Multi-enzyme system for efficiently synthesizing beta-1, 3-glucan oligosaccharide through one-pot method and application of multi-enzyme system

Abstract

The invention discloses a multienzyme system for efficiently synthesizing beta-1, 3-glucan oligosaccharide through a one-pot method and application, and belongs to the technical field of gene engineering and modern enzyme engineering. The key point of the method is that various high-activity G1P donor generating enzymes and beta-1, 3-gluco-oligosaccharide phosphorylase are systematically screened and optimally combined, and the beta-1, 3-gluco-oligosaccharide with specific polymerization degree distribution can be directly synthesized in a single reactor through a'one-pot method 'by taking cheap and easily available cane sugar, cellobiose or maltodextrin as raw materials. According to the invention, the molar conversion rate of sucrose can reach 98.24%, and beta-1, 3-glucan oligosaccharide in an enzyme reaction product in the 72h can reach DP18, so that the dependence on expensive nucleotide activated sugar is thoroughly eliminated. By adjusting reaction parameters and directionally enriching a product with a target polymerization degree, a green, economical, efficient and large-scale brand-new production path is provided for the functional sugar.

Description

Technical Field

[0001] This invention relates to a one-pot high-efficiency synthesis β The multi-enzyme system and applications of -1,3-glucan oligosaccharides, particularly involving the utilization of sucrose phosphorylase (SP), cellobiose phosphorylase (CBP), maltodextrin phosphorylase (MDP), and... β -1,3-glucosinolate phosphorylase ( β Different combinations of OGPs (oligomeric proton pumpases) were used as starting substrates, sucrose, cellobiose, or maltodextrin, to efficiently synthesize products with specific degree of polymerization distributions via a one-pot synthesis method. β -1,3-glucan oligosaccharide belongs to the fields of genetic engineering and modern enzyme engineering technology. Background Technology

[0002] β -1,3-glucan oligosaccharide ( β -1,3-oligosaccharides (COS) are oligomers with a degree of polymerization of 2 to 10. β -1,3-glucan is a type of... β Linear oligosaccharides linked by -1,3-glycosidic bonds (COS). Besides inducing plant resistance, COS has also been shown to possess various biological activities, including antiviral, anti-allergic, immunomodulatory, and antitumor potential. Due to its low toxicity, high stability, and good biocompatibility, it shows great application potential in medicine, food, and agricultural production.

[0003] However, β The preparation technology of -1,3-glucan oligosaccharides restricts their industrialization. Existing methods fall into two categories: degradation and synthesis. Degradation methods use thermogels as raw materials, prepared through acid hydrolysis or enzymatic hydrolysis. On the one hand, thermogel substrates are expensive; on the other hand, thermogels are insoluble in water and the degree of polymerization of the product is difficult to control. Chemical synthesis involves cumbersome steps and leaves residues, making it difficult to apply to pharmaceuticals and food. In the biosynthetic pathway, nature mainly relies on glycosyltransferases to catalyze the elongation of glucan chains, but the required nucleotide-activated sugars (such as UDP-) are problematic. The high cost of glucose limits its industrial application.

[0004] Phosphorylases provide a new pathway for oligosaccharide synthesis. β -1,3-glucosinolate phosphorylase ( β OGP can catalyze G1P and β Reversible reactions of -1,3-glucan oligosaccharides: In the presence of G1P, polymers are synthesized using glucose or oligomers as primers; conversely, in the presence of G1P, longer-chain oligosaccharides can be synthesized using glucose or short-chain oligosaccharides as primers. β 1,3 Oligosaccharides. This enzyme enables the controlled synthesis of oligosaccharides with varying degrees of polymerization.

[0005] To reduce the supply cost of G1P, other phosphorylases can be used to generate G1P in situ from inexpensive substrates. For example, sucrose phosphorylase (SP) catalyzes the production of G1P and fructose from sucrose; cellobiose phosphorylase (CBP) catalyzes the production of G1P and glucose from cellobiose; and maltodextrin phosphorylase (MDP) catalyzes the production of G1P and short-chain maltodextrin from maltodextrin. These donor-generating enzymes can be combined with… β OGP coupling, in theory, can enable one-pot synthesis from inexpensive raw materials. β 1,3 Glucosamine oligosaccharides enable efficient use of resources and lay the foundation for large-scale production.

[0006] However, phosphorylases from different sources vary in catalytic efficiency, substrate specificity, optimal reaction conditions, and interaction with... β Significant differences exist in the compatibility of OGP. These differences manifest as numerous specific drawbacks when constructing multi-enzyme systems: for example, the G1P supply enzyme and... β The catalytic rates of OGP (one-pot enzyme catalysis) are difficult to match, easily leading to the accumulation or scarcity of reaction intermediates, affecting the overall flow rate and yield. The incompatibility of optimal reaction conditions among different enzymes makes it difficult to find a common "one-pot" reaction condition that allows all enzymes to work efficiently simultaneously. Furthermore, potential substrate competition or unfavorable side reactions between enzymes can also reduce the yield and purity of the target product. Therefore, how to construct a stable, efficient, and controllable multi-enzyme catalytic system from the vast enzyme resources through systematic screening and combination strategies has become a key challenge. β The key technological bottlenecks that must be overcome for the low-cost, large-scale biomanufacturing of -1,3-glucan oligosaccharides.

[0007] Based on this, the present invention aims to develop a highly efficient multi-enzyme conjugation system, thereby overcoming the shortcomings of existing technologies and providing a basis for... β 1,3 The green manufacturing of glucose oligosaccharides offers a completely new solution. Summary of the Invention

[0008] To address the shortcomings of the existing technologies, this invention provides a method for the efficient synthesis of polymers with specific degrees of polymerization using a one-pot multi-enzyme coupling technique. β The catalytic system and method for the hydrolysis production of -1,3-glucan oligosaccharides aim to solve the current problems in the production of -1,3-glucan oligosaccharides. β 1,3 The insolubilities of glucose oligosaccharide raw materials in water and the uncontrollable degree of polymerization of the products, the difficulty and low safety of chemical synthesis, as well as the limited enzyme sources, high acquisition costs, and poor compatibility and efficiency between different enzymes in existing biocatalytic synthesis routes, have limited their widespread application.

[0009] The first technical solution provided by this invention is a method for synthesizing β -1,3-glucose oligosaccharide multienzyme composition, said composition comprising: (1) a first enzyme component: at least one selected from sucrose phosphorylase (SP), cellobiose phosphorylase (CBP) or maltodextrin phosphorylase (MDP), for catalyzing the conversion of said initial glycosyl donor substrate into glucose. 1 Phosphate (G1P); (2) Second enzyme component: β 1,3 Oligosaccharide phosphorylase ( β OGP), used to synthesize using the G1P as a glycosyl donor. β 1,3 Glucooligans.

[0010] In some embodiments, the sucrose phosphorylase (SP) in the first enzyme component is selected from any of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0011] In some embodiments, the cellobiose phosphorylase (CBP) in the first enzyme component is selected from any of the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0012] In some embodiments, the maltodextrin phosphorylase (MDP) in the first enzyme component is selected from the amino acid sequences shown in either SEQ ID NO.8 or SEQ ID NO.9.

[0013] In some embodiments, the second enzyme component β 1,3 Oligosaccharide phosphorylase ( β OGP is selected from amino acid sequences as shown in either SEQ ID NO.10 or SEQ ID NO.11.

[0014] In some embodiments, the nucleotide sequence of the sucrose phosphorylase is as shown in any one of SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15; the nucleotide sequence of the cellobiose phosphorylase is as shown in any one of SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18; and the nucleotide sequence of the maltodextrin phosphorylase is as shown in any one of SEQ ID NO.19 and SEQ ID NO.20. β 1,3 The nucleotide sequence of the oligosaccharide phosphorylase is shown in either SEQ ID NO.21 or SEQ ID NO.22.

[0015] This invention also provides sucrose phosphorylase, cellobiose phosphorylase, and / or maltodextrin phosphorylase in the production of glucose. 1 Phosphoric acid or glucose production 1 Application in products with phosphate as a glycosyl donor, wherein the sucrose phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4; the cellobiose phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7; and the maltodextrin phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9.

[0016] The second technical solution provided by this invention is a method for synthesizing β A multi-enzyme coupling reaction system for -1,3-glucan oligosaccharides, the system comprising: (a) At least one of sucrose, cellobiose, and maltodextrin is selected as the starting glycosyl donor substrate. In reaction systems where the substrate is sucrose, glucose is added as a primer. (b) The multi-enzyme composition described in the first technical solution; In this process, the first enzyme component and the second enzyme component work synergistically in the same reaction system to achieve a one-pot synthesis from the starting glycosyl donor substrate. β 1,3 Glucooligans.

[0017] In some embodiments, the reaction system also contains phosphates.

[0018] The third technical solution provided by this invention is a synthesis β 1,3 The method for producing oligosaccharides involves: utilizing the multi-enzyme coupling reaction system described in the second technical solution at pH 5.0–7.0 and 40–60°C to carry out the reaction, thereby generating… β 1,3 Glucooligans.

[0019] In some embodiments, the method uses sucrose and glucose as substrates, and utilizes a sucrose phosphorylase (SP) selected from any one of SEQ ID NO. 1-4 and a phosphorylase selected from SEQ ID NO. 10 or 11. β 1,3 Oligosaccharide phosphorylase ( β OGP was used as a catalyst in the reaction system to obtain... β -1,3-glucan oligosaccharide.

[0020] In some embodiments, the method uses cellobiose as a substrate and utilizes cellobiose phosphorylase (CBP) selected from any one of SEQ ID NO. 5-7 and CBP selected from SEQ ID NO. 10 or 11. β 1,3 Oligosaccharide phosphorylase ( β OGP was used as a catalyst in the reaction system to obtain... β -1,3-glucan oligosaccharide.

[0021] In some embodiments, the method uses maltodextrin as a substrate and utilizes maltodextrin phosphorylase (MDP) selected from any one of SEQ ID NO. 8-9 and [another enzyme] selected from SEQ ID NO. 10 or 11. β 1,3 Oligosaccharide phosphorylase ( β OGP was used as a catalyst in the reaction system to obtain... β -1,3-glucan oligosaccharide.

[0022] In some embodiments, the second enzyme component ( β The enzyme preparation, immobilized enzyme, recombinant bacteria expressing the enzyme, or recombinant enzyme prepared therefrom are the enzyme preparation, the first enzyme component (sucrose phosphorylase, cellobiose phosphorylase or maltodextrin phosphorylase) and the second enzyme component (1,3-glucose oligosaccharide phosphorylase).

[0023] In some embodiments, the amount of substrate sucrose, cellobiose, or maltodextrin added is 0.5-50% (w / v); the amount of primer glucose added is 0.5-5% (w / v); and the phosphate concentration is 10-200 mM.

[0024] In some embodiments, the β β-1,3-glucan oligosaccharides are unbranched, homogeneous polymers composed of glucose linked by β-1,3-glycosidic bonds, with a degree of polymerization of approximately 2 to 10. β -1,3-glucan.

[0025] The fourth technical solution provided by this invention contains specificβ Recombinant plasmids containing the 1,3-glucosamine phosphorylase gene, recombinant bacteria expressing the enzyme, microbial preparations containing the recombinant bacteria, or immobilized microorganisms. β -1,3-glucan oligosaccharide phosphorylase in phosphorylation β -1,3-glucan oligosaccharides and / or synthesis β The application of -1,3-glucan oligosaccharides, the β The amino acid sequence of -1,3-glucosamine phosphorylase is shown in SEQ ID NO.10 or SEQ ID NO.11.

[0026] The fifth technical solution provided by this invention is a method for synthesizing a specific degree of polymerization distribution. β A method for producing -1,3-glucan oligosaccharides, wherein the method utilizes the multi-enzyme coupling reaction system described in the second technical solution, and achieves the target degree of polymerization (DPn) in the product by adjusting at least one parameter among the following: reaction initiation substrate concentration, inorganic phosphate concentration, primer addition amount, enzyme addition amount, and reaction time. β Enrichment of -1,3-glucan oligosaccharides.

[0027] In some embodiments, the sucrose concentration is 50–800 mg / mL, and when the starting substrate concentration is 50 mg / mL, the degree of polymerization distribution of the product is DP. 2~4 When the initial substrate concentration is 100–200 mg / mL, the degree of polymerization distribution of the product is DP. 2~7 When the starting substrate concentration was 300 mg / mL, the degree of polymerization distribution of the product was DP. 2~8 When the initial substrate concentration is 400–800 mg / mL, the degree of polymerization distribution of the product is DP. 2~9 .

[0028] When the initial sucrose concentration was 500 mg / mL and the initial glucose concentration was 450 mg / mL, and the enzyme addition ratio was 1:1, the product distribution was DP. 2~10 When the enzyme ratio is 2:1 and 5:1, products with DP > 10 can be obtained; when the enzyme ratio is 10:1, the product distribution is as follows: DP > 10. 2~9 When the enzyme ratio is 20:1, the product distribution is DP. 2~8 When the enzyme ratio was 40:1 and 50:1, the product distribution was DP. 2~7 .

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention greatly expands the synthesis β -1,3-glucan oligosaccharide core tool enzyme library. Provides a source as shown in SEQ ID NO.10, derived from... Lewinella sp. of β OGP ( LspβOGP) and SEQ ID NO. 11 (from) Tenuifilaceae bacterium High activity β -1,3-glucosinolate phosphorylase ( Tbβ OGP), with Tbβ Taking OGP as an example, this β The molecular weight of 1,3-glucose oligosaccharide phosphorylase is about 100 kDa. The optimal temperature for synthesis is 40℃, the optimal pH is 4.0, and the enzyme activity is as high as 160.35 U / mg.

[0030] Meanwhile, through extensive screening, this invention has obtained multiple types of high-efficiency and compatible... β OGP-compatible donor phosphorylases, including but not limited to those derived from: Paenibacillus sp. Sucrose phosphorylase (SP) from various microorganisms such as JQZ6Y-1 (SEQ ID NO.2); derived from Paenibacillus athensensis Cellobiose phosphorylase (CBP) such as (SEQ ID NO.5); and derived from Thermotoga maritima Maltodextrin phosphorylase (MDP), such as (SEQ ID NO.8), has been identified. The acquisition and application of these enzymes have solved the problem of… β The limited availability of 1,3-glucosamine phosphorylase and its coupled chaperone enzymes is a key bottleneck.

[0031] (2) This invention constructs a highly efficient and switchable multi-enzyme coupled catalytic system. Based on the rich enzyme library mentioned above, this invention successfully constructs multiple one-pot synthetic pathways starting with inexpensive substrates (sucrose, cellobiose, maltodextrin). For example, using... Psp SP (SEQ ID NO.2) and Tbβ OGP (SEQ ID NO.11) coupling enables efficient synthesis using sucrose as a substrate. β -1,3-glucan oligosaccharide; using Pa CBP (SEQ ID NO.5) and Tbβ OGP (SEQ ID NO.11) conjugation enables the synthesis of cellobiose as a substrate. β -1,3-glucan oligosaccharide; using Dsp MDP (SEQ ID NO.9) and Tbβ OGP (SEQ ID NO.11) coupling enables efficient synthesis of maltodextrin as a substrate. β -1,3-glucan oligosaccharides provide an optimal solution for obtaining high value-added products.

[0032] (3) The preferred enzyme combination provided by this invention exhibits excellent catalytic performance and conversion efficiency. PspTaking SP (SEQ ID NO.2) as an example, its optimal temperature is 50℃, its optimal pH is 4.5, and its enzyme activity is as high as 207.94 U / mg. Comparing it with... Tbβ The coupling reaction was carried out using OGP (SEQ ID NO.11, optimum temperature 40℃, optimum pH 4.0, enzyme activity 154.99 U / mg). Experiments showed that the optimal concentrations were 30% sucrose, 3% glucose, and an enzyme ratio of 2:1 (PspSP:Tb). β Under the conditions of OGP, pH=5.0, and reaction at 50℃ for 12 h, when Psp When the SP enzyme dosage is 150 U / g sucrose β The conversion rate of -1,3-glucan was 98.24%, demonstrating the high efficiency of the conversion under the synergistic effect of the dual-enzyme system.

[0033] (4) The process of this invention is simple, controllable, and green and economical. The entire synthesis process is carried out in a single reactor under mild conditions (pH 5.0-7.0, 40-60℃), without the need for expensive activators and toxic reagents. By adjusting parameters such as substrate concentration, primer (glucose) addition, enzyme addition, and reaction time, the degree of polymerization distribution of the final product can be effectively controlled, achieving the conversion from DP2 to DP3. 18 The enrichment of target oligosaccharides on both sides meets the needs of different application scenarios.

[0034] In summary, this invention, by constructing a tool library containing multiple highly efficient and specific enzymes and a tunable multi-enzyme coupling system composed of them, achieves the efficient, green, and controllable synthesis of enzymes with specific degree of polymerization distributions from a variety of inexpensive raw materials. β -1,3-glucan fundamentally breaks through the technical and economic barriers to the large-scale preparation of this functional oligosaccharide, making it suitable for industrial applications in food, medicine, and other fields, and possessing significant industrial application value. Attached Figure Description

[0035] Figure 1 Recombinant Escherichia coli expression product β SDS-PAGE analysis of -1,3-glucose oligosaccharide / cellobiose / sucrose phosphorylase.

[0036] Figure 2 SDS-PAGE analysis of maltose phosphorylase, a product of recombinant Escherichia coli expression.

[0037] Figure 3 The effect of pH on sucrose phosphorylase activity: (A) SP_Ab, (B) SP_Psp, (C) SP_Pw, (D) SP_Gb.

[0038] Figure 4 For temperature and pH β-1,3-glucosinolate phosphorylase β The effect of OGP_Tb enzyme activity; (A) β OGP_Tb optimal pH, (B) β OGP_Tb optimal temperature.

[0039] Figure 5 For sucrose phosphorylase and β Preparation of -1,3-glucosinolate phosphorylase β Thin-layer chromatogram of -1,3-glucan oligosaccharide.

[0040] Figure 6 For cellobiose phosphorylase and β Preparation of -1,3-glucosinolate phosphorylase β Thin-layer chromatogram of -1,3-glucan oligosaccharide.

[0041] Figure 7 For maltodextrin phosphorylase and β Preparation of -1,3-glucosinolate phosphorylase β Thin-layer chromatogram of -1,3-glucan oligosaccharide.

[0042] Figure 8 Thin-layer chromatograms of product distribution at different sucrose concentrations.

[0043] Figure 9 For sucrose phosphorylase SP_Psp and β -1,3-glucosinolate phosphorylase β The effect of different enzyme ratios added to OGP_Tb on the molar conversion rate of sucrose.

[0044] Figure 10 Thin-layer chromatograms of product distribution with different enzyme addition ratios.

[0045] Figure 11 When the enzyme ratio is 2:1, sucrose is used as a substrate to synthesize... β -1,3-glucan oligosaccharide, anion exchange chromatograms of products at different reaction times. Detailed Implementation

[0046] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0048] Materials and reagents: 2×PrimeSTAR Max Premix, nucleic acid marker, restriction endonuclease, DpnI-enzyme PCR reagents were purchased from TaKaRa Biotechnology Co., Ltd.; primers were purchased from Anshengda Biotechnology Co., Ltd.; glucose-1-phosphate (1PG / G1P) assay kit (enzymatic method) was purchased from Shanghai Jianglai Industrial Co., Ltd.; cellobiose, ammonium molybdate, and zinc acetate were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; EDTA·2Na, citric acid (C6H8O7), sodium citrate (C6H7NaO7), tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), glycine, and other analytical grade and above reagents were purchased from Sinopharm Chemical Co., Ltd.; yeast extract, tryptone, agar, sodium chloride, glucose, sucrose, sodium dihydrogen phosphate, disodium hydrogen phosphate, electrophoresis buffer (TAE), nucleic acid dye (4S Green Plus), kanamycin (Kan), isopropyl- β -D-galactothioglycoside (IPTG), agarose, imidazole (C3H4N2), BCA method protein concentration assay kit for recombinant protein concentration determination, nickel ion chelate affinity chromatography packing material plasmid extraction kit, supercompetent cell preparation kit, agarose purification kit. E. coil DH5α, E. coil All BL21 (DE3) strains were purchased from Sangon Biotech (Shanghai) Co., Ltd.; other reagents were analytical grade reagents purchased domestically or internationally.

[0049] The culture media involved in the following examples are as follows: All culture media were prepared using ddH2O and sterilized at 121℃ for 15-20 min after preparation.

[0050] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0051] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L.

[0052] Example 1: Production of phosphorylase The specific steps are as follows: 1. Construction of recombinant plasmids (1) Enzyme screening Adopted from Alphaproteobacteria bacterium The target protein (protein sequence accession number MDE2112546.1) is derived from... Paenibacillus sp. The target protein of JQZ6Y-1 (protein sequence accession number WP_367060030.1) is derived from... Paenibacillus wynnii The target protein (protein sequence accession number WP_307585576.1) is derived from... Gammaproteobacteria bacteriumThe target protein (protein sequence accession number MDE1923982.1) is sucrase phosphorylase; Adopted from Paenibacillus athensensis The target protein (protein sequence accession number WP_134748827.1) is derived from... Bacteroidaceae bacterium The target protein (protein sequence accession number MBO4215563.1) is derived from... Segatella copri The target protein (protein sequence accession number WP_317608049.1) is cellobiose phosphorylase; Adopted from Thermotoga maritima Target protein, derived from Dictyoglomus sp. NZ13-RE01 targets the maltodextrin phosphorylase; Adopted from Tenuifilaceae bacterium The target protein (protein sequence accession number HRX31957.1) is derived from... Lewinella sp. The target protein (protein sequence accession number: MFK8162018.1) β -1,3-glucosinolate phosphorylase.

[0053] (2) Synthesis of the target gene The target protein gene fragments with the synthesized sequences shown in SEQ ID NO.12~22 were used as vectors for pET-28a(+) and the following PCR amplification reactions were performed: SP_Ab-F: 5'-AGCGGCCTGGTGCCGCGCGGCAGCCATATGAAAAACAAACTGCAGCTGATTGCGTA-3' SP_Ab-R: 5'-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCGCAAAAATGCGTTCGCCCAGGCCT-3' SP_Psp-F: 5'-AGCGGCCTGGTGCCGCGCGGCAGCCATATGAGCATTCAGAACAAAGCGATGCTGATTAC-3' SP_Psp-R: 5'-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCAGCACGCTCTGCCAGCCCGCGCCGT-3' SP_Pw-F: 5’-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGACCATTAAAAACAACGTGATGCT-3’ SP_Pw-R: 5’-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCACATCTTTCAGATAGCTTTC-3’ SP_Gb-F: 5’-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGACCGATATTGCGATTGATTGCAGCGGCACCGA-3’ SP_Gb-R: 5’-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCGCCGCGCCGCTCGCTTCCACG-3’ CBP_Pa-F: 5’-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAATTTGGCTATTTTGATGATGCGAGCAA-3’ CBP_Pa-R: 5’-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGGCCCAGCACCACTTCCACTTCATGGG-3’ CBP_Bb-F: 5’-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAAACCTTTGGCCATTTTGATGAT-3’ CBP_Bb-R: 5’-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCATAATCACTTTCACAATATGA-3’ CBP_Sc-F: 5’-AGCGGCCTGGTGCCGCGCGGCAGCCATATGGCGGGCAAAAAATATGGCCATTTTGATGATGCG-3’ CBP_Sc-R: 5’-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGCATTTCCACAACCACTTCATGTTTGC-3’ β OGP_Tb-F: 5'-GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAACAACCTGTATATTGGCAACACCAAAGCG-3' β OGP_Tb-R: 5'-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGGGTCAGTTTGTTTTTCTGAATATGCA-3' β OGP_Lsp-F: 5'-GCAGCGGCCTGGTGCCGCGCGGCAGCATATGCCGGAAATTCAGCAAGTGGAACTGAAAGG-3' β OGP_Lsp-R: 5'-GCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTTCAGCACTTCCGCCGGCACGT-3' The PCR reaction conditions are shown in Tables 1 and 2.

[0054] Table 1: Reaction System

[0055] Table 2: Reaction Conditions

[0056] Recombinant plasmids pET-28a(+)-SP_Ab, pET-28a(+)-SP_Psp, pET-28a(+)-SP_Pw, pET-28a(+)-SP_Gb, pET-28a(+)-CBP_Pa, pET-28a(+)-CBP_Bb, pET-28a(+)-CBP_Sc, pET-28a(+)-MDP_Tm, pET-28a(+)-MDP_Dsp, and pET-28a(+)- β OGP_Tb、pET-28a(+)- β OGP_Lsp.

[0057] 2. Construction of engineered bacteria The recombinant plasmids pET-28a(+)-SP_Ab, pET-28a(+)-SP_Psp, pET-28a(+)-SP_Pw, pET-28a(+)-SP_Gb, pET-28a(+)-CBP_Pa, pET-28a(+)-CBP_Bb, pET-28a(+)-CBP_Sc, pET-28a(+)-MDP_Tm, pET-28a(+)-MDP_Dsp, and pET-28a(+)- β OGP_Tb、pET-28a(+)- β OGP_Lsp uses a thermally activated chemical transformation method to transfer the synthesized recombinant plasmid into... E. coli BL21 (DE3) competent cells were used to obtain the corresponding recombinant engineered bacteria. E. coli BL21 (DE3) / pET-28a(+)-SP_Ab, E. coli BL21 (DE3) / pET-28a(+)-SP_Psp, E. coli BL21 (DE3) / pET-28a(+)-SP_Pw, E. coli BL21 (DE3) / pET-28a(+)-SP_Gb, E. coli BL21 (DE3) / pET-28a(+)-CBP_Pa, E. coli BL21 (DE3) / pET-28a(+)-CBP_Bb, E. coli BL21 (DE3) / pET-28a(+)-CBP_Sc, E. coli BL21 (DE3) / pET-28a(+)-MDP_Tm, E. coli BL21 (DE3) / pET-28a(+)-MDP_Dsp, E. coli BL21 (DE3) / pET-28a(+)- β OGP_Tb、 E. coli BL21 (DE3) / pET-28a(+)- β OGP_Lsp.

[0058] 3. Expression Induction (1) The recombinant engineered bacteria obtained in step 2 E. coli BL21 (DE3) / pET-28a(+)-SP_Ab, E. coli BL21 (DE3) / pET-28a(+)-SP_Psp, E. coli BL21 (DE3) / pET-28a(+)-SP_Pw, E. coli BL21 (DE3) / pET-28a(+)-SP_Gb, E. coli BL21 (DE3) / pET-28a(+)-CBP_Pa, E. coli BL21 (DE3) / pET-28a(+)-CBP_Bb, E. coli BL21 (DE3) / pET-28a(+)-CBP_Sc, E. coli BL21 (DE3) / pET-28a(+)-MDP_Tm, E. coli BL21 (DE3) / pET-28a(+)-MDP_Dsp, E. coli BL21 (DE3) / pET-28a(+)- β OGP_Tb、 E. coli BL21 (DE3) / pET-28a(+)- β OGP_Lsp was streaked from a glycerol tube to an LB solid medium plate (containing 50 μg / mL Kan, incubated upside down at 37 °C for 12 h).

[0059] (2) Pick a single colony from LB solid medium and culture it in 5 mL LB liquid medium containing 50 μg / mL Kan at 37 ℃ and 200 r / min for 12 h to prepare seed liquid. (3) The prepared seed culture was transferred to 50 mL LB liquid medium containing Kan at an inoculation rate of 2% (v / v) and cultured at 37 °C and 200 r / min until OD. 600 The pH value was around 0.6. IPTG was added to bring the final concentration of IPTG in the culture medium to 0.1 mmol / L. After induction at 16 ℃ and 200 r / min for 24 h, the fermentation broth was obtained.

[0060] (4) Centrifuge the fermentation broth at 4 ℃ and 6000 rpm for 10 min. Resuspend the cells in cell lysis buffer, then sonicate for 15 min (1 s supernatant, 2 s rest). Take 40 μL of crude enzyme solution, i.e., whole cells, lysed supernatant, and lysed precipitate, and perform protein gel electrophoresis at 120 V for 1 h. Figure 1 As shown, β -1,3-glucanase has a molecular weight of around 50 kDa.

[0061] 4. Protein purification The fermentation broth obtained in step 3 (3) was centrifuged at 4 ℃ and 6000 rpm for 10 min, the supernatant was removed, and the broth was resuspended in an equal volume of 50 mM phosphate buffer (pH 7.0). Then, it was sonicated for 15 min (1 s supernatant followed by a 2 s rest). The supernatant was collected by centrifugation, and impurities were removed by passing it through a 0.45 μm aqueous membrane. The membrane was then connected to the constant flow pump and Ni in the protein purification system. 2+ For the tubing connecting the affinity chromatography column, UV detector, and other components, set the constant flow pump to 1 mL / min and check for leaks using deionized water. After the protein purification system is ready, equilibrate Ni with two column volumes of binding buffer (50 mM Tris, 500 mM NaCl, pH=7.0). 2+ Affinity chromatography column was used, and the crude protein solution was pumped into the column at a flow rate of 0.5 mL / min. After all the crude protein solution had entered the column, unadsorbed protein and other impurities were washed away with binding buffer. After the detector reading stabilized, washing buffer (50 mM imidazole, 50 mM Tris, 500 mM NaCl, pH=7.0) was pumped in to wash away weakly binding proteins. After the detector reading stabilized again, elution buffer (500 mM imidazole, 50 mM Tris, 500 mM NaCl, pH=7.0) was pumped in to elute the adsorbed recombinant protein, and the eluent was collected according to the UV detector signal value, which is the target recombinant protein. The target recombinant protein solution obtained above was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. After clamping with dialysis clamps, the bag was placed in dialysis solution A (10 mM EDTA·2Na, 50 mM Tris, pH=7.0) and in a chromatography cabinet at 4 ℃ for dialysis for 18 h. Fresh dialysis solution was replaced every 6 h to remove imidazole and other metal ions from the target recombinant protein solution. The dialysis bag was then transferred to dialysis solution B (50 mM Tris, pH=7.0) and dialyzed for another 18 h, with fresh dialysis solution replaced every 6 h. After dialysis, the target recombinant protein solution was collected into a tube, which is the pure enzyme solution.

[0062] Example 2: Performance of phosphorylase (1) SDS-PAGE protein electrophoresis Gel preparation: Remove the pre-made protein gel from the refrigerator.

[0063] Sample loading: Mix the pure enzyme solution and protein loading buffer (5×) at a ratio of 4:1, boil in a water bath for 5 min, and centrifuge at 12000 r / min for 5 min. Carefully pipette 20 μL of sample supernatant and slowly add it to the loading well, using 5 μL of protein marker as a reference.

[0064] Electrophoresis: First, adjust the voltage to 80 V for 30 min, then adjust it to 120 V and wait for the protein bands to move to about 5 mm from the bottom edge, which takes about 60 min.

[0065] Staining / Destaining: Pour an appropriate amount of staining solution into a petri dish. Use the tip of a plastic plate to peel the gel apart from both sides and immerse it in the staining solution. After 40 minutes, remove the gel and replace it with the prepared destaining solution. First, quickly wash once with the destaining solution, then change the destaining solution every two hours until the protein bands become clear and the background becomes transparent. Observe and photograph the gel using a gel imaging system. Results are as follows: Figure 1 , Figure 2 As shown.

[0066] (2) Protein concentration was determined using the BCA method: a. Preparation of BCA working solution. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA Reagent B to 50 volumes of BCA Reagent A (50:1) and mix thoroughly.

[0067] b. Standard curve plotting. Take an ELISA plate and add reagents according to the data in the table below:

[0068] c. Sample preparation: Dilute the protein sample to be tested with deionized water to an appropriate concentration, take 20 μL of sample and add 200 μL of BCA working solution.

[0069] d. After shaking and mixing, place at 37°C for 20 to 30 minutes.

[0070] e. Measure the absorbance at A562 nm using an ELISA reader, with the absorbance value without BSA as a blank control.

[0071] f. Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis.

[0072] g. The protein content of the sample can be calculated from the standard curve based on the measured absorbance value.

[0073] h. Calculate protein concentration: Divide the protein content found by the enzyme solution volume of 20 μL, and then multiply by the corresponding dilution factor to obtain the actual concentration of the sample to be tested.

[0074] (3) The enzyme activity of sucrose / cellobiose / maltodextrin phosphorylase of the purified target recombinant protein prepared in Example 1 was determined. The specific steps are as follows: Sucrose phosphorylase: Prepare 5% (w / v) sucrose using acetic acid-sodium acetate solution (pH 4.5 / 5.0) and citric acid-sodium citrate solution (pH 5.5). Take 100 μL of sucrose, add 10 μL of 50 mM phosphate solution and 20 μL of pure enzyme, and react at 40 / 50℃ for 30 min. Measure the G1P content.

[0075] Cellobiose phosphorylase: Prepare 0.5% (w / v) cellobiose in a pH 6.5 Tirs-HCl solution. Take 60 μL, add 40 μL of 50 mM phosphate solution and 100 μL of pure enzyme, and react at 60℃ for 30 min. Measure the G1P content.

[0076] Maltodextrin phosphorylase: Prepare 0.5% (w / v) maltodextrin in an acetate-sodium acetate solution at pH 5.5. Take 60 μL of the solution, add 40 μL of 50 mM phosphate solution and 100 μL of pure enzyme, and react at 70℃ for 30 min. Measure the G1P content.

[0077] An enzyme activity unit (1U) is defined as the amount of enzyme required to produce 1 μg of glucose-1-phosphate in 1 minute under the above reaction conditions. The calculation formula is as follows:

[0078] The test results show that: The protein concentration of sucrose phosphorylase SP_Ab was 0.36 mg / mL, and the enzyme activity was 852.00 U / mg under the conditions of pH 5.5 and 40℃. The protein concentration of sucrose phosphorylase SP_Psp was 1.45 mg / mL, and the enzyme activity was 207.94 U / mg under the conditions of pH 4.5 and 50℃. The protein concentration of sucrose phosphorylase SP_Pw was 0.45 mg / mL, and the enzyme activity was 370.93 U / mg under the conditions of pH 5.5 and 50℃. The protein concentration of sucrose phosphorylase SP_Gb was 0.28 mg / mL, and the enzyme activity was 409.01 U / mg under the conditions of pH 5.0 and 40℃. The protein concentration of cellobiose phosphorylase CBP_Pa was 0.42 mg / mL, and the enzyme activity was 11.69 U / mg under the conditions of pH 6.5 and 60℃. The protein concentration of cellobiose phosphorylase CBP_Bb was 0.25 mg / mL, and the enzyme activity was 2.63 U / mg under the conditions of pH 6.5 and 60℃. The protein concentration of cellobiose phosphorylase CBP_Sc was 0.96 mg / mL, and the enzyme activity was 3.14 U / mg under the conditions of pH 6.5 and 60℃. The protein concentration of maltodextrin phosphorylase MDP_Tm was 0.35 mg / mL, and the enzyme activity was 26.45 U / mg under the conditions of pH 5.5 and 70℃. The protein concentration of maltodextrin phosphorylase MDP_Dsp was 0.36 mg / mL, and the enzyme activity was 22.68 U / mg under the conditions of pH 5.5 and 70℃.

[0079] (4) Determination of the purified target recombinant protein prepared in Example 1 β The enzyme activity of -1,3-glucan oligosaccharide phosphorylase is determined by the following steps: Prepare 250 mM glucose solution using acetic acid-sodium acetate solution at pH 4.0 and Tirs-HCl at pH 6.5. Take 20 μL of the glucose solution, add 100 μL of 160 mM G1P and 80 μL of pure enzyme diluted 10 times, and react at 40 / 55℃ for 10 min. Measure the free phosphorus content.

[0080] Enzyme activity unit (1U) is defined as: the amount of enzyme required to produce 1 μmol of free phosphorus in the reaction system within 1 minute under the above reaction conditions. The formula for calculating enzyme activity is as follows: ; The test results showed that the pure enzyme solution yielded the following results: β -1,3-glucosinolate phosphorylase β The protein concentration of OGP_Tb was 0.43 mg / mL, and the enzyme activity was 154.99 U / mg under the conditions of pH 4.0 and 40℃. β -1,3-glucosinolate phosphorylase β The protein concentration of OGP_Lsp was 0.36 mg / mL, and the enzyme activity was 22.68 U / mg under the conditions of pH 6.5 and 55℃.

[0081] Example 3: Determination of the properties of sucrose phosphorylase The activity of sucrose phosphorylase according to the present invention was measured as a function of temperature in the pH range of 4.0 to 8.0 and in the temperature range of 30°C to 60°C.

[0082] The specific steps are as follows: (1) Recombinant engineered bacteria obtained using the codon-optimized enzyme obtained in Example 1 E. coli BL21(DE3) / pET-28a(+)-SP_Ab、 E. coli BL21 (DE3) / pET-28a(+)-SP_Psp, E. coli BL21(DE3) / pET-28a(+)-SP_Pw、 E. coli BL21 (DE3) / pET-28a(+)-SP_Gb is a purified target protein sucrose phosphorylase obtained by fermentation according to the method in Example 1.

[0083] The obtained pure enzyme solutions were boiled and inactivated according to the protein concentration and enzyme activity determination methods in Example 2, and centrifuged to determine the G1P content and enzyme activity data in the reaction solution.

[0084] The preparation methods for 5% (w / v) sucrose solutions with pH = 4.0, pH = 4.5, pH = 5.0, and pH = 5.5 are as follows: dissolve 5 g of sucrose in 100 mL of acetate-sodium acetate buffer (at pH = 4.0, pH = 4.5, pH = 5.0, and pH = 5.5, respectively) to form a 50 mg / mL sucrose solution; the preparation methods for 5% (w / v) sucrose solutions with pH = 5.5, pH = 6.0, pH = 6.5, and pH = 7.0 are as follows: dissolve 5 g of sucrose in 100 mL of citrate-sodium citrate buffer (at pH = 5.5, pH = 6.0, pH = 6.5, and pH = 7.0, respectively) to form a 50 mg / mL sucrose solution; the preparation methods for 5% (w / v) sucrose solutions with pH = 7.0, pH = 7.5, pH = 8.0, and pH = 8.5 are as follows: dissolve 5 g of sucrose in 100 mL of acetate-sodium acetate buffer (at pH = 4.0, pH = 4.5, pH = 5.0, and pH = 7.0, respectively) to form a 50 mg / mL sucrose solution; the preparation methods for 5% (w / v) sucrose solutions with pH = 7.0, pH = 7.5, pH = 8.0, and pH = 8.5 are as follows: dissolve 5 g of sucrose in 100 mL of citrate-sodium citrate buffer (at pH = 5.5, pH = 6.0, pH = 6.5, and pH = 7.0, respectively) to form a 50 mg / mL sucrose solution; the preparation methods for 5% (w / v) sucrose solutions with pH Dissolve sucrose in Tris-HCl buffer (at pH 7.0, pH 7.5, and pH 8.0, respectively) to form 50 mg / mL sucrose solutions; prepare 5% (w / v) sucrose solutions at pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0, respectively.

[0085] The SP_Ab results show: In acetate-sodium acetate buffer, the relative enzyme activity was 50.33% at pH 4.5, 87.95% at pH 5.0, and 68.40% at pH 5.5. In citrate-sodium citrate buffer, the relative enzyme activity was 100.00% at pH 5.5, 87.14% at pH 6.0, 42.90% at pH 6.5, and 60.05% at pH 7.0. In Tris-HCl buffer, the relative enzyme activity was 45.22% at pH 7.0, 59.37% at pH 7.5, 27.39% at pH 8.0, and 6.72% at pH 8.5.

[0086] The SP_Psp results show: In acetate-sodium acetate buffer, the relative enzyme activity was 51.52% at pH 4.0; 100.00% at pH 4.5; 87.15% at pH 5.0; and 78.82% at pH 5.5. In citrate-sodium citrate buffer, the relative enzyme activity was 84.20% at pH 5.5; 79.22% at pH 6.0; 75.34% at pH 6.5; and 69.86% at pH 7.0. In Tris-HCl buffer, the relative enzyme activity was 54.34% at pH 7.0; 42.86% at pH 7.5; and 40.97% at pH 8.0.

[0087] The SP_Pw results show: In acetate-sodium acetate buffer, the relative enzyme activity was 4.81% at pH 4.0 and 73.71% at pH 4.5. At pH 5.0, the relative enzyme activity was 84.27%; at pH 5.5, the relative enzyme activity was 77.36%; in citrate-sodium citrate buffer, at pH 5.5, the relative enzyme activity was 100.00%; at pH 6.0, the relative enzyme activity was 85.06%; at pH 6.5, the relative enzyme activity was 85.61%; at pH 7.0, the relative enzyme activity was 75.41%; in Tris-HCl buffer, at pH 7.0, the relative enzyme activity was 61.41%; at pH 7.5, the relative enzyme activity was 80.51%; and at pH 8.0, the relative enzyme activity was 54.01%.

[0088] The SP_Gb results show: In acetate-sodium acetate buffer, the relative enzyme activity was 64.11% at pH 4.5, 100.00% at pH 5.0, and 97.79% at pH 5.5. In citrate-sodium citrate buffer, the relative enzyme activity was 71.76% at pH 5.5, 83.48% at pH 6.0, 66.14% at pH 6.5, and 94.47% at pH 7.0. In Tris-HCl buffer, the relative enzyme activity was 60.37% at pH 7.0, 44.65% at pH 7.5, and 27.82% at pH 8.0.

[0089] (2) The pure enzyme solution obtained in Example 1 was added to the reaction system of Example 2 and reacted at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 30 min respectively. After boiling to inactivate, the reaction solution was centrifuged to obtain the reaction solution. The G1P content and enzyme activity data in the reaction solution were measured.

[0090] The SP_Ab results show: The relative enzyme activity was 82.13% when reacting at 35℃; 100.00% when reacting at 40℃; 78.16% when reacting at 45℃; 24.79% when reacting at 50℃; 51.83% when reacting at 55℃; and 33.94% when reacting at 60℃.

[0091] The results showed that the optimal pH for G1P production by recombinant sucrose phosphorylase SP_Ab was 5.5, and the optimal temperature was 40 ℃.

[0092] The SP_Psp results show: The relative enzyme activity was 49.22% at 30℃, 78.12% at 35℃, 94.60% at 40℃, 85.20% at 45℃, 100.00% at 50℃, 67.16% at 55℃, and 64.47% at 60℃.

[0093] The results showed that the optimal pH for G1P production by recombinant sucrose phosphorylase SP_Psp was 4.5, and the optimal temperature was 50 ℃.

[0094] The SP_Pw results show: The relative enzyme activity was 58.85% at 30℃, 63.39% at 35℃, 77.89% at 40℃, 81.80% at 45℃, 100.00% at 50℃, 80.75% at 55℃, and 57.57% at 60℃.

[0095] The results showed that the optimal pH for G1P production by recombinant sucrose phosphorylase SP_Pw was 5.5, and the optimal temperature was 50 ℃.

[0096] The SP_Gb results show: The relative enzyme activity was 71.70% at 30℃, 60.33% at 35℃, 100.00% at 40℃, 49.89% at 45℃, 24.43% at 50℃, 36.50% at 55℃, and 19.83% at 60℃.

[0097] The results showed that the optimal pH for G1P production by recombinant sucrose phosphorylase SP_Gb was 5.0, and the optimal temperature was 40 ℃.

[0098] Example 4: Determination of the properties of oligosaccharide phosphorylase The enzyme activity of the sucrose phosphorylase according to the present invention was measured as a function of temperature in the pH range of 3.5 to 8.5 and in the temperature range of 30 °C to 70 °C.

[0099] The specific steps are as follows: (1) Recombinant engineered bacteria obtained using the codon-optimized enzyme obtained in Example 1 E. coli BL21(DE3) / pET-28a(+)- β OGP_Tb is a purified target protein sucrose phosphorylase obtained by fermentation according to the method in Example 1. The obtained pure enzyme solutions were boiled and inactivated according to the protein concentration and enzyme activity determination methods in Example 2, and centrifuged to determine the content of free phosphorus and enzyme activity data in the reaction solution.

[0100] The preparation methods for 250 mM glucose solutions with pH values ​​of 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 are as follows: 4.5 g of glucose is dissolved in 100 mL of acetate-sodium acetate buffer (at pH values ​​of 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0, respectively) to form a 45 mg / mL sucrose solution. The preparation methods for 250 mM glucose solutions with pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 are as follows: 5 g of sucrose is dissolved in 100 mL of Tris-HCl buffer (at pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively) to form a 45 mg / mL sucrose solution. Solutions with pH values ​​of 3.5, 4.0, 4.5, 5.0, 5.5, 5.5, and 6.0 are prepared accordingly. 250 mM glucose solution with pH values ​​of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5.

[0101] β The OGP_Tb results show: In acetate-sodium acetate buffer, the relative enzyme activity was 2.08% at pH 3.5; 100.00% at pH 4.0; 88.53% at pH 4.5; 83.35% at pH 5.0; 81.96% at pH 5.5; and 76.17% at pH 6.0. In Tris-HCl buffer, the relative enzyme activity was 77.70% at pH 6.0; 74.33% at pH 6.5; 69.86% at pH 7.0; 60.62% at pH 7.5; 41.11% at pH 8.0; and 22.96% at pH 8.5.

[0102] (2) The pure enzyme solution obtained in Example 1 was added to the reaction system of Example 2 and reacted at 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ for 10 min respectively. After boiling to inactivate, the reaction solution was centrifuged to obtain the reaction solution. The free phosphorus content and enzyme activity data in the reaction solution were measured.

[0103] β The OGP_Tb results show: The relative enzyme activity was 79.66% at 30℃; 88.71% at 35℃; 100.00% at 40℃; 78.80% at 45℃; 39.93% at 50℃; 26.53% at 55℃; 12.38% at 60℃; 11.06% at 65℃; and 0.84% ​​at 70℃.

[0104] The results are as follows Figure 4 As shown, recombinant sucrose phosphorylase β The optimal pH for OGP_Tb to produce G1P is 4.0, and the optimal temperature is 40℃.

[0105] Example 5: Sucrose / cellobiose / maltodextrin phosphorylase and β -1,3-glucosinolate phosphorylase coupled catalysis to prepare β -1,3-glucoligosaccharide (1) Sucrose phosphorylase and β -1,3-glucosinolate phosphorylase coupled catalysis to prepare β -1,3-glucoligosaccharide After mixing 300 μL of 50 mM phosphate with 400 μL of 50 mg / mL sucrose and 20 μL of 45 mg / mL glucose, 200 μL of sucrose phosphorylase prepared in Example 1 and 80 μL of [prepared solution] were added. β The purified enzyme solution of -1,3-glucosinolate phosphorylase was reacted for 30 min under the optimal conditions of sucrose phosphorylase described in Example 3. Then the reaction system was heated at 100°C for 10 min to stop the reaction. The supernatant was collected by centrifugation, and the composition of the product was analyzed by thin-layer chromatography (TLC).

[0106] The specific steps for thin-layer chromatography detection are as follows: High-performance silica gel thin-layer chromatography was performed using aluminum foil plates; the developing solvent was n-butanol-acetic acid-water (2:1:1, v / v / v). 2 μL of the reference standard and sample were spotted separately and placed in the developing solvent for chromatography. After chromatography, the plates were dried and then sprayed with a colorimetric reagent (0.2% w / v lichenin, 75% v / v anhydrous ethanol, 10% v / v concentrated sulfuric acid) and developed at 105 °C for 5 min.

[0107] Sucrose phosphorylase and β Different degree of polymerization distributions were prepared by coupled catalysis with 1,3-glucose oligosaccharide phosphorylase. β -1,3-glucan oligosaccharide. Results as follows: Figure 5 As shown.

[0108] The results showed that sucrose phosphorylases from all four sources could interact with... β -1,3-glucan oligosaccharide phosphorylase reaction produces β -1,3-glucan oligosaccharides, of which SP_Psp and β The molecular weight distribution (DP) of the OGP_Tb coupling product can reach 5 or higher.

[0109] (2) Cellobiose phosphorylase and β -1,3-glucosinolate phosphorylase coupled catalysis to prepare β -1,3-glucoligosaccharide After mixing 80 μL of 50 mM phosphate with 200 μL of 0.5% (w / v) cellobiose and 20 μL of 500 mg / mL glucose, 500 μL of cellobiose phosphorylase prepared in Example 1 and 200 μL of [prepared solution] were added. β The purified enzyme solution of 1,3-glucosamine phosphorylase was reacted at 6.5°C and 50°C for 12 h. The control group was treated with buffer solution instead of glucose solution. The reaction system was heated at 100°C for 10 min to stop the reaction. The supernatant was collected by centrifugation and the composition of the product was analyzed by thin-layer chromatography (TLC).

[0110] Cellobiose phosphorylase and β Different degrees of polymerization were prepared by coupled catalysis of -1,3-glucose oligosaccharide phosphorylase. β -1,3-glucan oligosaccharide. Results as follows: Figure 6 As shown.

[0111] The results showed that cellobiose phosphorylase CBP_Pa does not require glucose as a primer and can interact with... β -1,3-glucan oligosaccharide phosphorylase reaction produces β -1,3-glucan oligosaccharides were synthesized by reacting at 6.5°C and 50°C for 12 hours. β The degree of polymerization of -1,3-glucan oligosaccharides in DP 2~7 about.

[0112] (3) Maltodextrin phosphorylase and β -1,3-glucosinolate phosphorylase coupled catalysis to prepare β -1,3-glucoligosaccharide After mixing 80 μL of 50 mM phosphate with 200 μL of 0.5% (w / v) maltodextrin and 20 μL of 500 mg / mL glucose, 500 μL of maltodextrin phosphorylase prepared in Example 1 and 200 μL of glucose solution were added. βThe purified enzyme solution of 1,3-glucose oligosaccharide phosphorylase was reacted at 5.5°C and 50°C for 12 h. The control group was treated with buffer solution instead of glucose solution. The reaction system was heated at 100°C for 10 min to stop the reaction. The supernatant was collected by centrifugation and the composition of the product was analyzed by thin-layer chromatography (TLC).

[0113] Maltodextrin phosphorylase and β DP2~8 and above were prepared by coupled catalysis with -1,3-glucan oligosaccharide phosphorylase. β -1,3-glucan oligosaccharide. Results as follows: Figure 7 As shown.

[0114] The results showed that maltodextrin phosphorylases MDP_Tm and MDP_Dsp did not require glucose as a primer and could react with glucose at 6.5°C and 60°C for 12 hours. β -1,3-glucan oligosaccharides are generated by phosphorylation reaction to produce oligosaccharides with different degrees of polymerization. β -1,3-glucan oligosaccharide.

[0115] Example 6: Using sucrose as a substrate with SP_Psp and β OGP_Tb One-Pot Synthesis β Optimization of conditions for -1,3-glucan oligosaccharides The products of the coupled reaction were analyzed by high-performance liquid chromatography (HPLC) using an amino column at 30°C and a mobile phase of 75% acetonitrile. The molar conversion of sucrose was calculated as follows:

[0116] (1) Under the coupled reaction conditions of 5.0 and 50℃, the glucose concentration is kept constant at 10 mg / mL. The final sucrose concentration is changed (10~130 mg / mL). When the final sucrose concentration is 100 mg / mL, the sucrose molar conversion rate is the highest, that is, sucrose and glucose = 10:1, and the highest conversion rate is 98.06%. When the final sucrose concentration is 10 mg / mL (sucrose to glucose ratio 1:1), the highest conversion rate is 68.71%; when the final sucrose concentration is 20 mg / mL (sucrose to glucose ratio 2:1), the highest conversion rate is 82.00%; when the final sucrose concentration is 30 mg / mL (sucrose to glucose ratio 3:1), the highest conversion rate is 87.24%; when the final sucrose concentration is 40 mg / mL (sucrose to glucose ratio 4:1), the highest conversion rate is 91.87%; when the final sucrose concentration is 50 mg / mL (sucrose to glucose ratio 5:1), the highest conversion rate is 94.33%; when the final sucrose concentration is 60 mg / mL (sucrose to glucose ratio 6:1), the highest conversion rate is 95.26%; when the final sucrose concentration is 70 mg / mL (sucrose to glucose ratio 7:1), the highest conversion rate is 97.51%; when the final sucrose concentration is 80 mg / mL (sucrose to glucose ratio 80 mg / mL ... At a final sucrose concentration of 90 mg / mL (sucrose to glucose ratio of 8:1), the highest conversion rate was 97.02%; at 90 mg / mL (sucrose to glucose ratio of 9:1), the highest conversion rate was 96.90%; at 110 mg / mL (sucrose to glucose ratio of 11:1), the highest conversion rate was 98.02%; at 120 mg / mL (sucrose to glucose ratio of 12:1), the highest conversion rate was 97.73%; and at 130 mg / mL (sucrose to glucose ratio of 13:1), the highest conversion rate was 93.80%. The product distribution at different sucrose concentrations is shown in the figure. Figure 8 As shown, the starting substrate concentration of sucrose ranges from 50 to 800 mg / mL. When the starting substrate concentration is 50 mg / mL, the degree of polymerization of the product is DP2-4; when the starting substrate concentration is 100-200 mg / mL, the degree of polymerization of the product is DP2-7; when the starting substrate concentration is 300 mg / mL, the degree of polymerization of the product is DP2-8; and when the starting substrate concentration is 400-800 mg / mL, the degree of polymerization of the product is DP2-9.

[0117] (2) Under the coupled reaction conditions of 5.0 and 50℃, the ratio of sucrose concentration to glucose concentration was fixed at 10:1. Sucrose phosphorylase was added at 500 U / g sucrose with an enzyme ratio of 5:1. The conversion rate was highest when the final concentration of sucrose was 150 mg / mL and the final concentration of glucose was 15 mg / mL. The highest conversion rate of 98.95% was reached after 2 hours. The highest conversion rate (98.06%) was achieved when the final sucrose concentration was 90 mg / mL and the final glucose concentration was 9 mg / mL, reaching its peak after 5 hours. The highest conversion rate (97.63%) was achieved when the final sucrose concentration was 100 mg / mL and the final glucose concentration was 10 mg / mL, reaching its peak after 3 hours. The highest conversion rate (98.43%) was achieved when the final sucrose concentration was 110 mg / mL and the final glucose concentration was 15 mg / mL, reaching its peak after 3 hours. The highest conversion rate (98.43%) was achieved when the final sucrose concentration was 120 mg / mL and the final glucose concentration was 12 mg / mL, reaching its peak after 4 hours. The highest conversion rate (98.59%) was achieved when the final sucrose concentration was 130 mg / mL and the final glucose concentration was 13 mg / mL, reaching its peak after 6 hours. The highest conversion rate (98.66%) was achieved when the final sucrose concentration was 140 mg / mL and the final glucose concentration was 13 mg / mL. The highest conversion rate (98.89%) was achieved when the final glucose concentration was 14 mg / mL, reaching its maximum after 3 hours.

[0118] (3) Change the amount of enzyme added, but do not change the enzyme ratio, glucose, sucrose ratio and concentration. Sucrose concentration: 200 mg / mL, glucose concentration: 20 mg / mL, enzyme amount: 100~500 U / g sucrose.

[0119] When the enzyme dosage was 500 U / g sucrose, the reaction reached equilibrium in 3 hours and the highest conversion rate of 98.58% was reached in 8 hours. When the enzyme dosage was 300 U / g sucrose, the reaction reached equilibrium in 3 hours and the highest conversion rate of 98.39% was reached in 24 hours. When the enzyme dosage was 200 U / g sucrose, the reaction reached equilibrium in 6 hours and the highest conversion rate of 98.47% was reached in 24 hours. When the enzyme dosage was 150 U / g sucrose, the reaction reached equilibrium in 10 hours and the highest conversion rate of 98.43% was reached in 30 hours. When the enzyme dosage was 100 U / g sucrose, the reaction reached equilibrium in 24 hours and the highest conversion rate of 93.96% was achieved in 24 hours.

[0120] (4) The ratio of sucrose to glucose concentration was fixed at 10:1, the sucrose concentration was 300 mg / mL, and enzyme SP_Psp and enzymes were added at a total sucrose concentration of 180 U / g. β OGP_Tb samples were taken at different enzyme ratios and hydrolysis time intervals.

[0121] like Figure 9 As shown, the best overall results were achieved with enzyme ratios of 5:1 and 2:1. At a 2:1 enzyme ratio, the highest conversion rate was 98.24% after 12 hours; at a 5:1 ratio, the highest conversion rate was 98.09% after 30 hours; furthermore, the highest conversion rate was 98.28% after 48 hours at a 20:1 ratio; 98.38% after 24 hours at a 10:1 ratio; 98.20% after 48 hours at a 1:2 ratio; 98.52% after 48 hours at a 1:5 ratio; 96.19% after 72 hours at a 1:10 ratio; and 77.16% after 72 hours at a 1:20 ratio. The product distribution for different enzyme ratios is shown in the figure. Figure 10 As shown, when the enzyme ratio is 1:1, the product distribution is DP2~10; when the enzyme ratio is 2:1 and 5:1, products with DP>10 can be obtained; when the enzyme ratio is 10:1, the product distribution is DP2~9; when the enzyme ratio is 20:1, the product distribution is DP2~8; and when the enzyme ratio is 40:1 and 50:1, the product distribution is DP2~7.

[0122] Anion exchange chromatography was used to analyze different reaction times. β Distribution of degree of polymerization of -1,3-glucose oligosaccharides: HPAEC-PAD: Model DIONEX ICS-5000+SP-5, Thermo Fisher Scientific, USA; Analytical column: CarboPac PA1; Sample loading volume: 25 μL; Elution conditions: 250 mM sodium hydroxide solution containing 1 M sodium acetate for linear elution; Elution time: 20 min; Flow rate: 1 mL / min; Column temperature: 30 ℃.

[0123] The results are as follows Figure 11 As shown in the figure, the results indicate that different reaction times were detected. β The degree of polymerization distribution of -1,3-glucan oligosaccharides, when the reaction time is 4 h, DP 2~4 39.36%, DP 5~10 The proportion was 60.49%, and the proportion of DP≥10 was 0.16%; when the reaction time was 6 hours, DP 2~4 24.99%, DP 5~10 The proportion was 72.80%, and the proportion of DP≥10 was 2.20%; when the reaction time was 10h, DP 2~4 27.32%, DP 5~10 The proportion was 66.95%, and the proportion of DP≥10 was 5.72%; when the reaction time was 72h, DP 2~4 35.80%, DP5~10 The proportion was 45.80%, and the proportion of DP≥10 was 15.38%. As the reaction time increased, β The degree of polymerization of 1,3-glucan gradually increased, and at 72 hours, the product... β -1,3-glucan oligosaccharides reached a degree of polymerization of DP. 18 .

[0124] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for synthesis β The multi-enzyme composition of -1,3-glucan oligosaccharides is characterized by... The composition contains: (1) a first enzyme component: selected from at least one of sucrose phosphorylase, cellobiose phosphorylase or maltodextrin phosphorylase, for catalyzing the conversion of an initiating glycosyl donor substrate into glucose. 1 Phosphate; (2) Second enzyme component: β 1,3 Glucose oligosaccharide phosphorylase, used to utilize the glucose 1 Phosphate serves as a glycosyl donor for the synthesis of... β 1,3 Glucooligans.

2. The multi-enzyme composition according to claim 1, characterized in that, The sucrose phosphorylase in the first enzyme component is selected from any of the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4; the cellobiose phosphorylase in the first enzyme component is selected from any of the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7; the maltodextrin phosphorylase in the first enzyme component is selected from any of the amino acid sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9; and the second enzyme component... β 1,3 Oligosaccharide phosphorylase ( β OGP is selected from amino acid sequences as shown in either SEQ ID NO.10 or SEQ ID NO.

11.

3. Sucrose phosphorylase, cellobiose phosphorylase, and / or maltodextrin phosphorylase in glucose production 1 Phosphoric acid or glucose production 1 Its application in products where phosphate is a glycosyl donor is characterized by, The sucrose phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4; the cellobiose phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; and the maltodextrin phosphorylase is selected from any of the amino acid sequences shown in SEQ ID NO.8 and SEQ ID NO.

9.

4. A method for synthesis β The multi-enzyme coupling reaction system of -1,3-glucan oligosaccharides is characterized by... The reaction system comprises: (a) At least one of sucrose, cellobiose, and maltodextrin is selected as the starting glycosyl donor substrate. In reaction systems where the substrate is sucrose, glucose is added as a primer. (b) The multi-enzyme composition according to any one of claims 1 to 2.

5. The reaction system according to claim 4, characterized in that, The reaction system also contains phosphates.

6. A synthesis β 1,3 The method for using oligosaccharides is characterized by, The method is as follows: The multi-enzyme coupling reaction system according to any one of claims 4-5 is reacted at pH = 5.0-7.0 and 40-60℃ to generate… β 1,3 Glucooligans.

7. The method according to claim 6, characterized in that, The amount of substrate sucrose, cellobiose, or maltodextrin added is 0.5-50% (w / v); the amount of primer glucose added is 0.5-5% (w / v); and the phosphate concentration is 10-200 mM.

8. Contains specific β Recombinant plasmids containing the 1,3-glucosamine phosphorylase gene, recombinant bacteria expressing the enzyme, microbial preparations containing the recombinant bacteria, or immobilized microorganisms. β -1,3-glucan oligosaccharide phosphorylase in phosphorylation β -1,3-glucan oligosaccharides and / or synthesis β Its application in -1,3-glucan oligosaccharides is characterized by... The β The amino acid sequence of -1,3-glucosamine phosphorylase is shown in SEQ ID NO. 10 or SEQ ID NO.

11.

9. A method for synthesizing a specific degree of polymerization distribution β The method for using 1,3-glucan oligosaccharides is characterized by... The method utilizes the multi-enzyme coupling reaction system described in any one of claims 4-5, and achieves the target degree of polymerization in the product by adjusting at least one parameter among the following: the concentration of the initial substrate, the concentration of inorganic phosphate, the amount of primer added, the amount of enzyme added, and the reaction time. β Enrichment of -1,3-glucan oligosaccharides.

10. The method according to claim 9, characterized in that, The regulation includes any of the following: (1) The substrate is 50~800 mg / mL sucrose. When the initial substrate concentration is 50 mg / mL, the degree of polymerization distribution of the product is DP. 2~4 When the initial substrate concentration is 100–200 mg / mL, the degree of polymerization distribution of the product is DP. 2~7 When the initial substrate concentration was 300 mg / mL, the degree of polymerization distribution of the product was DP. 2~8 When the initial substrate concentration is 400–800 mg / mL, the degree of polymerization distribution of the product is DP. 2~9 ; (2) When the initial concentration of sucrose is 500 mg / mL and the initial concentration of glucose is 450 mg / mL, and the enzyme ratio is 1:1, the product distribution is DP. 2~10 When the enzyme ratio is 2:1 and 5:1, products with DP > 10 can be obtained; when the enzyme ratio is 10:1, the product distribution is as follows: DP > 10. 2~9 When the enzyme ratio is 20:1, the product distribution is DP. 2~8 When the enzyme ratio was 40:1 and 50:1, the product distribution was DP. 2~7 .

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