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CN-121991992-A - Double-enzyme expression plasmid, double-enzyme expression recombinant strain and whole-cell catalysis mannitol and gluconic acid co-production method

CN121991992ACN 121991992 ACN121991992 ACN 121991992ACN-121991992-A

Abstract

The invention provides a double-enzyme expression plasmid, a double-enzyme expression recombinant strain and a method for co-producing mannitol and gluconic acid by whole cell catalysis, wherein the plasmid co-expresses heat-resistant glucose dehydrogenase and heat-resistant mannitol dehydrogenase, the plasmid is transferred into escherichia coli engineering bacteria to obtain the double-enzyme expression recombinant strain, glucose is taken as a raw material, under the action of glucose isomerase, the double-enzyme expression recombinant strain is applied, and the whole cell biocatalysis system consisting of the co-expressed mannitol dehydrogenase and the glucose dehydrogenase is utilized for catalytic production of mannitol and gluconic acid. The method adopts a brand new catalytic path, has the advantages of low production cost, low pollution, high yield and the like, selects enzyme with high temperature source, has high reaction rate and high substrate conversion rate, is a new preparation path for co-producing mannitol and gluconic acid, and is suitable for large-scale production of mannitol and gluconic acid.

Inventors

  • ZHANG YIHENG
  • LIU XINYUE
  • HAN PINGPING

Assignees

  • 中国科学院天津工业生物技术研究所

Dates

Publication Date
20260508
Application Date
20241104

Claims (10)

  1. 1. A double enzyme expression plasmid is characterized in that mannitol dehydrogenase gene and glucose dehydrogenase gene are co-expressed on the same plasmid.
  2. 2. The double enzyme expression plasmid of claim 1, wherein the plasmid comprises a first promoter, a mannitol dehydrogenase gene, a first terminator, a second promoter, a glucose dehydrogenase gene and a second terminator, or comprises a first promoter, a glucose dehydrogenase gene, a first terminator, a second promoter, a mannitol dehydrogenase gene and a second terminator, or comprises a first promoter, a mannitol dehydrogenase gene, a second promoter, a glucose dehydrogenase gene, a terminator, or comprises a first promoter, a glucose dehydrogenase gene, a second promoter, a mannitol dehydrogenase gene, a terminator, preferably the promoter is a T7 promoter, and the terminator is a T7 terminator.
  3. 3. The double enzyme expression plasmid of claim 1 or 2, wherein the mannitol dehydrogenase is derived from Thermotoga maritima (Thermotoga maritima), thermotoga new Apollo (Thermotoganeapolitana) or Thermoanaerobacter thermophilus (Thermoanaerobacteriumthermosaccharolyticum) the glucose dehydrogenase is derived from sulfolobus (Sulfolobus solfataricus), thermoplasma acidophilum (Thermoplasmaacidophilum) or Thermoarchaea hyperthermophil (Sulfolobus tokodaii), preferably the mannitol dehydrogenase is derived from Thermotoga maritima, and the glucose dehydrogenase is derived from sulfolobus.
  4. 4. A recombinant strain with double enzyme expression, which is characterized in that the recombinant strain is obtained by transferring the double enzyme expression plasmid of one of claims 1-3 into engineering bacteria of escherichia coli.
  5. 5. The recombinant strain of claim 4, wherein the engineering bacterium is Escherichia coli BL21 (DE 3).
  6. 6. A method for co-producing mannitol and gluconic acid by whole cell catalysis is characterized in that glucose is used as a raw material, under the action of glucose isomerase, the double-enzyme expression recombinant strain in claim 4 or 5 is applied, and the whole cell biocatalysis system consisting of co-expressed mannitol dehydrogenase and glucose dehydrogenase is utilized for producing mannitol and gluconic acid in a catalysis way.
  7. 7. The method for whole cell catalytic co-production of mannitol and gluconic acid as claimed in claim 6, wherein the double-enzyme expression recombinant strain and glucose isomerase and glucose together perform whole cell catalytic reaction.
  8. 8. The method for the whole-cell catalytic co-production of mannitol and gluconic acid as claimed in claim 7, wherein glucose is taken as a single substrate, partial glucose is converted into glucose under the action of glucose isomerase, and then the double-enzyme expression recombinant strain reacts with glucose and fructose as substrates, namely the double-enzyme expression recombinant strain reacts with glucose and fructose together in a whole-cell catalytic manner.
  9. 9. The method for whole-cell catalytic co-production of mannitol and gluconic acid according to any one of claims 6 to 8, wherein the mannitol dehydrogenase is derived from Thermotoga maritima, thermotoga neo-Apollo or Thermotoga thermolytica, preferably the mannitol dehydrogenase is derived from Thermotoga maritima, the glucose dehydrogenase is derived from sulfolobus, thermoplasma acidophilum or Thermoarchaea, preferably the glucose dehydrogenase is derived from sulfolobus, the glucose isomerase is derived from Thermus thermophilus (Thermus thermophilus), thermoanaerobacterium ethanolicum (Thermoanaerobacterethanolicus) or Streptomyces flavus (Streptomyces flavogriseus), preferably the glucose isomerase is derived from Thermomyces thermophilus.
  10. 10. The method for the whole-cell catalytic co-production of mannitol and gluconic acid according to any one of claims 6 to 8, wherein glucose isomerase is added in a nickel column purification manner or in a permeabilization manner, wherein glucose isomerase and a double-enzyme expression recombinant strain are sequentially added to establish a whole-cell catalytic reaction system, wherein the concentration of glucose is 2-600g/L, preferably the concentration of glucose is 10-400g/L, further preferably the concentration of glucose is 100-300g/L; The temperature of the reaction system is 40-90 ℃, preferably, the temperature of the reaction system is 50-80 ℃, and further preferably, the temperature of the reaction system is 60-70 ℃; The cell amount of the double-enzyme expression recombinant strain is 0.001-0.3g DCW/mL, preferably, the cell amount of the double-enzyme expression recombinant strain is 0.005-0.2g DCW/mL, and more preferably, the cell amount of the double-enzyme expression recombinant strain is 0.01-0.05g DCW/mL.

Description

Double-enzyme expression plasmid, double-enzyme expression recombinant strain and whole-cell catalysis mannitol and gluconic acid co-production method Technical Field The invention relates to the technical field of mannitol and gluconic acid co-production, in particular to a double-enzyme expression plasmid, a double-enzyme expression recombinant strain and a method for co-producing mannitol and gluconic acid by whole cell catalysis. Background Mannitol (D-Mannitol) is a naturally occurring hexitol and is widely used in the industries of food, medicine, chemical industry, feed, etc. At present, the main production methods of mannitol comprise three methods of natural extraction, chemical synthesis and bioconversion. Mannitol is extracted from seaweed and is a commercial method for producing mannitol in small scale in China, but the technology has the problems of high water consumption, high energy consumption and the like. The main method for industrially producing mannitol is to chemically hydrogenate a mixture of about 50% fructose and about 50% glucose, wherein Raney nickel is used as a catalyst, hydrogen is used as a reducing agent, glucose is converted into sorbitol, and fructose generates a mixture of mannitol and sorbitol. In this method, there are problems such as extreme reaction conditions, complicated purification steps, low product yield, and the like. In recent years, the biotransformation method for producing mannitol, which has the advantages of high selectivity, low energy consumption and the like, has been attracting attention. Many microorganisms in nature can use carbohydrates to synthesize mannitol, such as yeasts, fungi, lactic acid bacteria, and the like. The technical route for producing mannitol by using a biological fermentation method is that glucose is converted into cheap acetic acid and lactic acid, and energy is provided to participate in the conversion of fructose into mannitol. However, the microbial fermentation method for producing mannitol has the disadvantages of long growth period, more byproducts, complex culture medium components and the like, which requires the development of a new strategy for producing mannitol in a simpler and more efficient manner. Many studies on mannitol production using whole cell catalytic or multi-enzyme co-catalytic systems are now available. However, cofactors are the main limiting factor for mannitol production. In Wichmann, 1981, a double enzyme system was proposed in which mannitol dehydrogenase and formate dehydrogenase were coupled to convert fructose and formate into mannitol and CO 2, respectively, to achieve continuous regeneration of cofactors. In 2004 Kaup et al, a whole cell catalytic system for converting fructose into mannitol was developed for the first time in E.coli, which strain expressed L.pseudomereteroides-derived mannitol-2-dehydrogenase and Mycobacterium vaccae-derived formate dehydrogenase, and which recombinant strain was able to form 362mM mannitol in 8h with a yield of 84mol%. In 2005 Kaup et al, extracellular glucose isomerase or co-expressed glucose isomerase was added to the recombinant E.coli strain to allow the cell to synthesize D-mannitol using D-glucose as a substrate. However, this method utilizes formic acid or formate as one of the substrates and generates carbon dioxide, which is not only environmentally friendly but also causes waste of raw materials. Gluconic acid is a naturally occurring organic acid and has wide application in various fields. The current production methods of gluconic acid at home and abroad mainly comprise a chemical oxidation method, an electrolytic oxidation method, a biological fermentation method, an enzyme conversion method and the like. The chemical oxidation method and the electrolytic oxidation method both take glucose as raw materials and produce the gluconic acid through sodium hypochlorite or electrolytic oxidation, and have the defects of high production cost, complex operation industry and the like. The biological fermentation method is to oxidize glucose into gluconic acid by using the oxidization of bacterial and fungal microorganism fermentation. At present, most of the Aspergillus niger fermentation is industrially used for preparing gluconic acid, pande et al, at the temperature of 33 ℃ and the pH value of=6, the conversion rate after 20 hours is 87%, and the selectivity is 38%. The microbial fermentation method has the problems of long production period, more byproducts, complicated sewage treatment and the like. The production of the gluconic acid by the enzyme method is mainly realized by using the coupling reaction of glucose oxidase and catalase, and has the advantages of mild reaction conditions, single product and the like, but H 2O2 generated in the oxidative decomposition process of the glucose has certain damage to the enzyme. At present, two approaches for co-production of mannitol and gluconic acid are mainly available, one is to take glucose and fruct