A method for synthesizing amylose from glucose by a three-enzyme cascade

Abstract

The present application relates to the field of biological catalysis engineering, and particularly relates to a method for synthesizing amylose by three-enzyme cascade catalysis of glucose. The method comprises: phosphorylating the substrate glucose into an intermediate G-6-P in the presence of polyphosphoric acid by a heat-resistant polyphosphate glucose kinase; shifting G-6-P into G-1-P by a heat-resistant phosphoglucomutase, and adding the intermediate G-1-P to the non-reducing end of oligosaccharide to synthesize amylose by a heat-resistant alpha-dextran phosphorylase. The present application optimizes the reaction conditions of the reaction system for synthesizing amylose by glucose, and explores the most suitable metal ion type and concentration, pH, and buffer type. In the reaction process of the three-enzyme cascade catalysis of glucose for preparing amylose after optimization, the conversion rate is 71.93%, the yield reaches 3.5 g / L, and the space-time yield is 2.33 g / L / h.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biocatalysis engineering, and particularly relates to a method for synthesizing amylose by three-enzyme cascade catalysis of glucose. BACKGROUND

[0002] The production of plant-derived starch is highly dependent on the photosynthetic carbon fixation pathway, which is limited by low light energy conversion efficiency and complex metabolic regulation, making it difficult to break through the production bottleneck.

[0003] In recent years, synthetic biology technology has made breakthrough progress in the synthesis of starch from straw, such as the construction of an in vitro enzyme method for cellulose to starch system, which has for the first time realized the conversion of non-food cellulose to amylose. This pathway partially hydrolyzes cellulose to cellobiose by endoglucanase (EG) and cellobiohydrolase (CBH), and then cellobiose is phosphorylated to glucose and glucose-1-phosphate (G-1-P) by cellobiose phosphorylase (CBP), and finally G-1-P is polymerized to a maltose chain to generate amylose by potato alpha-glucan phosphorylase (PGP) from potato. However, the actual starch yield of this pathway is only 2.1%. When cellulose is degraded to cellobiose by EG and CBH, a small part of glucose is generated, which cannot be directly utilized for the synthesis of starch; when cellobiose is phosphorylated by CBP, 1 molecule of glucose and 1 molecule of G-1-P are generated, and G-1-P is utilized for the synthesis of starch, and the equivalent amount of glucose cannot be utilized for the synthesis of starch, resulting in a theoretical conversion rate of less than 50% in the entire pathway.

[0004] The generation of starch depends on the activation of precursors, mainly including phosphorylated glucose derivatives (such as G-1-P and G-6-P) and ADPG specific to plants. Because the α-1,4-glycosidic bond formed between molecules needs to be generated in the in vitro enzyme method for cellulose to starch system, the generated glucose cannot directly participate in starch biosynthesis, and additional energy supply is required. In order to improve the efficiency of synthetic starch, glucose needs to be activated for the synthesis of starch. Hexokinase can be used to convert glucose to G-6-P, but this process consumes ATP, thereby increasing the cost of artificial synthesis of starch, which is not conducive to industrial production.

[0005] The key bottleneck for improving the efficiency of enzyme method for cellulose to starch is the activation of glucose, so it is urgent to develop a simple, low-cost and environmentally friendly method for synthesizing amylose from glucose. SUMMARY

[0006] Since glucose cannot directly participate in starch biosynthesis, the current enzymatic cellulose conversion starch efficiency is low, and therefore how to efficiently utilize glucose to synthesize amylose is a core block of enzymatic cellulose conversion starch.

[0007] The purpose of the present application is to provide a method for synthesizing amylose from glucose by three-enzyme cascade catalysis.

[0008] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present application comprises the following steps:

[0009] In a buffer system, the substrate glucose is phosphorylated to an intermediate G-6-P by polyphosphate glucose kinase in the presence of polyphosphate;

[0010] The G-6-P is mutarotated to an intermediate G-1-P by a heat-resistant phosphoglucomutase;

[0011] The intermediate G-1-P is added to the non-reducing end of an oligosaccharide to synthesize amylose by a heat-resistant alpha-glucan phosphorylase,

[0012] wherein the buffer system contains 1 mM to 5 mM Mg 2+ , 1 mM to 20 mM Co 2+ , or 1 mM to 50 mM Mn 2+ , and the pH of the buffer system is 7.75 to 8.0.

[0013] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present application, wherein the buffer system contains 5 mM Ca 2+ or Ni 2+ , 20 mM Mn 2+ , or 5 mM to 8 mM Co 2+ .

[0014] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present application, wherein the buffer system is a Tris-HCl buffer.

[0015] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present application, wherein the buffer system is a 50 mM Tris-HCl buffer with pH 7.75.

[0016] The method for synthesizing amylose from glucose by three-enzyme cascade catalysis according to the present application, wherein the polyphosphate glucose kinase is a polyphosphate glucose kinase (SEQ ID NO: 3) derived from Thermobifida fusca YX , and the phosphoglucomutase is a phosphoglucomutase derived from Phaeodactylibacter sp.the heat-resistant phosphoglucomutase (SEQ ID NO: 2) of the α-glucan phosphorylase (SEQ ID NO: 1) or a mutant thereof. Thermotoga petrophila the heat-resistant phosphoglucomutase (SEQ ID NO: 2) of the α-glucan phosphorylase (SEQ ID NO: 1) or a mutant thereof.

[0017] The method for synthesizing amylose from glucose by a three-enzyme cascade according to the present application, wherein, at 60°C, the substrate glucose is phosphorylated into the intermediate G-6-P by the polyphosphate glucose kinase in the presence of polyphosphate; the G-6-P is mutased into the intermediate G-1-P by the phosphoglucomutase; the intermediate G-1-P is added to the non-reducing end of the oligosaccharide one by one to synthesize amylose by the α-glucan phosphorylase.

[0018] The method for synthesizing amylose from glucose by a three-enzyme cascade according to the present application, wherein the mutant of the α-glucan phosphorylase is obtained by mutating the α-glucan phosphorylase with the amino acid sequence shown in SEQ ID NO: 1 as follows:

[0019] D774E / L775S / F776I / V777R / Y778L / T779H / Y780L / T781H / N782Q / G783W / V784C, i.e. mutant Tp αGPM3;

[0020] F669S / S670A, i.e. mutant Tp αGPM5,

[0021] the amino acid at position 669 is mutated from Phe to Ser, the amino acid at position 670 is mutated from Ser to Ala, the amino acid at position 774 is mutated from Asp to Glu, the amino acid at position 775 is mutated from Leu to Ser, the amino acid at position 776 is mutated from Phe to Ile, the amino acid at position 777 is mutated from Val to Arg, the amino acid at position 778 is mutated from Tyr to Leu, the amino acid at position 779 is mutated from Thr to His, the amino acid at position 780 is mutated from Tyr to Leu, the amino acid at position 781 is mutated from Thr to His, the amino acid at position 782 is mutated from Asn to Gin, the amino acid at position 783 is mutated from Gly to Trp, and the amino acid at position 784 is mutated from Val to Cys, to obtain mutant F669S / S670A / D774E / L775S / F776I / V777R / Y778L / T779H / Y780L / T781H / N782Q / G783W / V784C, i.e. mutant Tp αGPM7;

[0022] the amino acid at position 485 is mutated from Arg to Lys, to obtain mutant R485K, i.e. mutant Tp αGPR485K;

[0023] amino acid at position 671 is mutated from Pro to Thr, amino acid at position 672 is mutated from Pro to Thr, amino acid at position 673 is mutated from Ala to Arg, amino acid at position 674 is mutated from Cys to Met, and amino acid at position 675 is mutated from Cys to Leu, to obtain mutant P671T / P672T / A673R / C674M / C675L, i.e. mutant Tm αGPM6;

[0024] α-glucan phosphorylase Tp amino acid at position 673 of αGP is mutated from Arg to Ala to obtain mutant R673A, i.e. mutant Tp αGPR673A; or

[0025] amino acid at position 171 is mutated from Glu to Gly to obtain mutant E170G, i.e. mutant Tp αGPM1.

[0026] In an embodiment of the present application, the reaction conditions for the three-enzyme cascade to catalyze the synthesis of amylose from glucose are as follows: 200 μL of reaction system containing 50 mM Tris-Hcl buffer at pH 7.75, 30 mM glucose, 4 mM sodium hexametaphosphate, 20 mM Mn 2+ , 0.1 mM maltotetraose, 3.5 μM polyphosphate glucose kinase, 1 μM phosphoglucomutase and 9 μM α-glucan phosphorylase, and the reaction system is carried out at 60 ℃ for 2 h.

[0027] The sequence of α-glucan phosphorylase derived from Thermotoga petrophila is as follows (SEQ ID NO: 1):

[0028] MLEKLPENLKELESLAYNLWWSWSRPAQRLWRMIDSEKWEEHRNPVKILREVSKERLEELSKDEDFIALYELTLERFTDYMEREDTWFNVNYPEWDEKIVYMCMEYGLTKALPIYSGGLGILAGDHLKSASDLGLPLIAVGLLYKHGYFTQQIDSDGRQIEIFPEYDIEELPMKPLRDEDGNQVIVEVPIDNDTVKARVFEVQVGRVKLYLLDTDFEENEDRFRKICDYLYNPEPDVRVSQEILLGIGGMKLLKTLKIKPGVIHLNEGHPAFSSLERIKSYMEEGYSFTEALEIVRQTTVFTTHTPVPAGHDRFPFDFVEKKLTKFFEGFESKELLMNLGKDEDGNFNMTYLALRTSSFINGVSKLHADVSRRMFKNVWKGVPVEEIPIEGITNGVHMGTWINREMRKLFDRYLGRVWREHTDLEGIWYGVDRIPDEELWEAHLNAKKRFIDYIRESIKRRNERLGINEPLPEISENVLIIGFARRFATYKRAVLLFSDLERLKRIVNNSERPVYIVYAGKAHPRDEGGKEFLRRIYEVSQMPDFKNKIIVLENYDIGMARLMVSGVDVWLNNPRRPMEASGTSGMKAAANGVLNASVYDGWWVEGYNGRNGWVIGDESVLPETEADDPKDAEALYELLENEIIPTYYENREKWIFMMKESIKSVAPKFSTTRMLKEYTEKFYIKGLVNREWLERRENVEKIGAWKERILKNWENVSIERIVLEDSKSVEVTVKLGDLTPNDVIVELVAGRGEGMEDLEVWKVIHIRRYRKENDLFVYTYTNGVLGHLGSPGWFYAVRVIPYHPRLPIKFLPEVPVVWKKVL.

[0029] the phosphoglucomutase sequence (SEQ ID NO: 2) from Phaeodactylibacter sp. is:

[0030] MALHPQAGKTATPDQLVNIPRLITAYFTGQPDPSVREQRVSFGTSGHRGSSLNRSFNEQHILATTQAICLYRQKEGINGPVFMGIDSHALSEPAQATALEVLAANGVETMIAAGDEYTPTPAVSQAILAYNRGRAGGLADGIVITPSHNPPEDGGFKYNMTNGGPAESNVTAWIEAKANELLENGLREVKRIPFQRAMKASTTHRYDYLGAYVNGLGQVIDMDAIRSSGLEMGVDPLGGAGVHYWGHIADHYRLNLTVVDTEVDPTFRFMSLDWDGKIRMDPSSPYAMQRLIRLKDDYPVAFACDTDHDRHGIVTRSAGLMPPNHYLAVAIDYLFRHRPKWKPETGIGKTLVSSQMIDRVAARLGRKLVEVPVGFKWFVDGLFDGSLGFGGEESAGASFLDREGNAWSTDKDGIIAALLAGEITARTGKDPGEIYREFTREFGEPAYGRIDAPATPAQKDKLKKLSREQVTSSQLAGEKIEAILTEAPGNGASIGGLKAVTANGWFAARPSGTEDIYKIYAESFKGEEHLRQLQKEAQELVDRVIG.

[0031] Thermobifida fusca YX The enzyme sequence of the polyphosphate glucose kinase of the present application (SEQ ID NO: 3) is:

[0032] MASRGRVGLGIDIGGSGIKGAPVDLDRGTLVVDPVKIATPQPATPEAVAAVVAEIVTAFADDVPQDAPLGVAFPAVIQHGVARSAANMDRSWIGTNVEELLSAVTGRRVLVVNDADAAAMAEHRYGAASGVDGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETRASASAKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRTPIVPAKLRNTAGIVGAAVLAAERLGGDRVSA.

[0033] Advantages of the present application:

[0034] (1) The application first uses biosynthesis method to prepare amylose with glucose as substrate, compared with chemical method, the method has the advantages of simple operation, mild reaction condition, low cost, green and environmental protection and the like.

[0035] (2) The application optimizes the reaction conditions of the reaction system for synthesizing amylose from glucose, and explores the most suitable metal ion type and concentration, pH, and buffer type. After optimization, the conversion rate of the reaction process of three-enzyme cascade catalysis of glucose for preparing amylose is 71.93%, the yield reaches 3.5 g / L, and the space-time yield is 2.33 g / L / h. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The synthesis route of amylose synthesized from glucose of the application is shown, wherein Glucose: glucose; G-6-P: glucose-6-phosphate; G-1-P: glucose-1-phosphate; Amylose: amylose; PPGK: polyphosphate glucose kinase; PGM: phosphoglucomutase; alphaGP: alpha-glucan phosphorylase;

[0037] Figure 2 The yield of the reaction system“A”“B”“C” for preparing amylose from glucose by three-enzyme cascade catalysis is shown;

[0038] Figure 3 The SDS-PAGE analysis of PGM and other 12 screened PGMs is shown Tk (a) figure: SDS-PAGE analysis of soluble expressed PGM, wherein M: protein Marker; 1: Tk PGM; 2: Phs PGM; 3: Bb PGM; 4: At PGM; 5: Pa PGM; 6: Ds PGM; 7: Ob PGM; 8: Ke PGM; 9: Aa PGM; 10: Prs PGM; 11: Cf PGM, (b) figure and (c) figure Nib PGM and Neb PGM, wherein M: protein Marker; 1: whole cell lysate; 2: cell precipitate; 3: supernatant; 4: flow-through

[0039] Figure 4 The SDS-PAGE analysis of PGM and other 12 screened PGMs is shown TkPGM and other 10 kinds of PGM bi-directional enzyme specific activity which can be soluble expressed, wherein, (a) figure: PGM bi-directional enzyme specific activity detected at 37 DEG C, curve shows 60 DEG C and 37 DEG C forward enzyme specific activity ratio, (b) figure PGM bi-directional enzyme specific activity detected at 60 DEG C;

[0040] Figure 5 Show Tm SDS-PAGE analysis of αGP and other 8 kinds of screened αGP, wherein, (a) figure: SDS-PAGE analysis of αGP which can be soluble expressed, wherein, M: protein Marker; 1: Bb αGP; 2: Mc αGP; 3: Eb αGP; 4: Ar αGP; 5: Tp αGP; 6: Tm αGP, (b) figure, (c) figure, (d) figure: Ms αGP, Ta αGP and Cb SDS-PAGE analysis of αGP, wherein, M: protein Marker; 1: whole cell lysate; 2: cell precipitate; 3: supernatant; 4: flow-through;

[0041] Figure 6 Show Tm Bi-directional enzyme specific activity of αGP and other 5 kinds of αGP which can be soluble expressed, (a) figure αGP bi-directional enzyme specific activity detected at 37 DEG C, curve shows 60 DEG C and 37 DEG C forward enzyme specific activity ratio, (b) figure αGP bi-directional enzyme specific activity detected at 60 DEG C;

[0042] Figure 7 Show Thermotoga petrophila Comparison of bi-directional enzyme activity of α-glucan phosphorylase from different sources and mutants;

[0043] Figure 8 Show the influence of different metal ion concentrations and types on the efficiency of three-enzyme cascade catalysis of glucose to prepare amylose, wherein (a) figure is the influence of 1 mM different metal ion types on the efficiency of three-enzyme cascade catalysis of glucose to prepare amylose, (b) figure is the influence of 5 mM different metal ion types on the efficiency of three-enzyme cascade catalysis of glucose to prepare amylose, (c) figure is the influence of different concentrations of Mn 2+ on the efficiency of three-enzyme cascade catalysis of glucose to prepare amylose, (d) figure is the influence of different concentrations of Co 2+ on the efficiency of three-enzyme cascade catalysis of glucose to prepare amylose;

[0044] Figure 9The effects of different buffers and pH values on the efficiency of the three-enzyme cascade catalysis of glucose to prepare amylose are shown, wherein (a) is a graph showing the effects of different pH values on the efficiency of the three-enzyme cascade catalysis of glucose to prepare amylose, and (b) is a graph showing the effects of different Tris-HCl buffers on the efficiency of the synthesis of amylose;

[0045] Figure 10 The time course analysis of the yield and conversion rate of the three-enzyme cascade catalysis of glucose to prepare amylose after optimization is shown.

[0046] Figure 11 The product characterization is shown. DETAILED DESCRIPTION

[0047] Test materials and reagents

[0048] 1. Strains and vectors: the expression host is BL21 (DE3) Escherichia coli, and the expression plasmid vectors are pET-28a(+) and pET-30a(+).

[0049] 2. Enzymes: endoglucanase is purchased, and ligase is purchased.

[0050] 3. Culture medium:

[0051] (1) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH natural);

[0052] Note: In the following examples, the molecular biology experimental methods not specifically explained are performed according to the specific methods listed in the book “Molecular Cloning Experiment Guide” (third edition) J. Sambrook, or according to the reagent kit and product instructions.

[0053] As shown in Figure 1 , the catalytic pathway of amylose verified according to the present application includes: phosphorylation of the substrate glucose to the intermediate G-6-P in the presence of polyphosphate by the heat-resistant polyphosphate glucose kinase; translocation of G-6-P to G-1-P by the heat-resistant phosphoglucomutase; and synthesis of amylose by adding the intermediate G-1-P to the non-reducing end of oligosaccharide one by one by the heat-resistant α-glucan phosphorylase.

[0054] Example 1, Construction of Recombinant Engineering Bacteria of Polyphosphate Glucose Kinase, Phosphoglucomutase and α-Glucan Phosphorylase and Detection of Amylose

[0055] The specific steps are as follows:

[0056] 1. Construction of Recombinant Engineering Bacteria of Heat-Resistant Polyphosphate Glucose Kinase PPGK and Acquisition of Polyphosphate Glucose Kinase PPGK

[0057] (1) Induced expression of heat-resistant polyphosphate glucose kinase PPGK

[0058] Will come from Thermobifida fusca YX's PPGK F30L / R34P / T72A / V88M / A231T The corresponding gene (ZHOU et al., 2018) was inserted between the BamHI and XholI restriction sites of the pET-28a(+) vector to construct the plasmid pET-28a(+)- tfppgk The plasmid was synthesized, and the recombinant product was then transformed into *E. coli* Trans1-T1 competent cells and plated on LB filtrate (containing 50 μg / mL Kanamycin) for selection. After successful sequencing, the plasmid was transformed into the BL21(DE3) *E. coli* expression host to obtain the recombinant expression strain BL21(DE3)(pET-28a(+)). -tpαgp Positive clones obtained from transformation were picked and inoculated into 40 mL LB liquid medium containing a final concentration of 50 μg / mL Kana, and cultured overnight in a shaker at 37°C and 200 rpm.

[0059] On the second day, 4 mL of seed culture was taken from 40 mL of LB liquid medium and inoculated into 400 mL of LB liquid medium containing a final concentration of 50 μg / mL Kana. The culture was then expanded in a shaker at 37°C and 200 rpm.

[0060] When the bacterial solution OD 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.1 mM and incubate in a shaker at 16 ℃ and 200 rpm for 18 h.

[0061] After induction, the fermentation broth was centrifuged at 6000 rpm for 10 min at 4 °C to collect the cells, and the supernatant was removed. The cell pellet was resuspended in cell lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 10 mM imidazole, pH 7.4), centrifuged at 6000 rpm for 10 min, and the cells were collected again, removing the supernatant. This step was to remove impurities, culture medium components, dead cell debris, and other substances that might interfere with subsequent experiments. Finally, the cells were frozen and stored at -80 °C.

[0062] (2) Purification of heat-resistant polyphosphoglucosinolate kinase

[0063] The target protein encoded by the pET-28a(+) vector has His tags at both the N-terminus and C-terminus. Therefore, the PPGK protein was purified by nickel affinity chromatography, and the purified target protein was desalted by gel filtration chromatography.

[0064] (3) Determination of the enzyme activity of heat-resistant polyphosphate glucokinase

[0065] PPGK enzyme activity unit definition: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol G-6-P per minute using glucose and polyphosphate as substrates, and the unit is U / mg. For the quantitative detection of G-6-P, glucose-6-phosphate dehydrogenase (G6PDH) and nicotinamide adenine dinucleotide (NAD + ) are used as auxiliary reagents. In the specific reaction process, G6PDH catalyzes the reaction of G-6-P and NAD + , converts G-6-P into 6-phospho-D-gluconate-1,5-lactone, and generates reduced nicotinamide adenine dinucleotide (NADH) at the same time. Since NADH has a characteristic absorption peak at 340 nm, its formation will cause a significant change in absorbance at 340 nm. Therefore, by measuring the change in absorbance at 340 nm by spectrophotometry, the amount of G-6-P generated can be indirectly and accurately measured, and the enzyme activity of PPGK can be determined.

[0066] The detection process is divided into two main steps. The first step is to generate the target product G-6-P through an enzymatic reaction. 200 μL of reaction solution contains a final concentration of 12.5 mM glucose, 5 mM MgCl2, 1 mM sodium hexametaphosphate, and an appropriate amount of PPGK. The reaction is started by adding PPGK, and is carried out at a constant temperature of 60 ℃ for 10 min. The reaction is terminated by adding HClO4, and the pH is neutralized with KOH.

[0067] The second step is to generate NADH. 200 μL of reaction solution contains a final concentration of 5 mM MgCl2, 6 mM NAD + , 2 U / mL G6DPH, and an appropriate amount of the first step reaction product. The reaction is started by adding G6DPH, and the reaction kinetics is monitored at 340 nm wavelength. As the reaction proceeds, the generation of NADH will cause the absorbance (OD 340 ) at 340 nm to gradually increase. When the absorbance reaches a stable value and no longer changes significantly, it is determined that the reaction is terminated. All reactions are carried out in triplicate to ensure data accuracy and reliability.

[0068] 2. Construction of heat-resistant phosphoglucomutase PGM recombinant engineering bacteria and obtaining of phosphoglucomutase PGM

[0069] (1) Induced expression of heat-resistant phosphoglucomutase

[0070] The PPGK gene was obtained fromThermococcus kodakarensis of tkpgm The gene (RASHID et al., 2004) was inserted into the BamHI and XholI enzyme digestion sites of the pET-28a(+) vector to construct the plasmid, and the plasmid was synthesized, and other operations refer to polyphosphate glucose kinase.

[0071] (2) Purification of heat-resistant phosphoglucomutase

[0072] The N-terminus and C-terminus of the target protein encoded by the pET-28a(+) vector are both His-tagged, so nickel affinity chromatography is used to purify the PGM protein, and the desalting treatment of the purified target protein is carried out by gel filtration chromatography, and other operations refer to polyphosphate glucose kinase.

[0073] (3) Enzyme activity determination of heat-resistant phosphoglucomutase

[0074] PGM catalyzes the reversible conversion of G-6-P to G-1-P, and according to the demand for PGM in the synthesis of starch in the new pathway, the reaction direction of PGM catalyzing the conversion of G-6-P to G-1-P is defined as forward, and the reaction direction of PGM catalyzing the conversion of G-1-P to G-6-P is defined as reverse.

[0075] The PGM forward reaction enzyme activity unit is defined as: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol of G-1-P per minute using G-6-P as the substrate, and the unit is U / mg. HPAEC-PAD method is used to determine the G-1-P generated in the reaction.

[0076] The chromatographic conditions are as follows: In this experiment, Thermo Scientific Dionex Carbopac PA200 analytical column and guard column are used, the mobile phase A is 0.1 M NaOH solution, the mobile phase B is 0.1 M NaOH solution containing 1 M sodium acetate, the column temperature is set to 30 ℃, and the solute is eluted at a speed of 0.25 mL / min: The initial condition is set to 100% mobile phase A, and then the mobile phase B is increased in proportion to linear gradient elution; from the 8th min, the proportion of mobile phase B is linearly increased from 0% to 10%; from the 15th min, the proportion of mobile phase B is continuously linearly increased to 20%; from the 25th min, the proportion of mobile phase B is exponentially increased to 100% within 2 min, and after the elution is completed, 100% mobile phase A is used for 11 min to balance the column and prepare for the next injection.

[0077] The detection process consists of two steps. The first step involves generating the target product G-1-P through an enzymatic reaction. A 200 μL reaction solution contains a final concentration of 12.5 mM G-6-P, 5 mM MgCl2, and an appropriate amount of PGM. The reaction is initiated by adding PGM, carried out at 60 °C for 10 min, and terminated by a 100 °C water bath for 15 min.

[0078] The second step involved sample processing and analysis using high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD). After terminating the reaction, the sample was centrifuged at 12,000 rpm for 15 min in a 4 °C low-temperature centrifuge. The supernatant was carefully collected and filtered through a 0.22 μm microporous filter to further remove any small particles, prevent column clogging, and ensure sample clarity. The filtered sample was then analyzed using HPAEC-PAD, and the concentration of the target product was determined by comparison with G-1-P standards. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0079] The PGM reverse reaction enzyme activity unit is defined as follows: 1 U is defined as the amount of enzyme required per minute to catalyze the production of 1 μmol of G-6-P using G-1-P as a substrate, expressed in U / mg. The G-6-P generated in the reaction was measured using the high-performance anion exchange-pulse amperometric assay (HPAEC-PAD), following the same procedure as the PGM forward enzyme activity assay. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0080] 3. Construction of thermostable recombinant engineered bacteria for α-glucan phosphorylase (αGP) and obtaining α-glucan phosphorylase (αGP)

[0081] (1) Induction of expression of thermostable α-glucan phosphorylase

[0082] agpA The gene was derived from the work of the Bibel team (BIBEL et al., 1998). It was inserted between the BamHI and NotI restriction sites of the pET-30a(+) vector to synthesize the plasmid. Other operations were performed in accordance with the polyphosphate glucokinase method.

[0083] (2) Purification of thermostable α-glucan phosphorylase

[0084] The target protein encoded by the pET-30a(+) vector has His tags at both the N-terminus and C-terminus. Therefore, nickel affinity chromatography was used to purify the αGP protein, and gel filtration chromatography was used to desalt the purified protein. Tp Except for αGP, all αGPs can be purified to a high purity using nickel affinity chromatography; however, TpThe heat treatment step is still needed after nickel affinity chromatography to obtain relatively pure protein of aGP, and other operations refer to polyphosphate glucose phosphorylase.

[0085] (3) Enzymatic activity determination of thermostable a-glucan phosphorylase

[0086] aGP catalyzes the reversible phosphorolysis of a-1,4-glucan. According to the demand of aGP in the new starch synthesis pathway, the reaction direction of aGP catalyzing the addition of G-1-P to the non-reducing end of a-1,4-glucan to synthesize glycosidic bond is defined as forward, and the reaction direction of aGP catalyzing the attack of phosphate to the non-reducing end of a-1,4-glucan to break the glycosidic bond is defined as reverse.

[0087] The enzyme activity unit of aGP forward reaction is defined as: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol phosphate per minute in the presence of a-1,4-glucan primer, with G-1-P as the substrate, and the unit is U / mg. For the quantitative detection of phosphate, the method for determining inorganic phosphate in a mild pH range proposed by Saheki team (SAHEKI et al., 1985) is referred to.

[0088] 200 μL reaction solution contains maltodextrin with a final concentration of 9 g / L, 12.5 mM G-1-P, 0.1 M sodium acetate with pH 5.5 and appropriate amount of aGP, the reaction is started by adding aGP, 60 ℃ reaction for 10 min, the reaction is terminated by adding 800 μL molybdate reagent (15 mM ammonium molybdate, 100 mM zinc acetate, pH 5.0), followed by adding 200 μL ascorbic acid reagent (10% wt / vol, pH 5.0). The mixture is incubated at 30 ℃ for 20 min, and then the absorbance is measured at 850 nm to record the response of the reaction mixture. All reactions are performed in triplicate to ensure data accuracy and reliability.

[0089] The enzyme activity unit of aGP reverse reaction is defined as: 1 U is defined as the amount of enzyme required to catalyze the production of 1 μmol G-1-P per minute with a-1,4-glucan and phosphate as the substrate, and the unit is U / mg. For the quantitative detection of G-1-P, PGM and G6PDH (glucose-6-phosphate dehydrogenase) cascade is used to convert G-1-P into NADH (reduced nicotinamide adenine dinucleotide), the formation of NADH will cause the change of absorbance at 340 nm, and the production of G-1-P is indirectly measured by spectrophotometry (SUN et al., 2021; YOU et al., 2017).

[0090] The detection process is divided into two steps, the first step generates the target product G-1-P through an enzymatic reaction. 200 μL reaction solution contains final concentration of 20 mM phosphate, 9 g / L malt dextrin, 5 mM MgCl2 and appropriate amount of αGP, the reaction is started by adding αGP, 60 ℃ reaction for 10 min, the reaction is stopped by adding HCIO4 and neutralized pH with KOH.

[0091] The second step reaction, PGM catalyzes the conversion of G-1-P to G-6-P, followed by G6PDH using NAD + As a cofactor, G-6-P is oxidized to 6-phosphogluconolactone, while NADH is generated. 200 μL reaction solution contains 5 mM MgCl2, 6 mM NAD + , 2 U / mL PGM, 2 U / mL G6DPH and appropriate amount of the first step reaction product, the reaction is started by adding G6DPH, and the reaction kinetics is monitored at 340 nm wavelength. When the absorbance (OD 340 ) reaches a stable value and no longer changes, the reaction is determined to be terminated. All reactions are performed in triplicate to ensure data accuracy and reliability.

[0092] 4. Amylose detection

[0093] First, the enzyme cascade reaction system for starch synthesis is constructed: 200 μL reaction system contains 50 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 7.4), 30 mM glucose, 4 mM sodium hexametaphosphate, 5 mM MgCl2, 0.1 mM maltotetraose (Maltotetraose, M4) and appropriate amount of PPGK, PGM and αGP, the reaction system is carried out at 60 ℃ for 2 h.

[0094] After the enzyme cascade reaction, the glucan generated in the sample is treated using the Megazyme total starch assay kit, and the glucose generated in the reaction is determined using high performance anion exchange-pulse amperometric detection (HPAEC-PAD) method. All reactions are performed in triplicate to ensure data accuracy and reliability.

[0095] The chromatographic conditions are as follows: using Thermo Scientific Dionex Carbopac PA200 analytical column and guard column, mobile phase A is deionized water, mobile phase B is 0.1 M NaOH solution, column temperature is set to 30 DEG C, the solute is eluted at a speed of 0.25 mL / min: the initial condition is set to 98% mobile phase A, 2% mobile phase B, then linear gradient elution is carried out by increasing the proportion of mobile phase B; until the 8th min, the proportion of mobile phase B is linearly increased from 2% to 5%; then, the proportion of mobile phase B is increased to 100% within 17 min, and the proportion is maintained for 3 min; starting from the 28th min, the proportion of mobile phase B is decreased from 100% to 2% within 8 min; using 98% mobile phase A, 2% mobile phase B, running for 4 min to balance the chromatographic column, ready for the next sample injection.

[0096] As shown in "A" in the Figure 2 , 10 μM Thermobifida fusca YX-derived PPGK, Thermococcus kodakarensis derived PGM and Thermotoga maritima derived αGP were used, and 50 mM HEPES buffer (pH 7.4), 30 mM glucose, 4 mM sodium hexametaphosphate, 5 mM MgCl2, 0.1 mM maltotetraose were used to construct the initial reaction system "A", and the enzyme cascade reaction was carried out at 60 DEG C for 2 h, and 0.29 g / L starch was synthesized, and the conversion rate was 6.03%.

[0097] Example 2, mining and screening of phosphoglucomutase PGM and alpha-glucan phosphorylase αGP, and preparation of alpha-glucan phosphorylase mutant TpαGPM7

[0098] 1. Mining and screening of phosphoglucomutase

[0099] Twelve phosphoglucomutase sequences were selected, as shown in Table 1.

[0100] Table 1. Twelve different sources of PGM screened

[0101] Gene name Gene source Uniprot ID A0A959BHL9 A0A3D5X8C0 A0A2X0V701 A0A7J4GQ39 A0A2E1ZVD2 A0A7J0BJL5 A0A923U2U6 P38569 A0A1D8QV96 A0A928GH83 A0A386H0X2 A0A533ZIH4

[0102] The 12 PGMs screened and Thermococcus kodakarensis derived PGM ( Tk PGM) were induced for expression, and the experimental results are shown in Figure 3 , except that Nib PGM and Neb PGM exist in the form of inclusion bodies, all PGMs achieve soluble expression, and the soluble components are purified by nickel affinity chromatography.

[0103] Subsequently, 10 types of PGM and templates were analyzed. Tk PGM was used for bidirectional enzyme activity assays under different temperature conditions, such as... Figure 4 As shown, however, no detection was detected under test conditions of 37 ℃ and 60 ℃. At PGM and Cf The forward and reverse enzyme activities of PGM were measured. By comparing the bidirectional enzyme activity of PGM at 37℃ and 60℃ (the ratio of forward enzyme activity at 60℃ to that at 37℃ > 1), PGMs with good thermostability were screened. Phs PGM Bb PGM Ds PGM Ob PGM Ke PGM Aa PGM, ( Figure 4 (Figure (a)). In the new synthetic starch pathway, PGM is required to function in the forward reaction. However, during the screening process, no PGM was found to have a higher specific activity in the forward reaction than in the reverse reaction. Figure 4 (Figure b) Therefore, from the enzymes screened above, the one with the highest ratio of forward enzyme specific activity to reverse enzyme specific activity was selected. Phs PGM (0.065) was used for subsequent experiments.

[0104] 2. Discovery and screening of α-glucan phosphorylases

[0105] 9627 sequences homologous to αGP were retrieved, and 8 αGP sequences were selected, as shown in Table 2.

[0106] Table 2. αGP from 8 different sources obtained through screening.

[0107] Gene name Gene source Uniprot ID D2C4L9 UPI001AE22C4D A0A2N2EZ06 A0A832E8H0 A0A8T4DWL2 14B UPI000989D833 A0A357CTH3 UPI0021151EF9

[0108] For 8 types of αGP and Tm αGP was induced to express, and the experimental results are as follows: Figure 5 As shown, where Ms αGP, Ta αGP and Cb αGP mainly exists in the form of inclusion bodies. The remaining αGPs were expressed in soluble form, and the soluble components could be purified by nickel affinity chromatography. Tp After purification by nickel affinity chromatography, αGP requires heat treatment to obtain protein samples with a purity >90%.

[0109] Subsequently, the purified 5 αGPs and template were analyzed. Tm αGP was used for bidirectional enzyme specific activity assays under different temperature conditions. For example... Figure 6 As shown in Figure (a), only TpThe ratio of the forward enzyme specific activity of the αGP at 60 ℃ to the forward enzyme specific activity at 37 ℃ is > 1. In the new artificial synthetic starch pathway, the function of the forward reaction of the αGP is required. In the screening process, Ar αGP, Bb αGP and Tp The ratio of the forward enzyme specific activity of the αGP at 60 ℃ to the reverse enzyme specific activity is > 1 (b) in FIG. 1, so the αGP is selected Figure 6 for use in subsequent experiments. Tp

[0110] The pathway enzymes obtained by using the screening Phs PGM and Tp αGP, a reaction system "B" is constructed, and other conditions are the same as those of the reaction system "A", and 0.92 g / L of starch is synthesized, which is higher than that of the original pathway enzymes Tk PGM and Tm αGP, and the conversion rate is increased by about 3 times, to 18.87% (d) in FIG. 2. Figure 2 ).

[0111] 3. α-Glucan phosphorylase mutant Tp Preparation of αGPM7

[0112] A mutant primer is designed, and a plasmid pET-28a(+)- tpαgp is used as a template to introduce a mutant amino acid by a point mutation kit. The PCR product containing the mutant amino acid is treated with Dpn I to remove the template. The digested PCR product is transformed into E. coli Tans1-T1 competent cells, and sequencing verification is performed, to obtain E. coli containing the α-glucan phosphorylase mutant plasmid.

[0113] The recombinant plasmid is transformed into BL21(DE3) E. coli expression hosts, respectively, for induced expression, to obtain a recombinant expression strain.

[0114] The catalytic activity is increased by 36.24% at 60 ℃, Tp the forward enzyme activity of the αGPM1 is higher than that of the αGP, Tp the reverse enzyme activity of the αGPM1 is higher than that of the αGP. Tp Tp The catalytic activity is increased by 36.24% at 60 ℃, the forward enzyme activity of the αGPR673A is higher than that of the αGP,

[0115] the reverse enzyme activity of the αGPR673A is higher than that of the αGP. Tp Tp The catalytic activity is increased by 36.24% at 60 ℃, Tp the forward enzyme activity of the αGPR673A is higher than that of the αGP, Tp the reverse enzyme activity of the αGPR673A is higher than that of the αGP.

[0116] ​​At 60℃, the catalytic activity is enhanced. Tp The forward enzyme activity of αGPR485K is relatively high. Tp αGP increased by 9.08%. Tp The reverse enzyme activity of αGPR485K is relatively high. Tp αGP decreased by 2.72%. Tp Compared to αGPR485K Tp αGP prefers to consume G-1-P to synthesize glucans.

[0117] At 60℃, the catalytic activity is significantly improved. Tm The positive enzyme activity of αGPM6 is Tm 34 times that of αGP Tm The reverse enzyme activity of αGPM6 is Tm 37 times that of αGP.

[0118] The catalytic activity is significantly improved at 60℃. Tp The positive enzyme activity of αGPM3 is relatively high. Tp αGP increased by 149.78%. Tp The reverse enzyme activity of αGPM3 is relatively high. Tp αGP increased by 65.79%. Tp The positive enzyme activity of αGPM5 is relatively high. Tp αGP increased by 66.25%. Tp The reverse enzyme activity of αGPM5 is relatively high. Tp αGP increased by 35.81%. Tp The positive enzyme activity of αGPM7 is relatively high. Tp αGP increased by 354.04%. Tp The reverse enzyme activity of αGPM7 is relatively high. Tp αGP increased by 147.93%.

[0119] α-glucan phosphorylase parents and mutants Tp The bidirectional enzyme activity of αGPM7 was determined according to the enzyme activity determination of thermostable α-glucan phosphorylase in Example 1, and the results are as follows. Figure 7 As shown. Using Tp αGPM7 participates in starch synthesis. Reaction system "C" was constructed, and the reaction was continued for 2 hours under the same conditions as "B," synthesizing 1.75 g / L of starch, increasing the conversion rate from 18.87% to 35.98%. Figure 2 ).

[0120] Example 3: Optimization of reaction conditions and product identification were carried out using a glucose-to-amylose synthesis reaction system.

[0121] 1. Effects of different metal ion concentrations and types on the efficiency of amylose synthesis

[0122] The assay was performed at 60 °C and pH 5.5–9.0 [50 mM 2-morpholineethanesulfonic acid (MES) buffer at pH 5.5, 6.0, 6.5, and 7.0; 50 mM HEPES buffer at pH 7.0, 7.5, and 8.0; and 50 mM Tris-HCl buffer at pH 8.0, 8.5, and 9.0]. Phs PGM bidirectional enzyme specific activity was investigated to explore the effects of different pH conditions on its specific activity. Phs The effect of PGM two-way enzyme specific activity. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0123] like Figure 8 As shown in Figure (a), in the artificial starch synthesis system, no additional metal ions are added or monovalent metal ions (Na+) are used. + K + Ag + ) and trivalent metal ions (Fe 3+ ( ), cannot synthesize starch; among divalent metal ions, only the addition of Mg to the reaction system... 2+ Zn 2+ Co 2+ Mn 2+ It can promote starch synthesis. It is worth noting that the addition of Co to the reaction system... 2+ or Mn 2+ Compared to Mg 2+ This demonstrates higher synthesis efficiency.

[0124] like Figure 8 As shown in Figure (b), even increasing the concentrations of monovalent and trivalent metal ions to 5 mM, the artificial starch synthesis system still fails to synthesize starch. Among divalent metal ions, 5 mM Zn... 2+ The presence of [a substance] actually reduced starch synthesis efficiency; in contrast, the addition of 5 mM Ca [a substance] actually reduced starch synthesis efficiency. 2+ or Ni 2+ It can support starch synthesis, but its efficiency is lower than that of Mg. 2+ 5 mMCo 2+ or Mn 2+ Compared to Mg 2+ It still exhibits higher synthesis efficiency, and compared to 1 mM, the addition of 5 mM Co... 2+ or Mn 2+ This further improves starch synthesis efficiency.

[0125] Under the condition of adding 1 mM metal ions, adding Mn 2+ The efficiency of starch synthesis at that time is better than that of adding Co.2+ However, under the condition of adding 5 mM metal ions, the addition of Co 2+ The efficiency of starch synthesis at that time exceeded that of Mn. 2+ This discovery prompted further investigation into different concentrations of Mn. 2+ and Co 2+ The specific impact on starch synthesis efficiency. For example... Figure 8 As shown in Figure (c), add 8 mMCo 2+ At this point, starch synthesis efficiency reaches its highest level, at 2.45 g / L, while... Figure 8 In (d), when 20 mM Mn is added 2+ At this point, the starch synthesis efficiency was the highest, at 3.23 g / L, which was 132% of the former. Therefore, 20 mM Mn was selected. 2+ As a condition for subsequent reactions.

[0126] 2. Effects of different pH values ​​and buffer types on the efficiency of amylose synthesis

[0127] Starch yield from the enzyme cascade reaction was determined at 60 °C and pH 5.5–9.0 (50 mM MES buffer at pH 5.5, 6.0, 6.5, and 7.0; 50 mM HEPES buffer at pH 7.0, 7.5, and 8.0; and 50 mM Tris-HCl buffer at pH 8.0, 8.5, and 9.0) to investigate the effect of different pH conditions on starch synthesis efficiency. All reactions were performed in triplicate to ensure data accuracy and reliability.

[0128] Previous experimental results have confirmed that pH value has an effect on... Phs The specific activity of PGM enzymes was significantly affected. Based on this finding, the effect of different pH values ​​on starch synthesis efficiency was further investigated. Figure 9 As shown in Figure (a), the starch synthesis efficiency reached its highest level (3.39 g / L) under the condition of 50 mM Tris-HCl buffer at pH 8.0, which is 17% higher than that obtained using HEPES buffer at the same pH. This result indicates that, in addition to pH itself, the type of buffer also affects the starch synthesis efficiency. Therefore, the effect of different concentrations of Tris-HCl buffer on starch synthesis efficiency was further investigated in order to find the optimal buffer conditions, such as... Figure 9 As shown in Figure (b), the starch synthesis efficiency reached 3.44 g / L under the conditions of 50 mM Tris-HCl buffer at pH 7.75.

[0129] To utilize Tp The efficiency of starch synthesis by the αGPM7 mutant in the optimized reaction system was precisely determined. For example... Figure 10As shown, starch synthesis reached equilibrium after 90 min. This system can utilize 30 mM glucose to synthesize 3.50 g / L starch, with a conversion rate of 71.93% and a space-time yield of 2.33 g / L / h.

[0130] 3. Product identification

[0131] (1) Preparation and separation of starch

[0132] The enzymatic reaction was terminated by adding HClO4, and the pH of the reaction system was adjusted using KOH. The sample was then centrifuged at 10,000 rpm for 10 min to effectively remove impurities such as salt ions and proteins from the reaction system. The supernatant was collected, and 95% ethanol was added at a 1:2 ratio to promote starch precipitation. The wet starch powder was then washed once with 95% ethanol to ensure the removal of any residual soluble sugars, thus guaranteeing the purity of the final product. Finally, the washed starch was freeze-dried to obtain starch powder.

[0133] (2) Product characterization was performed using iodine staining and spectrophotometry.

[0134] Sodium carboxymethyl cellulose (CMCNa), amylose, maltodextrin, amylopectin, and artificial starch powders were systematically processed. After adding iodine solution to each sample to initiate a colorimetric reaction, the color changes generated by the reaction of different samples with iodine were observed and recorded. Subsequently, the iodine-containing sample mixtures were placed in a spectrophotometer for spectral scanning analysis in the wavelength range of 400 nm to 800 nm.

[0135] like Figure 11 As shown, when CMCNa reacts with iodine / potassium iodide, it displays the original color of the iodine reagent (test tubes 1 and 6). Maltodextrin and amylopectin standards, after reacting with iodine / potassium iodide, exhibit an orange-red color (test tubes 2 and 4). In contrast, amylose and artificial starch, after reacting with iodine / potassium iodide, exhibit a blue-green color (test tubes 3 and 5), and both amylose and artificial starch show a characteristic absorption peak at 600 nm during spectral scanning.

[0136] Based on the results of the iodine colorimetric reaction and spectral scanning, it is indicated that the product generated in the new pathway for artificial starch synthesis is amylose.

[0137] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method of a three-enzyme cascade catalyzing the synthesis of amylose from glucose, characterized in that, The method comprises the following steps: In a buffer system, in the presence of polyphosphoric acid, the substrate glucose is phosphorylated by polyphosphoglucomutase to the intermediate G-6-P, G-6-P is mutated by phosphoglucomutase to the intermediate G-1-P, and the intermediate G-1-P is added one by one to the non-reducing end of oligosaccharide by α-glucan phosphorylase to synthesize amylose, wherein the buffer system contains 1 mM to 5 mM Mg 2+ , 1 mM to 20 mM Co 2+ , or 1 mM to 50 mM Mn 2+ , and the pH of the buffer system is 7.75 to 8.0, The α-glucan phosphorylase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO. 1 as follows: the 774th amino acid is mutated from Asp to Glu, the 775th amino acid is mutated from Leu to Ser, the 776th amino acid is mutated from Phe to Ile, the 777th amino acid is mutated from Val to Arg, the 778th amino acid is mutated from Tyr to Leu, the 779th amino acid is mutated from Thr to His, the 780th amino acid is mutated from Tyr to Leu, the 781th amino acid is mutated from Thr to His, the 782th amino acid is mutated from Asn to Gin, the 783th amino acid is mutated from Gly to Trp, and the 784th amino acid is mutated from Val to Cys; the 669th amino acid is mutated from Phe to Ser, the 670th amino acid is mutated from Ser to Ala, the 774th amino acid is mutated from Asp to Glu, the 775th amino acid is mutated from Leu to Ser, the 776th amino acid is mutated from Phe to Ile, the 777th amino acid is mutated from Val to Arg, the 778th amino acid is mutated from Tyr to Leu, the 779th amino acid is mutated from Thr to His, the 780th amino acid is mutated from Tyr to Leu, the 781th amino acid is mutated from Thr to His, the 782th amino acid is mutated from Asn to Gin, the 783th amino acid is mutated from Gly to Trp, and the 784th amino acid is mutated from Val to Cys.

2. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. The buffer system contains 20 mM Mn 2+ .

3. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. The buffer system is a Tris-HCl buffer.

4. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. The buffer system is a Tris-HCl buffer.

5. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. The polyphosphate glucose kinase is a polyphosphate glucose kinase derived from Thermobifida fusca YX The phosphoglucomutase is a phosphoglucomutase derived from Phaeodactylibacter sp. The alpha-glucan phosphorylase is an alpha-glucan phosphorylase derived from Thermotoga petrophila The alpha-glucan phosphorylase is an alpha-glucan phosphorylase derived from 6. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. The following reaction is carried out at 60°C: The substrate glucose is phosphorylated to the intermediate G-6-P by polyphosphate glucose kinase in the presence of polyphosphate; the G-6-P is mutarotated to the intermediate G-1-P by phosphoglucomutase; and the intermediate G-1-P is added to the non-reducing end of the oligosaccharide one by one to synthesize amylose by α-glucan phosphorylase. The buffer system is a Tris-HCl buffer. The buffer system is a Tris-HCl buffer. Thermobifida fusca YX Phaeodactylibacter sp. Thermotoga petrophila The following reaction is carried out at 60°C: The substrate glucose is phosphorylated to the intermediate G-6-P by polyphosphate glucose kinase in the presence of polyphosphate; the G-6-P is mutarotated to the intermediate G-1-P by phosphoglucomutase; and the intermediate G-1-P is added to the non-reducing end of the oligosaccharide one by one to synthesize amylose by α-glucan phosphorylase.

7. The method of claim 1, wherein the three-enzyme cascade catalyzes the synthesis of amylose from glucose. In the buffer system 50 mM Tris-Hcl buffer pH 7.75, 30 mM glucose, 4 mM sodium hexametaphosphate, 20 mM Mn 2+ , 0.1 mM maltotetraose, 3.5 μΜ polyphosphate glucose kinase, 1 μΜ phosphoglucomutase and 9 μΜ a-glucan phosphorylase, for 2 h at 60 °C.

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