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CN-115806966-B - D-psicose 3-epimerase immobilized enzyme and preparation method and application thereof

CN115806966BCN 115806966 BCN115806966 BCN 115806966BCN-115806966-B

Abstract

The invention provides a D-psicose 3-epimerase immobilized enzyme, a preparation method and application thereof, wherein the D-psicose 3-epimerase immobilized enzyme comprises an enzyme carrier and D-psicose 3-epimerase immobilized on the enzyme carrier, the enzyme carrier is short-chain amino resin with 2-4 carbon atoms, and the D-psicose 3-epimerase is derived from Rhizobium freirei. The immobilized enzyme of the D-psicose 3-epimerase provided by the invention has the advantages of high enzyme activity under weak acid condition, short period for preparing the D-psicose, high recycling rate and the like.

Inventors

  • LIU XINRONG
  • FEI YANAN
  • WANG WEIQING
  • ZHAO ZONGPEI
  • XING ZHIQIANG

Assignees

  • 佳禾食品工业股份有限公司

Dates

Publication Date
20260505
Application Date
20221129

Claims (10)

  1. 1. A preparation method of D-psicose is characterized in that D-psicose 3-epimerase immobilized enzyme is added into fructose solution, the D-psicose is obtained through reaction at the pH of 6.0-6.4 and the temperature of 40-65 ℃, the D-psicose 3-epimerase immobilized enzyme comprises an enzyme carrier and D-psicose 3-epimerase immobilized on the enzyme carrier, the enzyme carrier is LX-1000EA, the D-psicose 3-epimerase is from Rhizobium freirei, and the amino acid sequence of the D-psicose 3-epimerase is shown as SEQ ID NO.1.
  2. 2. The method for producing D-psicose according to claim 1, wherein the method for producing D-psicose 3-epimerase immobilized enzyme comprises the steps of: (1) Synthesizing a nucleotide sequence shown as SEQ ID NO. 2; (2) Constructing the nucleotide sequence genetic engineering bacteria in the step (1); (3) Culturing the genetically engineered bacterium obtained in step (2) to obtain D-psicose 3-epimerase; (4) Mixing an enzyme carrier with the D-psicose 3-epimerase obtained in the step (3) to obtain the D-psicose 3-epimerase immobilized enzyme.
  3. 3. The method for preparing D-psicose as claimed in claim 2, wherein the genetically engineered bacterium is escherichia coli comprising a nucleotide sequence shown as SEQ ID No. 2.
  4. 4. The method for producing D-psicose according to claim 2, wherein the step (3) and the step (4) further comprise a step of purifying the D-psicose 3-epimerase to obtain a D-psicose 3-epimerase pure enzyme solution, and the D-psicose 3-epimerase pure enzyme solution is subjected to the step (4).
  5. 5. The method for producing D-psicose according to claim 4, wherein in the step (4), the D-psicose 3-epimerase pure enzyme solution is carried on the enzyme carrier in a covalent bonding manner, and the addition ratio of the D-psicose 3-epimerase pure enzyme solution to the enzyme carrier is 1g of the enzyme carrier per 3 mL-8 mL of the D-psicose 3-epimerase pure enzyme solution.
  6. 6. The method for producing D-psicose according to claim 2, wherein in the step (3), IPTG is added for induction in the culture of the genetically engineered bacterium, and the concentration of IPTG is 0.15mmol/L to 0.5mmol/L.
  7. 7. The method for preparing D-psicose according to claim 2, wherein in the step (4), glutaraldehyde with a concentration of 3% -6% is activated for 0.5-2 hours under the conditions of 22-26 ℃ and 150-200 r/min.
  8. 8. The method for producing D-psicose according to claim 1, wherein the reaction is controlled to be carried out at pH6.0 to 6.2.
  9. 9. The method for preparing D-psicose according to claim 1, wherein the pH of the reaction is controlled by using HEPES buffer with a concentration of 18-22 mmol/L, wherein the HEPES buffer contains 0.05-0.2 mmol/L Co 2+ .
  10. 10. The method for producing D-psicose according to claim 1, wherein the reaction is controlled to be carried out at 55 to 60 ℃.

Description

D-psicose 3-epimerase immobilized enzyme and preparation method and application thereof Technical Field The invention relates to a D-psicose 3-epimerase immobilized enzyme, a preparation method and application thereof. Background D-psicose (D-Psicose) is also called psicose, has obvious advantages in various aspects such as taste characteristics, physicochemical characteristics, physiological functions, edible safety and the like, has taste and safety advantages, and is a new generation of natural low-power sweetener which is attractive. The sweetness and taste of D-psicose are quite similar to those of sucrose, the sweetness of D-psicose is about 70% of that of sucrose, but the calorie of D-psicose is far lower than that of sucrose (the calorie value of D-psicose per gram is about 0.2-0.4 calorie, and the calorie of D-psicose per gram is 3.89 calorie). D-psicose is an ideal sucrose substitute for obese and diabetic patients. D-psicose is a weak inhibitor of alpha glucosidase, alpha amylase, maltase, sucrase and the like, can inhibit metabolism of starch and disaccharide in the gastrointestinal tract, and can inhibit elevation of blood sugar by protecting pancreatic beta cells, and has a potential antihyperglycemic effect, so D-psicose contributes to increase insulin sensitivity and reduce risk of developing type 2 diabetes. Toxicology experiments demonstrated that D-psicose has a median lethal dose (LD 50) of 16.3g/kg, fructose has an LD50 of 14.7g/kg, erythritol has an LD50 of 15.3g/kg, and D-psicose belongs to the "relatively harmless" class (lowest toxicity rating) according to toxicity rating. In 2014, the U.S. food and drug administration (U.S. food and Drug Administration, FDA) officially approved D-psicose as generally recognized safety (GENERALLY RECOGNIZED AS SAFE, GRAS), allowing its use in foods, dietary supplements, and pharmaceutical formulations. Currently, industrial processes for the production of D-psicose generally employ biological processes, i.e. the preparation of D-psicose under the catalysis of D-psicose-3-epimerase (DPE), but non-enzymatic browning occurs easily during the preparation process, also known as maillard reaction (Maillard reaction). Weak acid conditions are effective in inhibiting maillard reactions, but the enzyme activity of D-psicose-3-epimerase is low at this time. Therefore, how to maintain higher enzyme activity under weak acid condition, so that D-psicose can be produced efficiently and rapidly, and the method has important value in industrial production. Disclosure of Invention The invention aims to provide a D-psicose 3-epimerase immobilized enzyme, a preparation method and application thereof. The immobilized enzyme of the D-psicose 3-epimerase provided by the invention has the advantages of high enzyme activity under weak acid condition, short period for preparing the D-psicose, high recycling rate and the like. In order to solve the technical problems, the invention adopts the following technical scheme: The invention provides a D-psicose-3-epimerase immobilized enzyme, which comprises an enzyme carrier and D-psicose-3-epimerase immobilized on the enzyme carrier, wherein the enzyme carrier is short-chain amino resin with 2-4 carbon atoms, and the D-psicose-3-epimerase is derived from Rhizobium freirei. Preferably, the amino acid sequence of the D-psicose 3-epimerase is shown as SEQ ID NO.1. Preferably, the enzyme carrier is LX-1000EA. In a second aspect, the present invention provides a method for producing a D-psicose-3-epimerase immobilized enzyme, comprising the steps of: (1) Synthesizing a nucleotide sequence shown as SEQ ID NO. 2; (2) Constructing an expression vector and genetically engineered bacteria comprising the nucleotide sequence in the step (1); (3) Culturing the genetically engineered bacterium obtained in step (2) to obtain D-psicose 3-epimerase; (4) And (3) mixing an enzyme carrier with the D-psicose 3-epimerase obtained in the step (3) to obtain the D-psicose 3-epimerase immobilized enzyme, wherein the enzyme carrier is short-chain amino resin with 2-4 carbon atoms. Preferably, the genetically engineered bacterium is escherichia coli comprising a nucleotide sequence shown as SEQ ID NO. 2. Preferably, the step (3) and the step (4) further comprise a step of purifying the D-psicose 3-epimerase to obtain a D-psicose 3-epimerase pure enzyme solution, and the D-psicose 3-epimerase pure enzyme solution is subjected to the step (4). Further preferably, the purification step comprises the steps of collecting the genetically engineered bacteria in the step (3), carrying out ultrasonic crushing and centrifugation, taking a supernatant to obtain a D-psicose 3-epimerase crude enzyme solution, adding the D-psicose 3-epimerase crude enzyme solution into a gravity purification column, eluting by using an imidazole solution, and collecting an eluent to obtain the D-psicose 3-epimerase pure enzyme solution. Further preferably, under the procedures of 200W-5