CN-121992050-A - Method for synthesizing UDP-glucose by combining UDP-glucose pyrophosphorylase and cellobiose phosphorylase through one-pot method

Abstract

The invention discloses a method for synthesizing UDP-glucose by a one-pot method by combining UDP-glucose pyrophosphorylase and cellobiose phosphorylase, belonging to the technical field of enzyme engineering. According to the invention, UDP-glucose pyrophosphorylase (UGPase) from myceliophthora thermophila and cellobiose phosphorylase (CBP) from clostridium thermocellum are selected for catalytic reaction, and cellobiose and UTP are used as raw materials to obtain UDP-glucose. Experimental results show that the heat-resistant UGPase is coupled with CBP, and the substrate cellobiose and UTP can be efficiently and highly selectively converted into UDP-glucose under mild conditions, so that the accumulation of an intermediate glucose-1-phosphate is very small. The invention constructs a synergistic reaction system with matched conditions and provides a novel and efficient method for synthesizing UDP-glucose by a one-pot method.

Inventors

• QIN YUQI
• SONG XIN

Assignees

• 山东大学

Dates

Publication Date

20260508

Application Date

20260212

Claims (9)

1. 1. Use of an enzyme composition for the synthesis of UDP-glucose, said enzyme composition comprising a myceliophthora thermophila-derived UDP-glucose pyrophosphorylase and a clostridium thermocellum-derived cellobiose phosphorylase.
2. 2. The use according to claim 1, wherein the amino acid sequence of the UDP-glucose pyrophosphorylase is set forth in SEQ ID No. 1; the amino acid sequence of the cellobiose phosphorylase is shown as SEQ ID NO. 2.
3. 3. The one-pot method for catalytic synthesis of UDP-glucose is characterized by comprising the following steps: after cellobiose, phosphate, auxiliary agent and the enzyme composition in claim 1 or 2 are uniformly mixed to prepare a catalytic reaction system, the catalytic reaction is carried out to obtain UDP-glucose.
4. 4. A method according to claim 3, wherein the phosphate salt is uridine triphosphate.
5. 5. A method according to claim 3, wherein the adjuvant comprises sodium 3-morpholinopropane sulfonate, magnesium chloride, bovine serum albumin and phosphate buffer salt solution.
6. 6. The method of claim 3, wherein the catalytic reaction system comprises: 50 mM sodium 3-morpholinopropane sulfonate, 10 mM magnesium chloride, 2.0 mM uridine triphosphate, 2.0 mM cellobiose, 0.2 mg/mL bovine serum albumin, 6.8 mM phosphate buffer, 5 μg UDP-glucose pyrophosphorylase, and 5 μg cellobiose phosphorylase.
7. 7. A method according to claim 3, wherein the catalytic reaction is carried out at a temperature of 40 ℃ to 60 ℃ and at a pH of 6.0 to 8.0.
8. 8. The method of claim 3, wherein the catalytic reaction takes no less than 10 min a.
9. 9. The method of claim 3, further comprising the steps of centrifuging, collecting supernatant, boiling the supernatant, cooling, centrifuging again, collecting supernatant, and filtering with a filter membrane after completion of the catalytic reaction.

Description

Method for synthesizing UDP-glucose by combining UDP-glucose pyrophosphorylase and cellobiose phosphorylase through one-pot method Technical Field The invention relates to the technical field of enzyme engineering, in particular to a method for synthesizing UDP-glucose by a one-pot method by combining UDP-glucose pyrophosphorylase and cellobiose phosphorylase. Background UDP-glucose is an important sugar nucleotide in organisms and serves as a donor of glucosyl groups and plays a central role in glycogen synthesis, glycoside synthesis and various glycosylation reactions. In industry, UDP-glucose is a key intermediate in the synthesis of a variety of carbohydrate drugs, functional oligosaccharides (such as precursors to stevioside, ginsenoside, etc.), and glycosylated natural products. At present, the preparation method of UDP-glucose mainly comprises a chemical synthesis method, a microbial fermentation extraction method and an enzyme catalysis method. The chemical synthesis method has the advantages of complicated steps, low yield and large environmental pollution, and the microbial fermentation method has the disadvantages of difficult extraction and purification and high cost. The enzyme catalysis method is a research hot spot because of high efficiency, specificity and environmental protection. UDP-glucose pyrophosphorylase (UDP pyrophosphorylase, UGPase) (EC 3.2.1.4) is a key enzyme that catalyzes the reaction of "glucose-1-phosphate+UTP ↔ UDP-glucose+PPi". However, UGPase from most sources are poorly thermally stable and readily deactivated at higher temperatures. For example, UGPase derived from E.coli, saccharomyces cerevisiae and Arabidopsis thaliana all have an optimum temperature of 37℃and their activity decreases rapidly at 50℃which severely limits their use in industrial production. On the other hand, glucose-1-phosphate, which is a direct substrate for UGPase, is expensive, resulting in high production costs of UDP-glucose. Cellobiose phosphorylase (Cellobiose phosphorylase, CBP) (EC 2.4.1.20) can catalyze the reaction of cellobiose with inorganic phosphates to glucose-1-phosphate and glucose, thereby providing an economical source of substrate for the UGPase reaction. CBP derived from clostridium thermocellum (Clostridium thermocellum) has good thermal stability, and the optimal reaction temperature is about 50 ℃, and is a potential candidate enzyme in industrial application. In the prior art, no specific heat-resistant UGPase and CBP combination exists, and a synergistic enzyme catalytic system which is matched in temperature and pH and can efficiently synthesize UDP-glucose directly from cellobiose and inorganic phosphate by a one-pot method is designed and constructed. The specific enzyme combination and the technological innovation have important significance for reducing the production cost and promoting the development of the related sugar biological manufacturing industry. Disclosure of Invention The invention aims to provide a method for synthesizing UDP-glucose by combining UDP-glucose pyrophosphorylase and cellobiose phosphorylase through a one-pot method, which solves the problems in the prior art, the invention constructs a synergistic reaction system which is matched in condition and can directly start from cheap substrates, and provides a novel and efficient one-pot method for synthesizing UDP-glucose. In order to achieve the above object, the present invention provides the following solutions: The invention provides an application of an enzyme composition in synthesizing UDP-glucose, wherein the enzyme composition comprises UDP-glucose pyrophosphorylase (UGPase) from myceliophthora thermophila (Myceliophthora thermophila) and cellobiose phosphorylase (CBP) from clostridium thermocellum (Clostridium thermocellum). Further, the amino acid sequence of the UDP-glucose pyrophosphorylase is shown as SEQ ID NO. 1; the amino acid sequence of the cellobiose phosphorylase is shown as SEQ ID NO. 2. The invention provides a method for synthesizing UDP-glucose by one-pot catalysis, which comprises the following steps: The cellobiose (Cellobiose), phosphate, auxiliary agent and the enzyme composition are uniformly mixed to prepare a catalytic reaction system, and then the catalytic reaction is carried out to obtain UDP-glucose. Further, the phosphate is Uridine Triphosphate (UTP). Further, the adjuvants include sodium 3-morpholinopropane sulfonate (MOPS-NaOH), magnesium chloride (MgCl 2), bovine Serum Albumin (BSA), and Phosphate Buffered Saline (PBS). Further, the catalytic reaction system includes: 50 mM sodium 3-morpholinopropane sulfonate, 10 mM magnesium chloride, 2.0 mM uridine triphosphate, 2.0 mM cellobiose, 0.2 mg/mL bovine serum albumin, 6.8 mM phosphate buffer, 5 μg UDP-glucose pyrophosphorylase, and 5 μg cellobiose phosphorylase. Further, the temperature of the catalytic reaction is 40-60 ℃ and the pH is 6.0-8.0. Further, the time of the catalytic reaction is not less than 10 min