CN111100799A - Method for synthesizing (R) -1, 3-butanediol by using microorganism whole cells - Google Patents
Method for synthesizing (R) -1, 3-butanediol by using microorganism whole cells Download PDFInfo
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- CN111100799A CN111100799A CN202010029884.9A CN202010029884A CN111100799A CN 111100799 A CN111100799 A CN 111100799A CN 202010029884 A CN202010029884 A CN 202010029884A CN 111100799 A CN111100799 A CN 111100799A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/84—Pichia
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
Abstract
The invention discloses a method for synthesizing (R) -1, 3-butanediol by using microbial whole cells, belonging to the technical field of biocatalytic asymmetric transformation. The invention obtains a microorganism for synthesizing (R) -1, 3-butanediol by screening a high-selectivity whole cell, namely Pichia kudriavzevii CCTCC M2019329, takes the whole cell as a catalyst, takes 4-hydroxy-2-butanone as a substrate, and adopts reaction pH, temperature, rotating speed and auxiliary substrate obtained by single-factor optimization, wherein the yield of the (R) -1, 3-butanediol reaches more than 80 percent, the optical purity reaches more than 99 percent, and the product concentration reaches more than 100 g/L.
Description
Technical Field
The invention relates to a method for synthesizing (R) -1, 3-butanediol by using microbial whole cells, belonging to the technical field of biocatalytic asymmetric transformation.
Background
The chemical structure of (R) -1, 3-butanediol ((R) -1,3-BDO) is as follows:
in industrial production, it is generally used as an intermediate for the synthesis of pheromones and pesticides, particularly in the preparation of azetidinone derivatives, azetidinone derivatives are starting materials for the synthesis of penems and carbapenem antibiotics, which are important β -lactam antibiotics having advantages of strong bactericidal activity, low toxicity, wide clinical indications and excellent efficacy.
Currently, (R) -1,3-BDO can be synthesized by chemical synthesis and biotransformation. In the chemical synthesis process, more environmental pollution exists, which is not beneficial to large-scale production. The biological conversion method for synthesizing (R) -1,3-BDO can overcome the problem of environmental pollution, and the biological method for preparing chiral alcohol has the advantages of high efficiency, specificity, good stereoselectivity, mild reaction conditions and the like, and becomes an important means for preparing chiral alcohol. Some reports of biotransformation method synthesis of (R) -1,3-BDO have appeared at present, for example, atsuyama et al respectively utilize K.lactis IFO 1267 and C.utilis IAM 4277 to realize catalytic conversion of 4-hydroxy-2-butanone (4H2B) as a substrate in a 10L fermentation tank scale, the optical purity of the product reaches 93% and 94%, and the optical purity of the product does not reach 99%, and the product can be used as an intermediate raw material of medicines by complicated separation and purification; zheng and Rao et al catalytically convert 4H2B with C.krusei ZJB-09162 and P.jadinii HBY61, respectively, and the optical purity of the product reaches 99%, but the substrate concentration can only reach 45g/L, and the yield is 83.9% and 85.1%; yamamoto et al prepared (R) -1,3-BDO by enantioselective oxidation using a fully recombinant E.coli cell expressing a specific secondary alcohol dehydrogenase of (S) -1,3-BDO with racemate (R, S) -1,3-BDO as a substrate, with an optical purity of 95%, but at a yield of only 43%; glucose is used as a catalytic substrate, recombinant bacteria are constructed in a genetic engineering mode, the recombinant bacteria are subjected to metabolic regulation and synthesis to synthesize (R) -1,3-BDO, Kataoka and the like, the glucose is catalytically converted by the method, the product concentration can only reach 9.05g/L, and the industrial requirement can not be met. Currently, the efficiency of biochemical conversion and the scale of the catalytic system are still limited in the synthesis of (R) -1, 3-BDO.
Disclosure of Invention
In order to solve the above problems, the first objective of the present invention is to provide a strain of Pichia kudriavzevii (Pichia kudriavzevii) QC-1, which has been deposited in the chinese typical culture collection at 7.5 months in 2019 with the collection number of CCTCC NO: m2019329.
The second object of the present invention is to provide a microbial preparation containing the above Pichia kudriavzevii.
The third purpose of the invention is to provide a method for synthesizing (R) -1, 3-butanediol, which takes the whole cells of the Pichia kudriavzevii as a catalyst and 4H2B as a substrate to synthesize the (R) -1, 3-butanediol.
In one embodiment of the invention, the catalyst is wet bacteria obtained by inoculating the Pichia kudriavzevii single colony into a YPD liquid culture medium, culturing at 28-32 ℃ and 200-220 rpm for 16-18 h, transferring the cultured bacteria into a new YPD culture medium at 1-5% (V/V), culturing at 28-32 ℃ and 200-220 rpm for 40-60 h, and centrifuging.
In one embodiment of the invention, the amount of catalyst added is 2 to 10g/g substrate.
In one embodiment of the invention, the substrate concentration is 15-25 g/L and the final catalyst concentration is 80-120 g/L.
In one embodiment of the invention, the substrate concentration is 100-150 g/L and the final catalyst concentration is 300-500 g/L.
In one embodiment of the present invention, the pH in the catalytic reaction is 7.0 to 8.5.
In one embodiment of the invention, the temperature in the catalytic reaction is 30-40 ℃.
In one embodiment of the present invention, the rotation speed in the catalytic reaction is 180 to 220 rpm.
In one embodiment of the invention, the co-substrate in the catalytic reaction is one or more of glucose, ethanol, isopropanol.
In one embodiment of the present invention, the mass concentration ratio of the auxiliary substrate to the substrate is maintained at 0.5 to 1.5.
Has the advantages that: the invention obtains a microorganism for synthesizing (R) -1, 3-butanediol by screening high selectivity whole cells, namely Pichia kudriavzevii CCTCC M2019329, and takes the whole cells as a catalyst, 4H2B as a substrate and reaction pH, temperature, rotating speed and auxiliary substrate obtained by single-factor optimization, wherein the yield of the (R) -1, 3-butanediol reaches more than 80%, the optical purity reaches more than 99%, and the product concentration reaches more than 100 g/L.
Biological material preservation
Pichia kudriavzevii (Pichia kudriavzevii) QC-1, depository organization: china center for type culture Collection, collection address: china, wuhan university, accession number: CCTCC NO: m2019329, date of deposit: year 2019, month 5 and day 7.
Drawings
FIG. 1: the effect of different pH on the catalysis of asymmetric reduction of 4H2B by Pichia kudriavzevii CCTCC M2019329.
FIG. 2: effect of different temperatures on the catalytic asymmetric reduction of 4H2B by Pichia kudriavzevii CCTCC M2019329.
FIG. 3: the effect of different rotation speeds on the catalysis of asymmetric reduction of 4H2B by Pichia kudriavzevii CCTCC M2019329.
FIG. 4: the effect of different co-substrates on the catalysis of asymmetric reduction of 4H2B by Pichia kudriavzevii CCTCC M2019329.
FIG. 5: pichia kudriavzevii CCTCC M2019329 fed-batch catalytic substrate 4H 2B.
Detailed Description
The screening culture medium is YPD culture medium (g/L): yeast extract 5.0, peptone 10.0, glucose 10.0, pH 7.0 (solid medium plus 20.0 agar). Sterilization conditions of the medium: sterilizing at 115 deg.C for 30 min.
The optical purity of the product was determined by Chiralcel OB-H (250 mm. times.4.6 mm, Daicel chemical industries), using V (n-hexane): V (isopropanol) (19: 1) as the mobile phase and a flow rate of 1.0 mL/min. The column temperature was 30 ℃ and the detector was a spectral detector (UV, λ 220nm.
Product quantification was determined by GC-FID, the column was Econo Cap-Wax (30 m.times.250. mu.m.times.0.25. mu.m), the inlet and detector temperatures were 230 ℃ and 220 ℃ respectively, the sample size was 1.0. mu.L, the program temperature: the temperature is maintained at 90 ℃ for 0.5 min and the temperature is raised to 200 ℃ at 50 ℃/min for 4 min.
Calculation of the enantiomeric excess of the product: enantiomeric excess (e.e.%) is [ (C)S-CR)/(CS+CR)]×100％
Calculation of product yield: yield (%) ═ CR/C0×100％
In the formula CSAs the concentration of the (S) -enantiomer after the reaction, CRAs the concentration of the (R) -enantiomer after the reaction, C0Is the concentration of the substrate before the reaction.
Example 1 screening and identification of high Performance catalytic strains
In the strain screening process, 4H2B is used as a substrate, glucose is used as an auxiliary substrate, more than 200 strains are obtained by separating and screening from soil, the strains obtained by screening are cultured for 48 hours by adopting a 48-pore plate, the strains are collected by centrifugation, the substrate and the auxiliary substrate are added into the 48-pore plate, after reaction for 48 hours, the products after the reaction are detected and analyzed, the high optical purity and yield of the products are used as selection standards, the strains with good catalytic efficiency and high specificity on the substrate 4H2B are obtained, finally, gene identification is carried out, and after identification, the strains are Pichia pastoris, and are sent to a preservation organization for preservation, wherein the preservation number is CCTCC M2019329.
Example 2 preparation of Pichia kudriavzevii CCTCC M2019329 Whole cells
A single colony of Pichia kudriavzevii strain CCTCC M2019329 is selected and inoculated into a test tube containing 5mL of YPD liquid culture medium, cultured at 30 ℃ and 200rpm for 16h, transferred into a 500mL triangular shaking flask (containing 100mL of YPD liquid culture medium) with the inoculum size of 2% (V/V), cultured at 30 ℃ and 200rpm for 48h with shaking, the fermentation broth is collected, centrifuged at 6000rpm for 10min, and thalli are collected after being washed for 3 times by physiological saline and used for asymmetric transformation reaction as a catalyst.
Example 3 Effect of different pH on Pichia kudriavzevii CCTCC M2019329 catalysis of asymmetric reduction of 4H2B
The catalytic reaction system is 10mL, the concentration of the substrate 4H2B is 20g/L, the final concentration of the catalyst is 1g/10mL, the temperature is 30 ℃, the rotation speed is 200rpm, the auxiliary substrate is glucose, and the reaction is carried out for 12H.
The catalytic activity of the enzymes showed large differences under different pH conditions. By comparing the optimal buffer intervals of different buffer solutions, the optimal pH (2.0-10.0) range is divided into 4 gradients: 0.1mol/L citrate buffer solution (pH 2.0-6.0), 0.1mol/L phosphate buffer solution (pH 6.0-8.0), 0.1mol/L Tris-HCl buffer solution (pH 8.0-9.0) and 0.1mol/L carbonate buffer solution (pH 9.0-10.0).
As shown in fig. 1, when the pH of the reaction is below 5, the yield of the product is below 40%; when the pH of the reaction is greater than 8, the yield decreases rapidly with increasing pH; at a pH of 10, the yield of product was only 20%. Therefore, the enzyme has low catalytic reaction efficiency in a peracid and high-alkaline environment and good catalytic efficiency in a neutral and slightly alkaline environment, and when the pH is 8.0, the reaction yield reaches 88.23%, and the optical purity of the product is more than 99%.
Example 4 Effect of different temperatures on Pichia kudriavzevii CCTCC M2019329 catalysis of asymmetric reduction of 4H2B
The catalytic reaction system is 10mL, the concentration of the substrate 4H2B is 20g/L, the final concentration of the catalyst is 1g/10mL, a phosphate solution with 0.1mol/L and pH of 8.0 is used as a buffer solution, the rotating speed is 200rpm, the auxiliary substrate is glucose, and the reaction is carried out for 12H.
The biotransformation process by using microbial cells as a catalyst in the catalytic reaction process is actually a biotransformation process using enzyme catalysis, and temperature is a very important influence factor concerning the efficiency of the enzyme-catalyzed reaction.
As shown in FIG. 2, the optimized temperature range is 15 ℃ to 50 ℃, a temperature gradient is set every 5 ℃, when the reaction temperature is lower than 30 ℃, the yield of the catalytic reaction is lower than 70%, and the analysis is that the temperature is too low, so that the activity of the intracellular enzyme is influenced, and the efficiency of the catalytic reaction and the final product yield are influenced. Increasing the temperature of the catalytic reaction can increase the catalytic activity of the enzyme and accelerate the conversion efficiency of the reaction, but when the temperature is higher than 35 ℃, the yield of the reaction is reduced, and when the temperature is 35 ℃, the yield of the catalytic reaction is 85.06%, and the optical purity of the product is more than 99%.
Example 5 Effect of different rotational speeds on Pichia kudriavzevii CCTCC M2019329 catalysis of asymmetric reduction of 4H2B
The catalytic reaction system is 10mL, the concentration of the substrate 4H2B is 20g/L, the final concentration of the catalyst is 1g/10mL, the temperature is 30 ℃, a phosphate solution with 0.1mol/L and pH of 8.0 is used as a buffer solution, an auxiliary substrate is glucose, and the reaction is carried out for 12 hours.
No matter the substrate 4H2B enters the yeast cells or the product (R) -1,3-BDO is separated from the cells, a certain mass transfer resistance exists, and the mass transfer resistance of substances in a reaction liquid can be influenced by changing the rotating speed of the reaction, so that the catalytic effect of the reaction is influenced. Four rotation speeds of 100rpm, 150rpm, 200rpm and 250rpm were selected for the investigation.
As shown in fig. 3, when the rotation speed is less than 200rpm, the yield of the reaction increases with the increase of the rotation speed, but the yield decreases with the further increase of the rotation speed, that is, the excessively high rotation speed inhibits the efficiency of the catalysis, and when the rotation speed is 200rpm, the yield of the product and the optical purity of the product are 85.26%, 99.4%, respectively.
Example 6 Effect of different co-substrates on the catalysis of asymmetric reduction of 4H2B by Pichia kudriavzevii CCTCC M2019329
The catalytic reaction system is 10mL, the concentration of the substrate 4H2B is 20g/L, the final concentration of the catalyst is 1g/10mL, the temperature is 30 ℃, the rotation speed is 200rpm, and 0.1mol/L phosphate solution with the pH value of 8.0 is used as a buffer solution for reaction for 12 hours.
During the asymmetric reduction of the catalytic substrate 4H2B by the strain Pichia kudriavzevii CCTCC M2019329, the reduced coenzyme as hydrogen donor will be continuously consumed. When the whole cell catalyzes the reaction, the cell can regenerate the coenzyme by oxidizing the co-substrate to obtain reduced hydrogen using various enzymes of the cell itself, thereby improving the catalytic efficiency of the reaction and ultimately the yield of the product.
Glucose, n-butanol, isopropanol, glycerol, xylan and ethanol are respectively selected as auxiliary substrates for research. As shown in FIG. 4, when the concentration of the co-substrate was low, the yield increased with the increase in the concentration of the co-substrate, but when the concentration of the co-substrate was continuously increased, reactions involving several other co-substrates were observed in addition to xylan, and the yield of the product was decreased. In particular, with respect to n-butanol, when the concentration of n-butanol reached 3% (w/v), the yield dropped almost to zero, from which analysis concluded that high concentrations of n-butanol completely inhibited the reaction. For the auxiliary substrate xylan, the yield is kept constant with the increase of the concentration, when glucose is used as the auxiliary substrate and the mass concentration ratio of the glucose to the 4H2B is kept at a ratio of 4:5, the yield reaches more than 80 percent, and the optical purity of the product is 99 percent.
Example 7 Strain Pichia kudriavzevii CCTCC M2019329 fed-batch catalysis substrate 4H2B
The catalytic reaction system is amplified, the concentration of a catalyst is increased from a 10mL shake flask system to a 100mL reactor system, the final concentration of the catalyst is 35g/100mL, the temperature is 30 ℃, the rotation speed is 200rpm, a phosphate solution with the pH value of 8.0 and 0.1mol/L is used as a buffer solution, an auxiliary substrate is glucose, the substrate concentration is increased from 20g/L to 120g/L in a fed-batch manner, the substrates are added in three batches in mass ratios of 0h, 6h, 16h, 32h and the like, the product concentration reaches 100.40g/L after 96h of reaction, and the yield reaches 83.66% (see FIG. 5).
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A Pichia kudriavzevii (Pichia kudriavzevii) QC-1 strain is deposited in the China center for type culture Collection in 5 months and 7 days in 2019, and the preservation number is CCTCC NO: m2019329.
2. A microbial preparation comprising the Pichia kudriavzevii strain of claim 1.
3. A method for synthesizing (R) -1, 3-butanediol, characterized in that (R) -1, 3-butanediol is synthesized by using the whole cells of the Pichia kudriavzevii yeast of claim 1 as a catalyst and 4-hydroxy-2-butanone as a substrate.
4. The method according to claim 3, wherein the catalyst is wet bacteria obtained by inoculating a single colony of the Pichia kudriavzevii yeast of claim 1 into a YPD liquid culture medium, culturing at 28-32 ℃ and 200-220 rpm for 16-18 h, transferring the single colony into a new YPD liquid culture medium at 1-5% of the inoculation amount, culturing at 28-32 ℃ and 200-220 rpm for 40-60 h, and centrifuging.
5. The method of claim 3 or 4, wherein the catalyst is added in an amount of 2 to 10g/g substrate.
6. The method of any one of claims 3 to 5, wherein the pH in the catalytic reaction is 7.0 to 8.5.
7. A process according to any one of claims 3 to 6, wherein the temperature during the catalytic reaction is in the range of 30 to 40 ℃.
8. The method of any of claims 3 to 7, wherein the speed of the catalytic reaction is 180 to 220 rpm.
9. The method according to any one of claims 3 to 8, wherein in the catalytic reaction, the co-substrate is one or more of glucose, ethanol and isopropanol.
10. The method according to claim 9, wherein the mass concentration ratio of the co-substrate to the substrate is 0.5 to 1.5.
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Application publication date: 20200505 |