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CN-121991916-A - Multi-enzyme system for efficiently synthesizing beta-1, 3-glucan by one-pot method and application

CN121991916ACN 121991916 ACN121991916 ACN 121991916ACN-121991916-A

Abstract

The invention discloses a multienzyme system for efficiently synthesizing beta-1, 3-glucan by a one-pot method and application thereof, belonging to the technical fields of genetic engineering and modern enzyme engineering. The core of the invention is that a plurality of high-activity G1P donor generating enzymes and beta-1, 3-glucan phosphorylase are systematically screened and optimally combined, and the beta-1, 3-glucan with specific polymerization degree distribution can be directly synthesized in a single reactor through a one-pot method by taking low-cost and easily available sucrose, cellobiose or maltodextrin as raw materials. The sucrose molar conversion rate can reach 98.24%, and the beta-1, 3-glucan in the 72h enzyme reaction product can reach DP 18 , so that the dependence on expensive nucleotide activating sugar is thoroughly eliminated. By adjusting reaction parameters, the directional enrichment of the target polymerization degree product provides a brand new production path which is green, economical, efficient and can be scaled for functional sugar.

Inventors

  • ZHANG TAO
  • ZHANG YILING

Assignees

  • 江南大学

Dates

Publication Date
20260508
Application Date
20260128

Claims (10)

  1. 1. A multi-enzyme composition for synthesizing beta-1, 3-glucan is characterized in that the composition contains (1) a first enzyme component, which is at least one selected from sucrose phosphorylase, cellobiose phosphorylase and maltodextrin phosphorylase, and is used for catalyzing an initial glycosyl donor substrate to generate glucose 1 Phosphate, (2) a second enzyme component beta 1,3 The enzyme phosphorylase of the glucosan and the phosphorylase of the glucosan, for utilizing said glucose 1 Synthesis of beta using phosphoric acid as glycosyl donor 1,3 And (3) glucan oligosaccharides.
  2. 2. The multi-enzyme composition of claim 1, wherein the sucrose phosphorylase in the first enzyme component is selected from the group consisting of amino acid sequences shown in any one of 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 the group consisting of amino acid sequences shown in any one of SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, and the maltodextrin phosphorylase in the first enzyme component is selected from the group consisting of amino acid sequences shown in any one of SEQ ID NO.8, and SEQ ID NO.9, and the beta-cyclodextrin phosphorylase in the second enzyme component is selected from the group consisting of beta-cyclodextrin phosphorylase in the first enzyme component 1,3 The gluco-oligosaccharide phosphorylase (beta OGP) is selected from any one of SEQ ID NO.10 and SEQ ID NO. 11.
  3. 3. Sucrose phosphorylase, cellobiose phosphorylase and/or maltodextrin phosphorylase in the production of glucose 1 Phosphoric acid or glucose production 1 The application of the product with phosphoric acid as a glycosyl donor is characterized in that the sucrose phosphorylase is selected from any one of amino acid sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, the cellobiose phosphorylase is selected from any one of amino acid sequences shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the maltodextrin phosphorylase is selected from any one of amino acid sequences shown as SEQ ID NO.8 and SEQ ID NO. 9.
  4. 4. A multienzyme coupling reaction system for synthesizing beta-1, 3-glucan, the reaction system comprising: (a) At least one of sucrose, cellobiose and maltodextrin is taken as an initial glycosyl donor substrate, and glucose is added as a primer in a reaction system of sucrose as the substrate; (b) The multi-enzyme composition according to any one of claims 1 to 2.
  5. 5. The reaction system of claim 4, further comprising a phosphate.
  6. 6. Synthetic beta 1,3 A method for producing a glucan by reacting a multi-enzyme-coupled reaction system according to any one of claims 4 to 5 at pH=5.0 to 7.0 and 40 to 60 ℃ to produce β 1,3 And (3) glucan oligosaccharides.
  7. 7. The method according to claim 6, wherein the substrate sucrose, cellobiose or maltodextrin is added in an amount of 0.5 to 50% (w/v), the primer glucose is added in an amount of 0.5 to 5% (w/v), and the phosphate concentration is 10 to 200mM.
  8. 8. The application of recombinant plasmid containing specific beta-1, 3-glucan phosphorylase gene or recombinant bacterium expressing the enzyme, microbial preparation containing the recombinant bacterium or immobilized beta-1, 3-glucan phosphorylase in the process of phosphorylating beta-1, 3-glucan and/or synthesizing beta-1, 3-glucan is characterized in that the amino acid sequence of the beta-1, 3-glucan phosphorylase is shown as SEQ ID NO.10 or SEQ ID NO. 11.
  9. 9. A method for synthesizing beta-1, 3-glucan with specific polymerization degree distribution is characterized in that the method utilizes the multi-enzyme coupling reaction system of any one of claims 4-5 to realize enrichment of beta-1, 3-glucan with target polymerization degree in a product by adjusting at least one parameter of reaction initial substrate concentration, inorganic phosphate concentration, primer addition amount, enzyme addition amount and reaction time.
  10. 10. The method of claim 9, wherein the modulation comprises any one of: (1) The substrate is sucrose with the concentration of 50-800 mg/mL, the polymerization degree of the product is distributed to be DP 2~4 when the initial substrate concentration is 50-mg/mL, the polymerization degree of the product is distributed to be DP 2~7 when the initial substrate concentration is 100-200 mg/mL, the polymerization degree of the product is distributed to be DP 2~8 when the initial substrate concentration is 300-mg/mL, and the polymerization degree of the product is distributed to be DP 2~9 when the initial substrate concentration is 400-800 mg/mL; (2) When the initial concentration of sucrose is 500mg/mL and the initial concentration of glucose is 450mg/mL, the product distribution is DP 2~10 when the enzyme addition amount is 1:1, the product with DP >10 can be obtained when the enzyme addition amounts are 2:1 and 5:1, the product distribution is DP 2~9 when the enzyme addition amount is 10:1, the product distribution is DP 2~8 when the enzyme addition amount is 20:1, and the product distribution is DP 2~7 when the enzyme addition amounts are 40:1 and 50:1.

Description

Multi-enzyme system for efficiently synthesizing beta-1, 3-glucan by one-pot method and application Technical Field The invention relates to a multienzyme system for efficiently synthesizing beta-1, 3-glucan by a one-pot method and application thereof, in particular to a method for efficiently synthesizing beta-1, 3-glucan with specific polymerization degree distribution by using different combinations of Sucrose Phosphorylase (SP), cellobiose phosphorylase (CBP), maltodextrin phosphorylase (MDP) and beta-1, 3-glucan phosphorylase (beta OGP) and using sucrose, cellobiose or maltodextrin as initial substrates, belonging to the technical fields of genetic engineering and modern enzyme engineering. Background Beta-1, 3-glucan (COS) is oligomeric beta-1, 3-glucan with a degree of polymerization of 2-10, and is a linear glucan connected by beta-1, 3-glycosidic bonds. In addition to the function of inducing plant resistance, COS has been demonstrated to have various biological activities such as antiviral, antiallergic, immunomodulating, and tumor-inhibiting potential. The preparation has low toxicity, high stability and good biocompatibility, and has great application potential in the fields of medicine, food, agricultural production and the like. However, the technology for preparing beta-1, 3-glucan is limited in industrialization. The existing method is divided into two types, namely degradation and synthesis, wherein the degradation method takes thermal gel as a raw material and adopts acidolysis or enzymolysis to prepare the thermal gel, on one hand, the thermal gel has high substrate price, on the other hand, the thermal gel is insoluble in water and the polymerization degree of the product is difficult to control, the chemical synthesis steps are complicated, residues are easy to leave, the chemical synthesis steps are difficult to apply to medical foods, and in the biological synthesis path, the natural world mainly depends on glycosyltransferase to catalyze the dextran chain to extend, but the required nucleotide activates sugar (such as UDPGlucose) is high in cost and restricts industrial application. Phosphorylase provides a new pathway for oligosaccharide synthesis. Beta-1, 3-glucan phosphorylase (beta OGP) can catalyze reversible reaction of G1P and beta-1, 3-glucan, wherein glucose or oligomer is used as a primer to synthesize a high polymer in the presence of G1P, and conversely, glucose or short-chain oligosaccharide is used as a primer to synthesize longer-chain beta in the presence of G1P1,3And (3) glucan oligosaccharides. The enzyme provides the possibility for controlled synthesis of the degree of polymerization. To reduce the cost of G1P supply, other phosphorylases may 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 maltooligosaccharides from maltodextrin. The donor-producing enzyme is coupled with beta OGP, so that the one-pot synthesis of beta from cheap raw materials can be theoretically realized1,3The gluco-oligosaccharide realizes the high-efficiency utilization of resources and lays a foundation for large-scale production. However, phosphorylases from different sources have significant differences in catalytic efficiency, substrate specificity, optimal reaction conditions, and compatibility with βogp. These differences represent specific drawbacks in the construction of multi-enzyme systems, such as the difficulty in matching the catalytic rates of the G1P-donating enzyme and the βOGP, the tendency to build up or starve reaction intermediates, affecting overall flow rates and yields, the incompatibility of the optimal reaction conditions for each enzyme, the difficulty in finding a common "one-pot" reaction condition for all enzymes to work efficiently at the same time, and the potential for substrate competition or adverse side reactions between enzymes, which can reduce the yield and purity of the target product. Therefore, how to construct a stable, efficient and controllable multienzyme catalytic system from vast enzyme resources through screening and combination strategies of the system becomes a key technical bottleneck which is necessary to be overcome for realizing low-cost and large-scale biological manufacture of beta-1, 3-glucan. Based on the above, the invention can develop a set of efficient multienzyme coupling synthesis system, aiming at overcoming the defects of the prior art and being beta1,3Green manufacture of glucooligosaccharides provides a completely new solution. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a set of catalytic system and a method for efficiently synthesizing beta-1, 3-glucan with specific polymerization degree based on a