CN114480459A

CN114480459A - Oxidase whole-cell catalyst and method for preparing high-optical-purity R-type 1,3 butanediol by using same

Info

Publication number: CN114480459A
Application number: CN202210048908.4A
Authority: CN
Inventors: 王建军; 吴胜
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2022-05-13
Anticipated expiration: 2042-01-17
Also published as: CN114480459B

Abstract

An oxidase whole-cell catalyst and a method for preparing high-optical-purity R-type 1,3 butanediol by using the same. The invention relates to the technical field of preparing R type 1,3 butanediol by biocatalysis. The invention provides an oxidase whole-cell catalyst, and a preparation method of the oxidase whole-cell catalyst comprises the following steps: inserting the Ad5SSDH gene into a pET30a vector after PCR amplification to obtain a plasmid pET30Ad5 SSDH; transforming the plasmid pET30Ad5SSDH into recipient bacterium escherichia coli BL21(DE3) to obtain high-yield oxidase gene engineering bacteria; purifying the high-yield oxidase gene engineering bacteria to obtain the oxidase whole-cell catalyst. The invention completely adopts the biological enzyme technology to carry out the enzymatic production of R-1,3-BDO, and compared with a chemical method, the invention reduces the pollution to the environment in the production process. The feeding reaches 6 percent of concentration, the conversion time is 36 hours, the conversion rate is 80 percent, and the chiral purity is 99.2 percent.

Description

Oxidase whole-cell catalyst and method for preparing high-optical-purity R-type 1,3 butanediol by using same

Technical Field

The invention relates to the technical field of preparing R type 1,3 butanediol by biocatalysis.

Background

R-type 1, 3-butanediol (abbreviated as R-1,3-BDO) is colorless, tasteless, low-toxicity, slightly bitter and sweet viscous liquid with a taste, a boiling point of 207.5 ℃, a relative density of 1.01, a flash point of 121 ℃, an ignition temperature of 393.9 ℃, slightly soluble in ether, very easy to dissolve in water, ethanol, acetone and the like, and insoluble in aliphatic hydrocarbon, toluene, mineral oil and the like.

R-1,3-BDO is an important chiral compound and can be used for synthesizing information hormone, spice, carbapenem parent nucleus-azetidinone, pesticide and the like.

R-1,3-BDO can be synthesized by organic chemistry, but the steps in the synthesis process are complicated. For example, Larchevsque uses threonine as a substrate, and R-1,3-BDO is finally obtained by four steps of nitrosation deamination, esterification, hydrogenolysis debromination and reduction, and the like, with the yield of 64 percent. Meanwhile, inorganic lithium is used as a catalyst in the reaction, and the raw material threonine in the method has high price, high control difficulty in each step, low yield and environmental pollution caused by toxic auxiliary materials.

The search of a biocatalytic method for synthesizing R-1,3-BDO is particularly necessary.

Disclosure of Invention

The invention aims to provide a preparation method of R-1,3-BDO, which achieves the purposes of reducing impurities and saving cost. The invention is realized by the following technical scheme:

the first purpose of the invention is to provide an oxidase whole-cell catalyst, and the preparation of the oxidase whole-cell catalyst comprises the following steps: inserting the Ad5SSDH gene into a pET30a vector after PCR amplification to obtain a plasmid pET30Ad5 SSDH; transforming the plasmid pET30Ad5SSDH into recipient bacterium escherichia coli BL21(DE3) to obtain high-yield oxidase gene engineering bacteria; purifying the high-yield oxidase gene engineering bacteria to obtain the oxidase whole-cell catalyst.

In a specific embodiment of the invention, the sequence of the Ad5SSDH gene is shown in a sequence table SEQ NO. 1.

The second purpose of the invention is to provide the application of the oxidase whole-cell catalyst in the oxidation of S-1, 3-butanediol to generate 4-hydroxy-2-butanone.

The third purpose of the invention is to provide a method for preparing R-type 1,3 butanediol by using an oxidase whole-cell catalyst, which comprises the following steps: oxidizing the racemate 1, 3-butanediol by using an oxidase whole-cell catalyst to obtain an oxidation product; reducing the oxidation product with a reductase whole-cell catalyst to obtain R-1, 3-butanediol to obtain an oxidation product; the preparation of the reductase whole-cell catalyst comprises the following steps: after the LnSSFabG gene is amplified by PCR, inserting the LnSSFabG gene into a pET30a vector to obtain a plasmid pET30 LnSSFabG; transforming the plasmid pET30LnSSFabG into recipient bacterium escherichia coli BL21(DE3) to obtain high-yield reductase genetic engineering bacteria; purifying the high-yield reductase genetic engineering bacteria to obtain the reductase whole-cell catalyst.

In a specific embodiment of the invention, the mass ratio of the oxidase whole-cell catalyst to the racemate 1, 3-butanediol is 50: 1.

in a specific embodiment of the invention, the temperature of the oxidation is 37 ℃.

In a specific embodiment of the present invention, the mass ratio of the oxidase whole-cell catalyst to the reductase whole-cell catalyst is 1: 1.

in a specific embodiment of the invention, the temperature of the reduction is 37 ℃.

By adopting the technical scheme, 80g/L of racemate 1,3-BDO can be completely converted into R-1,3-BDO with the optical purity of 99 percent within 24 hours, no by-product is generated, and the purposes of reducing reaction impurities and saving cost are achieved.

By adopting the technical scheme, the whole-cell catalyst has the characteristics of high activity and high substrate concentration tolerance, so that the substrate conversion reaction efficiency is high, and the substrate conversion rate is 95%.

By adopting the technical scheme, the target protein expressed by the genetic engineering bacteria for high-yield production of oxidase or reductase can reach 20-40% of total protein, and high-efficiency heterologous expression of the target protein is realized.

In conclusion, the invention has the following beneficial effects: the conversion rate of the substrate catalyzed by the whole-cell catalyst is 95%, and the cheap racemate 1,3-BDO can be converted into R-1,3-BDO with higher optical purity. Compared with the prior method for industrially preparing R-1,3-BDO by a chemical method, the method has the advantages of simple process, environmental protection, no byproduct generation, great reduction of the use of toxic compounds and organic solvents, and high production efficiency.

Drawings

Figure 1 is a catalytic scheme.

FIG. 2 is a graph showing the protein purity of Ad5SSDH and LnSSFabG.

FIG. 3 is a diagram of gas phase analysis of Ad5SSDH catalyzed 5% oxidative conversion of 1, 3-BDO.

FIG. 4 is a chiral analysis of Ad5SSDH catalyzed 5% oxidative conversion of 1, 3-BDO.

FIG. 5 is a diagram of gas phase analysis of LnSSFabG catalyzing the reduction conversion of 8% 1, 3-BDO.

FIG. 6 is a chiral analysis diagram of LnSSFabG catalyzing 8% reduction conversion of 1, 3-BDO.

FIG. 7 is a diagram of gas phase analysis of a two-step process for catalyzing the 6% reductive conversion of 1, 3-BDO.

FIG. 8 is a chiral analysis of a two-step process for catalyzing the 6% reduction conversion of 1, 3-BDO.

Detailed Description

The present invention will be described in further detail with reference to examples.

The reagents and consumables used in the present invention are all conventional products commercially available.

The invention adopts a two-step method to carry out enzymatic production of R type 1, 3-butanediol (abbreviated as R-1,3-BDO), and utilizes two-step selective catalytic reaction to improve the optical purity of a target product (figure 1).

Firstly, S-type dehydrogenase Ad5SSDH is adopted to carry out preoxidation of a substrate, the enzyme catalyzes oxidation of S-1,3-BDO configuration, and in the second step, LnSSFabG is adopted to reduce 4-hydroxy-2-butanone in the product in the previous step, so that the conversion rate can reach 95% in 18 hours, and the chiral purity of R-1,3-BDO is 99.2%.

The invention completely adopts the biological enzyme technology to carry out the enzymatic production of R-1,3-BDO, and compared with a chemical method, the invention reduces the pollution to the environment in the production process. The feeding reaches 6 percent of concentration, the conversion time is 36 hours, the conversion rate is 80 percent, and the chiral purity is 99.2 percent.

Example 1

Preparing a genetic engineering bacterium for producing oxidase or reductase with high yield:

primers were designed based on the sequences of the Ad5SSDH and LnSSFabG genes of Saccharomyces solfataricus:

the sequence of the Ad5SSDH is shown in a sequence table SEQ NO.1, and an amplification primer thereof:

a forward primer: CGCCATATGagagcagttagattggtagaaataggaa, the underlined sequence indicates the cleavage site NdeI.

Reverse primer: CC (challenge collapsar)GGATCCtggtattaatacttgccttcc, the underlined sequence is the HindIII site. The sequence of the gene product is shown in a sequence table SEQ NO. 2.

The two ends of the gene sequence obtained after PCR amplification are provided with NdeI and HindIII sites, the gene sequence obtained after PCR amplification is inserted into a pET30a vector, the obtained high-expression genetic engineering vector plasmid is named as pET30Ad5SSDH, and then the plasmid pET30Ad5SSDH is transformed into recipient bacterium escherichia coli BL21(DE3) to obtain the high-yield oxidase gene engineering bacterium. The protein expressed by the obtained high-yield oxidase gene engineering bacteria is provided with His-Tag label protein at the C end.

The sequence of the LnSSFabG is shown as the sequence table SEQ NO.3, and the amplification primer comprises the following components:

a forward primer: CGCCATATGatgcgtaaattctgcgatttaagcg, the underlined sequence indicates the cleavage site NdeI.

Reverse primer: CC (challenge collapsar)GGATCCtcataacactaaacccccggtaacac, the underlined sequence is the HindIII site. The sequence of the gene product is shown in a sequence table SEQ NO. 4.

The gene sequence obtained after PCR amplification is inserted into a pET30a vector, the obtained high-expression genetic engineering vector is named as a plasmid pET30LnSSFabG, and then the plasmid pET30LnSSFabG is transformed into a receptor bacterium escherichia coli BL21(DE3) to obtain the high-yield reductase genetic engineering bacterium. The protein expressed by the obtained genetic engineering bacteria has His-Tag label protein at the C end.

The donor bacteria for the oxidase and reductase are of the genus Saccharomyces solfataricus, with the strain name ATCC35091 ATCC35091, the strain name DSM 1616[ P1], the strain type Saccharomyces solfataricus, stored as DSM, isolated from volcanic hot spring, catalog number 35091, and culture medium ATCC 1304, Sulfolobus solfataricus.

Obtaining of high expression strain:

the prepared recombinant vector is introduced into escherichia coli BL21(DE3) by a conventional method to construct genetically engineered bacteria of which recombinase exists in a bacterial body in a soluble form, and the successfully constructed genetically engineered bacteria are screened out.

Engineering bacteria with the target protein expression amount not less than 30% are used as engineering bacteria strains for production and are preserved in the form of glycerol bacteria or milk freeze-dried strains.

The specific transformation method is as follows:

100 μ L of the competent cells of the above-mentioned engineering bacteria strain was taken out from the refrigerator. Cells were thawed on ice for 2-5 minutes. After thawing, flick the tube wall 1-2 times to resuspend the cells.

mu.L of pET30Ad5SSDH or pETLnSSFabG plasmid was added to the competent cells. Mix with gentle shaking and then replace the tube in ice. Ice-cooling for 30 min. Water bath at 42 ℃ for 90 seconds. The tubes were then quickly transferred to ice for 2 minutes to allow the cells to cool. Add 500. mu.L sterile and antibiotic-free LB medium into each centrifuge tube, mix well and put into 37 ℃ shaking table to shake and culture for 45min (150rpm/min) to recover the thallus. mu.L of the mixture was pipetted into LB solid medium containing kanamycin (Kan 100. mu.g/mL), gently spread with a glass spatula, the plate was left at room temperature until the liquid was absorbed, inverted, and cultured at 37 ℃ for 12 to 16 hours.

The LB culture medium has the following formula: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract; adjusting the pH value of NH3 & H2O to 7.0, adding 15g/L agar powder into a plate culture medium which is LB culture medium, and sterilizing at 121 ℃ for 20 min.

Obtaining high-expression genetic engineering bacteria: coli BL21(DE3) pET30Ad5SSDH and E.coli BL21(DE3) pETLnSSFabG.

Gas phase analysis and chiral analysis of reaction conversion process

Gas phase analysis; after the reaction was terminated, 200. mu.l of the reaction solution was extracted with the same volume of ethyl acetate, and the extraction was followed by analysis by gas chromatography.

The analysis conditions were HP7890 gas chromatograph, Agilent HP-5(0.2 mm. times.30 m) column chromatography, initial temperature of 90 deg.C, heating rate of 20 deg.C/min, and sample injection of 2 μ L. Under the condition, the peak time of the 4-hydroxy-2 butanone is between 1 and 1.05 minutes, and the peak time of the 1, 3-butanediol is between 1.2 and 1.3 minutes.

Performing chiral analysis; mu.l of the reaction mixture was extracted with the same volume of ethyl acetate, and 25. mu.l of the extract was added to 50. mu.l of dimethylformamide, and 375. mu.l of diethyltrimethylsilylamine was added to the mixture, and the mixture was incubated at 80 ℃ for 15 minutes, followed by chiral analysis.

The analysis conditions were HP7890 gas chromatography, and the column chromatography was CP-Cyclodextrin-. beta. -236-N19, (0.2 mm. times.30 m), isothermal temperature, and 2. mu.L of sample injection. The peak time for R-1, 3-butanediol ranged from 18 to 19 minutes and for S-1, 3-butanediol ranged from 26 to 27 minutes when analyzed with this addition.

Example 2

Expression and purification of oxidase Ad5SSDH or reductase LnSSFabG

(1) Respectively activating colonies of the high-yield oxidase gene engineering bacteria and the high-yield reductase gene engineering bacteria on a culture dish in a seed culture medium, and activating the colonies overnight at the temperature of 37 ℃ and the rpm of 200 in 5mL LB (Kan100 mu g/mL) culture medium.

(2) Fermentation culture the overnight activated seed solution was inoculated into 800mL LB fermentation medium (kan100g/mL) at a ratio of 10%, and cultured at 37 ℃ and 200rpm until the mid-log OD600 became about 0.6.

(3) And (3) adding lactose for induction at the middle logarithmic phase of the induction culture to ensure that the final concentration is 1mM, and carrying out induction culture at 25-30 ℃ and 180rpm for 4-6 hours.

(4) SDS-PAGE was performed by centrifuging 4mL of the fermentation broth after induction at 12000rpm for 90sec, adding 800. mu.L of deionized water, and sonicating for 5 min. Centrifuge at 12000rpm for 10 min.

Adding 20 mu L of centrifuged supernatant into 5 mu L of loading buffer solution; after centrifugation, the precipitate was washed with deionized water 1 time, and after adding 800. mu.L of deionized water, 20. mu.L of the precipitate was added to 5. mu.L of the loading buffer.

SDS-PAGE gels were loaded at 12.5% concentration in 10. mu.L per well.

(5) The purification was carried out in an AKTApurifier 10 apparatus under the following conditions: the column HisTrap HP-5 was equilibrated with buffer A (50mM Tris-HCl, 0.5M NaCl, 5% glycerol, 20mM imidazole, pH7.5), the enzyme-containing supernatant prepared in (4) was applied to the column, eluted to remove foreign proteins, the target protein was eluted with buffer B (50mM Tris-HCl, 0.5M NaCl, 5% glycerol, 0.5M imidazole, pH7.5), desalted by dialysis, and the enzyme solution was concentrated with an ultrafiltration membrane to obtain the Ad5SSDH oxidase whole cell catalyst and LnSSFabG reductase whole cell catalyst, which were stored at-80 ℃ for use.

(6) Purity of the Ad5SSDH oxidase whole-cell catalyst and LnSSFabG reductase whole-cell catalyst (FIG. 2).

Wherein M is a protein molecular weight standard.

Example 3

Method for preparing Ad5SSDH oxidase whole-cell catalyst and experiment for transforming racemate 1,3-BDO by using the whole-cell catalyst to generate R-1, 3-BDO:

(1) seed Medium activation Single colonies on the plates were picked up in 5mL LB (Kan 100. mu.g/mL) medium and activated overnight at 37 ℃ at 200 rpm.

The seed culture medium is LB culture medium, the formula is as follows: 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract; adjusting the pH value of NH3 & H2O to 7.0, adding 15g/L agar powder into a plate culture medium which is LB culture medium, and sterilizing at 121 ℃ for 20 min.

(2) Fermentation culture the overnight activated seed solution was inoculated into 1L TB fermentation medium (Kan 100. mu.g/mL) at 10% ratio, and cultured at 37 ℃ and 200rpm until the logarithmic mid-phase OD600 was 0.6-1.

The fermentation medium is a TB medium, and the formula is as follows: peptone 12g/L, yeast extract 24g/L, glycerin 4mL/L, constant volume to 900mL, simultaneously preparing 100mL 0.17M potassium dihydrogen phosphate-dipotassium hydrogen phosphate solution, sterilizing at 115 deg.C for 20min, mixing the sterilized two solutions for later use.

(3) And (3) adding lactose for induction at the middle logarithmic phase of the induction culture to ensure that the final concentration is 1mM, and carrying out induction culture at 25-30 ℃ and 180rpm for 4-6 hours. Centrifuging at 8000rpm for 10min to collect bacteria.

(4) 0.6g of the cells were weighed, resuspended in 2mL of 50mM TrisHCl buffer (pH 7.0), and then substrate racemate 1,3-BDO was added to the resulting mixture to a final concentration of 5g/L, and the mixture was converted at 37 ℃ and 200rpm for 18 hours to give 4-hydroxy-2-butanone and R-1, 3-BDO.

The dosage of the whole-cell catalyst in each milliliter of reaction system is 0.3-0.5 g.

The final conversion of the reaction was 75% (FIG. 3) and the ee value of R-1,3-BDO was 90% (FIG. 4).

Example 4

A method for preparing LnSSFabG reductase whole-cell catalyst and an experiment for converting 4 hydroxy 2 butanone to produce R-1,3-BDO by using the whole-cell catalyst are disclosed:

(3) Lactose is added in the middle logarithmic phase of the induction culture for induction, so that the final concentration is 1mM, 25 ℃ to

Performing induction culture at 30 ℃ and 180rpm for 4-6 hours. Centrifuging at 8000rpm for 10min to collect bacteria.

(4) 0.6g of the cells were weighed, resuspended in 2mL of 50mM TrisHCl buffer (pH 7.0), and the substrate, 4-hydroxy-2-butanone, was added to the cells to a final concentration of 8g/L, and the cells were transformed at 37 ℃ and 200rpm for 18 hours to give R-1, 3-BDO.

The final conversion of the reaction was 85% (FIG. 5), and the ee value of R-1,3-BDO was 92% (FIG. 6).

Example 5

Two-step method for converting racemate 1,3-BDO by using a whole-cell catalyst to generate R-1,3-BDO experiment:

(1) the cultivation and preparation of the oxidase whole-cell catalyst and the reductase whole-cell catalyst were the same as in the above examples.

(2) First step oxidation

0.6g of the oxidase whole-cell catalyst cells were weighed, resuspended in 2mL of 50mM TrisHCl buffer (pH 7.0), and 0.012g of the substrate racemate 1,3-BDO was added thereto, and the reaction temperature was 37 ℃ and the reaction was carried out at 200rpm for 18 hours.

The reaction was terminated and centrifuged, and the cell oxidation supernatant was discarded and adjusted to pH 7.0 for use.

(3) Second reduction step

0.6g of reductase whole-cell catalyst thalli was weighed and added to the oxidation supernatant after completion of the previous step, and the conversion reaction temperature was 37 ℃ and conversion was carried out at 200rpm for 10 hours.

The final conversion after termination of the reaction was 92%, and the ee value of R-1,3-BDO was 99.2% (FIG. 7).

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.

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Claims

1. The preparation method of the oxidase whole-cell catalyst is characterized by comprising the following steps:

inserting the Ad5SSDH gene into a pET30a vector after PCR amplification to obtain a plasmid pET30Ad5 SSDH;

transforming the plasmid pET30Ad5SSDH into recipient bacterium escherichia coli BL21(DE3) to obtain high-yield oxidase gene engineering bacteria;

purifying the high-yield oxidase gene engineering bacteria to obtain the oxidase whole-cell catalyst.

2. The oxidase whole-cell catalyst according to claim 1, wherein the sequence of the Ad5SSDH gene is shown as SEQ ID No.1 of the sequence Listing.

3. Use of the oxidase whole-cell catalyst according to claim 1 for the oxidation of S-1, 3-butanediol to 4-hydroxy-2-butanone.

4. The method for preparing R-type 1,3 butanediol by using the oxidase whole-cell catalyst according to claim 1, comprising the steps of:

oxidizing the racemate 1, 3-butanediol by using an oxidase whole-cell catalyst to obtain an oxidation product;

reducing the oxidation product with a reductase whole-cell catalyst to obtain R-1, 3-butanediol to obtain an oxidation product;

the preparation of the reductase whole-cell catalyst comprises the following steps:

after the LnSSFabG gene is amplified by PCR, inserting the LnSSFabG gene into a pET30a vector to obtain a plasmid pET30 LnSSFabG;

transforming the plasmid pET30LnSSFabG into recipient bacterium escherichia coli BL21(DE3) to obtain high-yield reductase genetic engineering bacteria;

purifying the high-yield reductase genetic engineering bacteria to obtain the reductase whole-cell catalyst.

5. The method for preparing R-type 1, 3-butanediol by using the oxidase whole-cell catalyst according to claim 4, wherein the mass ratio of the oxidase whole-cell catalyst to racemate 1, 3-butanediol is 50: 1.

6. the method for preparing R-type 1,3 butanediol by using the oxidase whole-cell catalyst according to claim 4, wherein the temperature of the oxidation is 37 ℃.

7. The method for preparing R-type 1,3 butanediol by using the oxidase whole-cell catalyst according to claim 4, wherein the mass ratio of the oxidase whole-cell catalyst to the reductase whole-cell catalyst is 1: 1.

8. the method for preparing R-type 1,3 butanediol by using the oxidase whole-cell catalyst according to claim 4, wherein the temperature of the reduction is 37 ℃.