SEQ ID NO. 3.

[0042] MVFSHKDRPLRPGEPYPLGANWEEEDDGVNFSIFSENATKVELLIYSPTNQKYPKEVIEVKQRSGDIWHVFVPGLGPGTLYAYRIYGPYKPDQGLRFNPNKVLIDPYAKAINGTLNWNDAVFGYKIGDSNQDLSFDDRPDDEFIPKGVVINPYFEWDDDHFFRRKKIPLKDTIIYEVHVKGFTKLRPDLPENIRGTYKGFASRQMIEYLKDLGVTTVEIMPVQQFVDDRFLVEKGLRNYWGYNPINYFSPECRYSSSGCMGEQVNEFKEMVNELHNAGFEVIIDVVYNHTAEGNHLGPTLSFRGIDNLAYYMLVPDNKRYYLDFTGTGNTLNLSHPRVLQMVLDSLRYWVLEMHVDGFRFDLAAALARQLYSVNMLSTFFVAIQQDPVLSQVKLIAEPWDVGPGGYQVGNFPYLWAEWNGKYRDTIRRFWRGEAIPYEELANRLMGSPDLYAGNNKTPFASINYITSHDGFTLEDLVSYNQKHNEANGFNNQDGMNENYSWNCGVEGETNDANVIQCREKQKRNFIITLFVSQGVPMILGGDELSRTQRGNNNAFCQDNEISWFNWNLDERKQRFHDFVRSMIYFYRAHPIFRRERYFQGKKLHGMPLKDVTFLKPDGNEADEQTWKSPTNFIAYILEGSVIDEVNDRGERIADDSFLIILNGSPNNIKFKFPQGKWSLVVSSYLRELRDDERVVDGGKELEIEGRTAMVYRRIEY; SEQ ID NO. 3.

[0043] The nucleotide sequence of isoamylase is shown in SEQ ID NO. 4.

[0044]

[0045] The amino acid sequence of glucan phosphorylase is shown in SEQ ID NO.5.

[0046] MLEKLPENLKELESLAYNLWWSWSRPAQRLWRMIDSEKWEEHRNPVKILREVSKERLEELSKDEDFIALYELTLERFTDYMEREDTWFNVNYPEWDEKIVYMCMEYGLTKALPIYSGGLGILAGDHLKSASDLGLPLIAVGLLYKHGYFTQQIDSDGRQIEIFPEYDIEGLPMKPLRDEDGNQVIVEVPIDNDTVKARVFEVQVGRVKLYLLDTDFEENEDRFRKIADYLYNPEPDVRVSQEILLGIGGMKLLKTLKIKPGVIHLNEGHPAFSSLERIKSYMEEGYSFTEALEIVRQTTVFTTHTPVPAGHDRFPFDFVEKKLTKFFEGFESKELLMNLGKDEDGNFNMTYLALRTSSFINGVSKLHADVSRRMFKNVWKGVPVEEIPIEGITNGVHMGTWINREMRKLFDRYLGRVWREHTDLEGIWYGVDRIPDEELWEAHLNAKKRFIDYIRESIKRRNERLGINEPLPEISENVLIIGFARRFATYKRAVLLFSDLERLKRIVNNSERPVYIVYAGKAHPRDEGGKEFLRRIYEVSQMPDFKNKIIVLENYDIGMARLMVSGVDVWLNNPRRPMEASGTSGMKAAANGVLNASVYDGWWVEGYNGRNGWVIGDESVLPETEADDPKDAEALYELLENEIIPTYYENREKWIFMMKESIKSVAPKFSTTRMLKEYTEKFYIKGLVNREWLERRENVEKIGAWKERILKNWENVSIERIVLEDSKSVEVTVKLGDLTPNDVIVELVAGRGEGMEDLEVWKVIHIRRYRKENDLFVYTYTNGVLGHLGSPGWFYAVRVIPYHPRLPIKFLPEVPVVWKKVLE;SEQ ID NO.5.

[0047] The nucleotide sequence of glucan phosphorylase is shown in SEQ ID NO.6.

[0048]

[0049] The amino acid sequence of glucose phosphate mutase is shown in SEQ ID NO.7.

[0050] MGKLFGTFGVRGIANEEITPEFALKIGMAFGTLLKREGRERPLVVVGRDTRVSGEMLKDALISGLLSTGCDVIDVGIAPTPAIQWATNHFNADGGAVITASHNPPEYNGIKLLEP NGMGLKKEREAIVEELFFSEDFHRAKWNEIGELRKEDIIKPYIEAIKNRVDVEAIKKRRPFVVVDTSNGAGSLTLPYLLRELGCKVVSVNAHPDGHFPARNPEPNEENLKGFMEI VKALGADFGVAQDGDADRAVFIDENGRFIQGDKTFALVADAVLRENGGGLLVTTIATSNLLDDIAKRNGAKVMRTKVGDLIVARALLENNGTIGGEENGGVIFPDFVLGRDGAMT SEQ ID NO.7.

[0051] The nucleotide sequence of glucose phosphate mutase is shown in SEQ ID NO.8.

[0052]

[0053] The amino acid sequence of glucose phosphate isomerase / mannose-6-phosphate isomerase is shown in SEQ ID NO.9.

[0054] MHHHHHHVEKLDQIEEVKKLDPKDMYSAVYNLPEQIEKAAEVAYKAEIKNISDKIDKVLVLGMGGSAIGGDVLQSVLFKEAKFPVFVNRDYDLPNWVDERTLVFAVSYSGHTVETVNSAKLSYSRGASVVAVTSGGVLKEFAKEKNLTLVEIPGGMAPRAAIGYVTIPSFAILERLGLISPKR SEQ IDNO.9.

[0055] The nucleotide sequence of glucose phosphate isomerase / mannose-6-phosphate isomerase is shown in SEQ ID NO.10.

[0056]

[0057] Example 1 Enzyme mining with mannose-6-phosphate phosphatase activity The reaction catalyzed by mannose-6-phosphate phosphatase is as follows Figure 1 As shown, through structural similarity retrieval and sequence similarity clustering analysis, [the following data was discovered]. Bacterium The M6PP gene (Genebank No.: MBN2028871.1) was named M6PP, and Zhonghe Gene Technology Co., Ltd. was commissioned to design a polynucleotide (DNA) sequence encoding the polypeptide based on the amino acid sequence (as shown in SEQ ID NO.1). Codon optimization was performed on the E. coli expression series, and the optimized sequence is shown in SEQ ID NO.2.

[0058] The sequence shown in SEQ ID NO.2 was cloned between NcoI and XhoI in the pET28a vector (commercial grade) to form the pET28a-M6PP plasmid. Figure 2 In the pET28a-M6PP expression vector, the expression of M6PP is handled by elements such as the T7 promoter and T7 terminator. The expressed M6PP has a 6-terminal C-terminus. His-labeled proteins are purified using Ni-NTA resin.

[0059] Example 2 Preparation of mannose-6-phosphate phosphatase The pET28a-M6PP plasmid was transferred into the protein expression strain *Escherichia coli* BL21(DE3). Single colonies were picked and cultured overnight at 37°C and 220 rpm in 3 ml of LB broth containing 50 μg / ml kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride). 1 ml of the overnight culture was transferred to 200 ml of LB broth containing 50 μg / ml kanamycin and cultured at 37°C and 220 rpm until the OD600 value reached approximately 0.8. IPTG (Isopropylβ-D-1-thiogalactopyranoside) was added to a final concentration of 100 μM, and protein expression was induced at 18°C ​​for 20 hours. After induction, the bacterial cells were collected by centrifugation, resuspended in Tris-HCl (pH 7.5) buffer, and sonicated to obtain cell lysate (T). Enzyme expression levels were detected by SDS-PAGE. The cell lysate was centrifuged at high speed (12000 rpm, 10 min), and the supernatant (S) was also analyzed by SDS-PAGE. The results are shown below. Figure 3The results showed that M6PP (24.5 kDa) was solublely expressed. The collected supernatant was then passed through a chromatography column packed with Ni-NTA resin. The column was flowed through a 10-column volume of 50 mM Tris-HCl (pH 7.5) buffer containing 20 mM imidazole and 50 mM NaCl to remove proteins non-specifically attached to the packing material. Finally, M6PP was eluted and purified by passing the column through a 50 mM Tris-HCl (pH 7.5) buffer containing 250 mM imidazole and 50 mM NaCl. To remove imidazole from the purified enzyme, it was ultrafiltered several times with 50 mM Tris-HCl (pH 7.5) buffer until the imidazole concentration was less than 0.1 mM. The concentration of the purified enzyme was determined using Bradford reagent.

[0060] Example 3: Determination of the activity of mannose-6-phosphate phosphatase To measure the activity of the enzyme obtained in Example 2, the purified enzyme (final concentration 0.5 g / L) was added to: 10 g / L mannose-6-phosphate, 5 mM Mg 2+ The reaction was carried out in 10 mM pH 7.0 PBS at 60 °C for 10 min. After the reaction, the reaction was terminated by boiling, and mannose was present as a product in the reaction system. Based on the 10-min reaction, the enzyme activity of peptide M6PP in catalyzing the conversion of mannose-6-phosphate to mannose at 60 °C was calculated to be 53.5 U / mg. One unit of enzyme activity represents the amount of enzyme required to produce 1 μmol of product per minute. Therefore, it can be concluded that the protein with the amino acid sequence shown in SEQ ID NO.1 possesses the activity to catalyze the conversion of mannose-6-phosphate to mannose at high temperatures, and this protein is defined as mannose-6-phosphate phosphatase.

[0061] Example 4: Preparation of mannose from maltodextrin catalyzed by novel M6PP catalyst An in vitro multi-enzyme catalytic system was established to convert maltodextrin to mannose. The key enzymes in the in vitro multi-enzyme catalytic system include: (1) Isoamylase IA, which catalyzes the hydrolysis of branched chains in maltodextrin; the amino acid sequence of IA is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; (2) αGP, an enzyme that catalyzes the conversion of maltodextrin to glucose-1-phosphate; the amino acid sequence of αGP is shown in SEQ ID NO.5 and the nucleotide sequence is shown in SEQ ID NO.6; (3) Glucose phosphate mutase PGM, which catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate; the amino acid sequence of PGM is shown in SEQ ID NO.7 and the nucleotide sequence is shown in SEQ ID NO.8; (4) Glucose phosphate isomerase / mannose-6-phosphate isomerase PMI / PGI, which catalyzes the conversion of glucose-6-phosphate to mannose-6-phosphate; the amino acid sequence of PMI / PGI is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10; (5) Mannose-6-phosphate phosphatase M6PP, which catalyzes the dephosphorylation of mannose-6-phosphate to produce mannose; the amino acid sequence of M6PP is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2.

[0062] The sequences shown in SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, and SEQ ID NO.10 were cloned into the NcoI and XhoI spaces of the pET28a vector (commercial grade) to form pET28a-IA, pET28a-αGP, pET28a-PGM, and pET28a-PMI / PGI plasmids, respectively. Purified enzymes of IA, αGP, PGM, and PMI / PGI were then prepared using the method described in Example 2.

[0063] The purified enzyme from Example 2 (final concentration 1 U / mL) was added to: 10 g / L maltodextrin, 5 mM Mg 2+ Add 1 U / mL each of IA, αGP, PGM, and PMI / PGI to 10mM pH6.5 PBS and react at 55℃ for 24h.

[0064] The purified enzyme from Example 2 (final concentration 5 U / mL) was added to: 100 g / L maltodextrin, 5 mM Mg 2+ 10mM pH6.5 PBS, with 5 U / mL each of IA, αGP, PGM and PMI / PGI added, and reacted at 55℃ for 24h.

[0065] The purified enzyme from Example 2 (final concentration 10 U / mL) was added to: 300 g / L maltodextrin, 5 mM Mg 2+ Add 10 mM pH 6.5 PBS, 10 U / mL each of IA, αGP, PGM and PMI / PGI, and react at 55℃ for 24 h.

[0066] After the reaction was complete, the reaction was stopped by boiling, centrifuged at 12,000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for liquid chromatography to determine the mannose concentration. Figure 4As shown, the concentration of mannose increases with increasing reaction time. After the reaction, the final concentration of mannose produced from 10 g / L maltodextrin substrate is 7.7 g / L, with a conversion rate of 77.0%; the final concentration of mannose produced from 100 g / L maltodextrin substrate is 74.7 g / L, with a conversion rate of 74.7%; and the final concentration of mannose produced from 300 g / L maltodextrin substrate is 207.6 g / L, with a conversion rate of 69.2%.

[0067] As can be seen, M6PP in this invention has the activity to catalyze the production of mannose from mannose-6-phosphate at high temperatures, and can be combined with other enzymes to form an in vitro multi-enzyme molecular system for use in the reaction of producing various mannose from maltodextrin.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A novel mannose-6-phosphate phosphatase, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. The application of the novel mannose-6-phosphate phosphatase as described in claim 1 in catalyzing the production of mannose from mannose-6-phosphate.

3. The application of the novel mannose-6-phosphate phosphatase as described in claim 1 in catalyzing the production of mannose from maltodextrin.

4. The application of the novel mannose 6-phosphate phosphatase as described in claim 1 in increasing mannose yield.

Images