Alpha-glucan phosphorylase and use thereof

Abstract

The application provides an alpha-glucan phosphorylase and application thereof, the amino acid sequence of the alpha-glucan phosphorylase is shown in the sequence table SEQ ID NO.1, can start from the non-reducing end of amylose, and release glucose-1-phosphate one by one by phosphorolysis, and can generate glucose-1-phosphate by using the alpha-glucan phosphorylase catalysis with starch as substrate, and then is converted into mannose, tagatose, allulose, mannitol, cellobiose, laminaribiose, trehalose, sophorose, fructose-1,6-bisphosphate and special starch and other products by multi-enzyme cascade catalysis.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biocatalysis, and in particular to an α-glucan phosphorylase and its applications. Background Technology

[0002] α-Glucose-1-phosphate (G1P) is the first metabolite in the glycogenolysis pathway, produced by the catalytic reaction of glycogen, starch, or maltodextrin with inorganic phosphate. Its synthesis is catalyzed by glycogen (starch, maltodextrin) phosphorylases or α-glucan phosphorylases (α-GP, EC 2.4.1.1). Upon entering glycolysis and other metabolic pathways, G1P can be converted to glucose-6-phosphate by phosphoglucomutases. As an activated glucose, G1P is an important precursor for the synthesis of complex carbohydrates (such as glycolipids, oligosaccharides, or nucleotide sugars). It can be produced by various oligosaccharides catalyzed by their respective glycoside phosphorylases, such as sucrose and sucrose phosphorylases, cellobiose and cellobiose phosphorylases, cellodextrin and cellodextrin phosphorylases, and the combination of maltodextrin or soluble starch with α-GP. However, disaccharides (such as sucrose and cellobiose) are generally more expensive than starch, and each disaccharide molecule produces only one molecule of G1P, with a theoretical yield of only 50% in hexose units, potentially leading to higher product separation costs. Although maltodextrin and soluble starch are less expensive, the presence of their α-1,6 glycosidic bonds results in low efficiency of α-GP-catalyzed stepwise phosphorylation. Furthermore, α-GP is far more efficient at catalyzing long-chain maltooligosaccharides (such as maltotetraose or even longer chains) than maltose or even maltotriose, leading to significant loss of glucose units in maltodextrin and soluble starch.

[0003] Starch, as one of the lowest-cost sources of hexoses, is widely used in industrial fermentation and biocatalysis. Starch is typically hydrolyzed to glucose in a two-step process: first, starch liquefaction is catalyzed by α-amylase at approximately 100°C; then, saccharification is catalyzed by pullulanase and glucosylamylase or β-amylase at approximately 60°C. Because different enzymes have different optimal temperatures and pH values, these two hydrolysis steps must be performed sequentially. To obtain high-yield G1P from starch, amylopectin must be debranched to cleave the α-1,6 glycosidic bonds, generating linear dextrins. These linear dextrins are then synthesized through multiple enzymatic reactions, including phosphorylation, isomerization, and dephosphorylation, to produce mannose, mannitol, allulose, tagatose, trehalose, fructose-1,6-bisphosphate, sophorose, and other specialty starches. In this process, α-glucan phosphorylase (EC 2.4.1.1) plays a crucial role: it catalyzes the non-reducing end phosphorylation of starch to generate glucose-1-phosphate, providing a key activation intermediate for the subsequent synthesis of functional sugars.

[0004] Therefore, developing a novel α-glucan phosphorylase that exhibits high activity against starch substrates, good compatibility in multi-enzyme cascade systems, and efficient participation in the synthesis of functional sugars is of great significance for overcoming existing technological bottlenecks and achieving efficient biotransformation from starch to functional sugars. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an α-glucan phosphorylase.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned α-glucan phosphorylase.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An α-glucan phosphorylase (α-GP) derived from the thermophilic bacterium *Chloroflexota* bacterium, having the amino acid sequence shown in SEQ ID NO. 1; or a protein having the amino acid sequence with one or more amino acid residues substituted, deleted, or added, and having the activity of releasing glucose-1-phosphate sequentially from the non-reducing end of amylose at high temperature; or a derivative protein having the above-mentioned amino acid sequence; or a derivative protein having ≥60% (preferably ≥80%, more preferably ≥90%) the same amino acid sequence as shown in SEQ ID NO. 1, and having the activity of releasing glucose-1-phosphate sequentially from the non-reducing end of amylose at high temperature.

[0008] Preferably, the amino acid sequence of the above-mentioned α-glucan phosphorylase is shown in SEQ ID NO.1 of the sequence listing.

[0009] The construction method of the above-mentioned α-glucan phosphorylase is as follows: 1) Obtain double-stranded DNA sequences from genomic DNA amplification; 2) Chemically synthesize DNA sequences to obtain double-stranded DNA of the polypeptide.

[0010] The biological material related to the above-mentioned α-glucan phosphorylase is any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the above-mentioned α-glucan phosphorylase; (a2) An expression cassette containing 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).

[0011] Preferably, in the above-mentioned biological material, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2; or a nucleotide sequence that has ≥50% (preferably ≥80%, more preferably ≥90%) the same as the nucleotide sequence shown in SEQ ID NO.2; or a nucleotide sequence that is complementary (preferably completely complementary) to the above-mentioned nucleotide sequence.

[0012] Preferably, the nucleotide sequence of the above-mentioned biological material is shown in SEQ ID NO.2.

[0013] Preferably, in the above-mentioned biological material, the host cell is a prokaryotic cell or a eukaryotic cell.

[0014] Preferably, the host cell of the above-mentioned biological material is a prokaryotic cell, such as a bacterial cell; more preferably, it is Escherichia coli.

[0015] The application of the above-mentioned α-glucan phosphorylase or biological materials in the catalytic production of glucose-1-phosphate.

[0016] Preferably, in the above application, the α-glucan phosphorylase or biomaterial releases glucose-1-phosphate sequentially from the non-reducing end of the amylose chain at high temperature.

[0017] Preferably, in the above applications, the enzyme catalytic system uses starch or maltodextrin as a substrate to prepare functional sugars.

[0018] Preferably, in the above application, when starch or maltodextrin is used as the substrate, the reaction system is a reaction system of starch or maltodextrin and multiple enzymes to synthesize various functional sugars; the functional sugars are mannose, tagatose, allulose, disaccharides, fructose-1,6-bisphosphate or special starches.

[0019] It should be noted that, in this invention, the polynucleotide sequence encoding the heat-resistant α-glucan phosphorylase (α-GP) 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 expressed in bacteria (Gene, 1987, 56:125), such as the pET20b vector; the pMSXND expression vector expressed in mammalian cells (J BioChem. 263:3521, 1988); and vectors derived from baculoviruses expressed 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 TiT4E polynucleotide sequence and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). The polynucleotide sequence can be efficiently linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. 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 gene expression in prokaryotic or eukaryotic cells or their viruses.

[0020] 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.

[0021] 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.

[0022] In this invention, a polynucleotide encoding α-glucan phosphorylase (α-GP) or a vector containing such polynucleotide can be transformed or introduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the vector. The term "host cell" refers to prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells such as *Salmonella typhimurium*; fungal cells such as yeast; plant cells; insect cells such as *Drosophila S2* or *Sf9*; and animal cells such as CHO, COS, or Bowes melanoma cells.

[0023] Transformation of host cells using the nucleotide sequence described in this invention or a vector containing the nucleotide sequence described herein can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. MgCl2 is also an option. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation or conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used.

[0024] Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant α-glucan phosphorylase (α-GP), generally involving the following steps: (1) transforming or transducing suitable host cells with the nucleotide encoding α-glucan phosphorylase (α-GP) 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.

[0025] 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.

[0026] In step (3), the protein may be encapsulated within the cell, expressed on the cell membrane, or secreted extracellularly. If desired, the recombinant protein may be separated and purified using various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional refolding treatment, protein precipitation (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0027] "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.

[0028] "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.

[0029] Beneficial effects: The aforementioned α-glucan phosphorylase, through enzyme molecule modification and optimization, possesses the characteristic of being able to release glucose-1-phosphate sequentially from the non-reducing end of amylose at high temperatures. It can be used to catalyze the production of glucose-1-phosphate by using starch as a substrate, and then, through multi-enzyme cascade catalysis, convert it into products such as mannose, tagatose, allulose, mannitol, cellobiose, laminabiose, trehalose, sophorose, fructose-1,6-bisphosphate, and special starches. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the conversion of starch / maltodextrin into functional sugars, involving the reaction of α-GP catalyzing the non-reducing end phosphorylation of starch to generate glucose-1-phosphate.

[0031] Figure 2 This is the pET28a-CbαGP plasmid map.

[0032] Figure 3 This shows the expression of Cbα-GP.

[0033] Figure 4 This is an HPLC pattern of mannose production using maltodextrin as a substrate via multi-enzyme catalysis. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0035] Unless otherwise stated, all units used in this manual are international standard units.

[0036] 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.

[0037] 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.

[0038] In this instruction manual, “α-glucan phosphorylase”, “α-starch phosphorylase”, and “α-GP” can be used interchangeably. They can phosphorylate the non-reducing ends of starch to produce glucose-1-phosphate.

[0039] 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.

[0040] 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.

[0041] The following materials are used in the embodiments of this invention: Starch, a product of Sigma-Aldrich; Glucose-1-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 can be obtained by prokaryotic expression using genetic engineering methods.

[0042] Example 1 Enzyme mining with α-glucan phosphorylase activity The reaction catalyzed by α-glucan phosphorylase is as follows Figure 1 As shown, the non-reducing ends of starch are phosphorylated to produce glucose-1-phosphate. This is based on the method described in the paper "An In Vitro Synthetic Biology Platform for the Industrial Biomanufacturing of Myo-Inositol From Starch" published by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. Thermotoga maritimaBased on α-glucan phosphorylase (KEGG ORF: TM1168), the α-GP gene (uniprot: A0A7C1F2Q1) of the thermophilic archaea Chloroflexotabacterium was identified through structural similarity search and sequence similarity clustering analysis. Named Cbα-GP, the gene was developed by Zhonghe Gene Technology Co., Ltd., which designed the polynucleotide (DNA) sequence encoding this polypeptide based on the amino acid sequence. Codon optimization was performed on an E. coli expression series, and the optimized sequence is shown in SEQ ID NO.2 of the sequence listing. Zhonghe Gene cloned the codon-optimized sequence into the pET28a vector (commercial grade), forming the pET28a-CbαGP plasmid. Figure 2 In this expression vector, the T7 promoter and T7 terminator are responsible for the expression of Cbα-GP. The expressed Cbα-GP has a 6XHis tag at its C-terminus, and the protein is purified using Ni-NTA resin.

[0043] Example 2 Preparation of α-glucan phosphorylase The pET28a-Cbα-GP plasmid was transferred into the protein expression strain *Escherichia coli* BL21(DE3). Single colonies were picked and transferred to 3 ml of liquid LB medium containing 50 μg / ml kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride), and incubated overnight at 37°C and 220 rpm. 1 ml of the overnight culture was transferred to 200 ml of LB medium containing 50 μg / ml kanamycin, and incubated 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. Enzyme expression levels were detected by SDS-PAGE. The cell lysate was centrifuged at high speed (12000 rpm, 10 min), and the supernatant was also analyzed by SDS-PAGE. Figure 3As shown in the figure, Cbα-GP can be expressed solublely. Subsequently, the collected supernatant was passed through a chromatography column packed with Ni-NTA resin. The column was flowed through a 10-column volume of 50mM Tris-HCl (pH 7.5) buffer containing 20mM imidazole and 50mM NaCl to remove proteins non-specifically attached to the packing material. Finally, Cbα-GP was eluted and purified by passing the column through a 50mM Tris-HCl (pH 7.5) buffer containing 250mM imidazole and 50mM NaCl. To remove imidazole from the purified enzyme, it was ultrafiltered several times with 50mM Tris-HCl (pH 7.5) buffer until the imidazole concentration was less than 0.1mM. The concentration of the purified enzyme was determined using Bradford reagent.

[0044] Example 3 Enzyme activity assay of α-glucan phosphorylase To measure the activity of the enzyme obtained in Example 2, the purified enzyme (0.5 g / L) was added to: 10 g / L maltodextrin, 5 mM Mg 2+ 10 mM pH 6.5 PBS was added, and the reaction was carried out at 55 °C for 10 min. After the reaction was completed, the reaction was terminated by boiling. One product, glucose-1-phosphate, was present in the reaction system. 1 U / ml of phosphogluconomutase (PGM, purchased from Sigma), 5 U / ml of glucose-6-phosphate dehydrogenase (G6PDH, purchased from Sigma, product number G6378), and 5 mM NAD+ were added to the final sample. + The reaction was carried out at 37°C until OD340 no longer increased. The amount of glucose-6-phosphate generated was calculated from the increased OD340, and then the amount of glucose-1-phosphate generated was calculated. After a 10-minute reaction, the enzyme activity of peptide Cbα-GP in releasing glucose-1-phosphate sequentially from the non-reducing end of amylose at 60°C was calculated to be 19.3 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 No. 1 has the enzyme activity of releasing glucose-1-phosphate sequentially from the non-reducing end of amylose at high temperature, and this protein is defined as α-glucan phosphorylase. It can also be concluded that the protein with the amino acid sequence shown in SEQ No. 1 has the enzyme activity of releasing glucose-1-phosphate sequentially from the non-reducing end of maltose at high temperature.

[0045] Normally, α-glucan phosphorylase can only phosphorylate maltodextrin up to a chain length of at least 4, meaning it will phosphorylate amylose or maltodextrin down to maltotetrasaccharide. After that, it cannot continue phosphorylation, leaving a large amount of maltotetrasaccharide in the system, resulting in insufficient substrate utilization. Therefore, the enzyme activities of Cbα-GP at 60℃ for maltose, maltotriose, maltotetrasaccharide, and maltopentose were measured to be 1.6, 3.2, 5.1, and 10.7 U / mg, respectively. This demonstrates that Cbα-GP can completely phosphorylate maltodextrin or amylose down to a single glucose unit before stopping, significantly improving substrate utilization and product yield.

[0046] Example 4: Novel Cbα-GP-catalyzed preparation of mannose from maltodextrin The purified enzyme (1 U / mL) from Example 2 was added to: 10 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 1 U / mL each of isoamylase IA, glucose phosphate mutase PGM, bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase PGI / PMI and mannose-6-phosphate dephosphorase M6PP. The mixture was reacted at 55 °C for 6 h.

[0047] The purified enzyme (5 U / mL) from Example 2 was added to: 100 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 5 U / mL each of isoamylase IA, glucose phosphate mutase PGM, bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase PGI / PMI and mannose-6-phosphate dephosphorase M6PP. The mixture was reacted at 55 °C for 24 h.

[0048] The purified enzyme (10 U / mL) from Example 2 was added to: 300 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 10 U / mL each of isoamylase IA, glucose phosphate mutase PGM, bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase PGI / PMI, and mannose-6-phosphate dephosphorase M6PP. The reaction was carried out at 55 °C for 24 h. After the reaction was completed, the reaction was stopped by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for mannose concentration detection by liquid chromatography.

[0049] 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 and analyzed by liquid chromatography for 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 8.1 g / L; the final concentration of mannose produced from 100 g / L maltodextrin substrate is 80.2 g / L; and the final concentration of mannose produced from 300 g / L maltodextrin substrate is 241 g / L.

[0050] Example 5: Novel Cbα-GP-catalyzed preparation of tagatose from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 5 U / mL each of isoamylase IA, glucose phosphate mutase PGM, glucose phosphate isomerase PGI, tagatose-6-phosphate epimerase TPE, and tagatose-6-phosphate dephosphorase T6PP. The reaction was carried out at 55 °C for 24 h. After the reaction was completed, the reaction was terminated by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for liquid chromatography to determine the tagatose concentration. After the reaction, 107.6 g / L tagatose was generated from 150 g / L maltodextrin substrate.

[0051] Example 6: Novel Cbα-GP-catalyzed preparation of allulose from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 5 U / mL each of isoamylase IA, glucose phosphate mutase PGM, glucose phosphate isomerase PGI, allulose-6-phosphate epimerase APE, and allulose-6-phosphate dephosphorase T6PP. The reaction was carried out at 55 °C for 24 h. After the reaction was completed, the reaction was terminated by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for allulose concentration detection by liquid chromatography. After the reaction, 110.9 g / L allulose was generated from the 150 g / L maltodextrin substrate.

[0052] Example 7: Novel Cbα-GP-catalyzed preparation of mannitol from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 5 U / mL each of isoamylase IA, glucose phosphate mutase PGM, glucose phosphate isomerase PGI, mannitol-1-phosphate dehydrogenase M1PDH, and mannitol-1-phosphate dephosphorase M1PP. The reaction was carried out at 55 °C for 24 h. After the reaction was completed, the reaction was terminated by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for liquid chromatography to determine the mannitol concentration. After the reaction, 128.3 g / L mannitol was generated from 150 g / L maltodextrin substrate.

[0053] Example 8: Novel Cbα-GP-catalyzed preparation of disaccharides from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and isoamylase IA, glucose, and one of the following disaccharide phosphorylases (choose one from cellobiose phosphorylase, laminarin phosphorylase, trehalose phosphorylase, and sophorose phosphorylase) at 5 U / mL. The reaction was carried out at 55 °C for 24 h. After the reaction was completed, the reaction was terminated by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for liquid chromatography to detect the concentrations of cellobiose, laminarin, trehalose, and sophorose. After the reaction, 150 g / L maltodextrin substrate could generate 119.1 g / L cellobiose, 118.5 g / L laminarin, 90.2 g / L trehalose, and 101.4 g / L sophorose, respectively.

[0054] Example 9: Novel Cbα-GP-catalyzed preparation of fructose-1,6-diphosphate from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg2+, 10 mM pH 6.5 PBS, and 5 U / mL each of isoamylase IA, glucose phosphate mutase PGM, glucose phosphate isomerase PGI, and polyphosphate-dependent phosphofructokinase PPi-PFK. The reaction was carried out at 70 °C for 24 h. After the reaction was completed, the reaction was terminated by boiling, centrifuged at 12000 rpm for 20 min, and the supernatant was passed through an aqueous membrane for liquid chromatography to determine the concentration of fructose-1,6-bisphosphate. After the reaction, 95.2 g / L fructose-1,6-bisphosphate was generated from the 150 g / L maltodextrin substrate.

[0055] Example 10: Novel Cbα-GP-catalyzed preparation of special starch from maltodextrin The purified enzyme (5 U / mL) from Example 2 was added to: 150 g / L maltodextrin, 5 mM Mg 2+ 10 mM pH 6.5 PBS was prepared with 5 U / mL each of isoamylase IA and potato-derived α-glucan phosphorylase PGP, and the reaction was carried out at 37°C for 24 h. After the reaction was completed, the reaction was terminated by centrifugation at 12000 rpm for 20 min. After the reaction, 150 g / L maltodextrin substrate yielded 100.6 g / L of specific starch.

[0056] As can be seen, Cbα-GP in this invention has the activity of releasing glucose-1-phosphate one by one from the non-reducing end of amylose at high temperature, achieving complete phosphate hydrolysis of the substrate, and can be combined with other enzymes to form an in vitro multi-enzyme molecular machine for use in the reaction of producing various functional sugars from starch.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An α-glucan phosphorylase, characterized in that: Its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. A biomaterial related to the α-glucan phosphorylase of claim 1, characterized in that: It can be any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the above-mentioned α-glucan phosphorylase; (a2) An expression cassette containing 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).

3. The biomaterial according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in the sequence listing SEQ ID NO.

2.

4. The biomaterial according to claim 2, characterized in that: The host cell is Escherichia coli.

5. The use of the α-glucan phosphorylase of claim 1 or the biomaterial of any one of claims 2-4 in the catalytic generation of glucose-1-phosphate.

6. The application according to claim 5, characterized in that: The α-glucan phosphorylase or biological material releases glucose-1-phosphate sequentially from the non-reducing end of the amylose chain at high temperature.

7. The application according to claim 5 or 6, characterized in that: The enzyme catalytic system prepares functional sugars using starch or maltodextrin as substrates.

8. The application according to claim 7, characterized in that: The functional sugar is mannose, tagatose, allulose, disaccharide, fructose-1,6-bisphosphate, or a special starch.

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