CN116064495B - THDP dependent decarboxylase and application thereof - Google Patents

Abstract

The present invention belongs to the field of bioengineering, and is particularly related to a THDP-dependent decarboxylase and its application in the preparation of furan ammonium salt precursors, and the specific application is that the above-mentioned decarboxylase can efficiently catalyze furfural and formaldehyde C-C bond formation reaction to prepare 2-furanyl hydroxymethyl ketone. The decarboxylase of the present invention is prepared by the following steps: synthesizing the coding gene of the decarboxylase, selecting an expression vector, transforming the protein expression host bacteria, and inducing protein expression. The decarboxylase of the present invention has extremely high catalytic efficiency and substrate tolerance concentration, and the concentration of the tolerated substrate furfural concentration can reach 60g/L, and the furfural concentration of 60g/L can be completely reacted within 6h under the whole cell catalysis of 20g/L, and the product liquid phase purity can reach 97.26%, and the maximum yield of 2-furanyl hydroxymethyl ketone reaches 80g/L.

Description

A THDP-dependent decarboxylase and its application

Technical Field

The invention belongs to the field of bioengineering, and in particular relates to a THDP-dependent decarboxylase and application thereof in preparing a furanammonium salt precursor.

Background technique

Furan ammonium salt is one of the key intermediates of the widely used antibiotic cefuroxime in clinical practice. Cefuroxime is a semi-synthetic broad-spectrum cephalosporin antibiotic for parenteral administration. At present, the mainstream production process of furan ammonium salt is to use acetylfuran as a raw material to first oxidize to obtain furanone acid, and then oxime it with methoxyamine and introduce ammonia to form a salt to obtain furan ammonium salt. The acetylfuran currently sold on the market basically uses furan to be decarbonylated by furfural, and then acylated with acetic anhydride to prepare acetylfuran. The raw material price of acetylfuran is relatively high. With the increasingly fierce market competition, the product profit of furan ammonium salt has gradually declined, and the price has dropped from about 300,000 yuan per ton in 2020 to about 180,000 yuan per ton at present. If furfural is used as a raw material to obtain 2-furanyl hydroxymethyl ketone through a decarboxylase step, and then the furan ammonium salt intermediate furanone acid is obtained by chemical oxidation, using 2-furanyl hydroxymethyl ketone instead of acetylfuran for the production of furan ammonium salt will significantly reduce the production cost of furan ammonium salt. The process route is as follows (schematic diagram of the preparation of 2-furanyl hydroxymethyl ketone by enzyme-catalyzed furfural):

The following documents have disclosed relevant reports on this process:

CN114591938A discloses a carboxylase mutant and its preparation method and application. The amino acid sequence of the carboxylase mutant is SEQ ID NO:3. SEQ ID NO:3 is obtained by mutating the 492nd amino acid of the amino acid sequence shown in SEQ ID NO:1 from Val to Pro. The highly efficient catalysis of the production of furan ammonium salt precursor 1-(2-furanyl)-2-hydroxyethanone using furfural and formaldehyde as raw materials significantly reduces the production cost of furan ammonium salt, and the purity of the product 1-(2-furanyl)-2-hydroxyethanone is greatly improved; wherein, the concentration of furfural is 20-60g/L, the concentration of formaldehyde is 20-60g/L, and the dosage of the carboxylase mutant is 8-12g/L; the catalytic reaction conditions are: the temperature is 20-37°C, the pH of the catalytic reaction is 6.0-80, and the catalytic reaction time is 8-20h.

The method disclosed in the above patent document CN114591938A, although the furan ammonium salt precursor 1-(2-furanyl)-2-hydroxyethanone finally obtained has a high purity, but the catalysis time is slightly longer. For example, the document discloses that to obtain 1-(2-furanyl)-2-hydroxyethanone with a purity of 97.1%, a reaction time of 20 hours is required, which is a long reaction time.

Summary of the invention

In order to further improve the catalytic efficiency and effect and shorten the catalytic time, the present invention provides a decarboxylase that can more efficiently catalyze the C-C bond formation reaction of furfural and formaldehyde to prepare 2-furanyl hydroxymethyl ketone, and the catalytic time is shorter than that of the products in the prior art.

In the present invention, the coding gene (SEQ ID NO: 2) of decarboxylase is obtained by molecular cloning technology, and the enzyme protein is obtained by constructing a 6×His fusion expression vector of the enzyme gene and introducing it into genetic engineering bacteria OverExpress C43 (DE3) to induce expression. The enzyme catalyzed reaction is carried out under appropriate conditions with decarboxylase as catalyst and furfural and formaldehyde as substrates. The results show that the 2-furanyl hydroxymethyl ketone synthesized by the enzyme has high purity, which can reach more than 97.26%, and the maximum yield reaches 60g/L. It has great application potential and value in the industrial production of furan ammonium salts, and its catalytic time is only 6 hours, which greatly saves catalytic time and improves catalytic efficiency compared to the carboxylase mutant disclosed in CN114591938A mentioned in the background technology.

The amino acid sequence of the THDP-dependent decarboxylase provided by the present invention is shown in SEQ ID NO: 1; the nucleotide sequence encoding the decarboxylase is shown in SEQ ID NO: 2.

The expression cassette, vector or recombinant bacteria containing the above nucleotide sequence are also within the scope of protection of the present invention.

The above-mentioned method for preparing the decarboxylase specifically comprises the following steps: synthesizing the coding gene of the decarboxylase, selecting an expression vector, transforming a protein expression host bacterium, and inducing protein expression.

The application of the above-mentioned THDP-dependent decarboxylase in catalyzing the C-C formation reaction of furfural and formaldehyde is the key content to be protected by the present invention.

Specifically, the above decarboxylase, when catalyzing, performs a catalytic reaction with furfural and formaldehyde as substrates, and can be used to catalyze the synthesis of 2-furanyl hydroxymethyl ketone. The above catalytic reaction is performed under the conditions of 25-40°C and pH=6.0-7.0.

The catalyst containing the above decarboxylase, and the use of the catalyst in catalyzing the C-C formation reaction between furfural and formaldehyde, are also within the scope of the present invention. Specifically, the application method of the above catalyst is also to carry out a catalytic reaction with furfural and formaldehyde as substrates to synthesize 2-furanyl hydroxymethyl ketone. The above catalytic reaction is also carried out under the conditions of 25-40°C and pH=6.0-7.0.

Beneficial effects of the present invention:

(1) The present invention obtains a decarboxylase through biological engineering, realizes the efficient catalysis of producing 2-furanyl hydroxymethyl ketone, a precursor of furan ammonium salt, using furfural and formaldehyde as raw materials, has extremely high catalytic efficiency and substrate tolerance concentration, and the tolerated substrate furfural concentration can reach 60 g/L. The 60 g/L furfural concentration can be completely reacted within 6 hours under the catalysis of 20 g/L whole cells, and the liquid phase purity of the product can reach 97.26%, and the maximum yield of 2-furanyl hydroxymethyl ketone reaches 80 g/L; if it is applied to industrial production, the production cost of furan ammonium salt will be significantly reduced, and it has extremely high economic benefits and value;

(2) Compared with the catalysts in the prior art, the decarboxylase provided by the present invention is used in catalysis. Compared with the catalyst in the prior art CN114591938A, its catalytic efficiency is higher and the catalytic time is shorter, which is only 1/3 of the catalytic time in CN114591938A. Compared with the catalyst in CN114591938A, the present invention has significant progress.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an SDS-PAGE electrophoresis diagram of decarboxylase; wherein BL21 (DE3) and Overexpress C43 (DE3) are two E. coli protein expression hosts; Marker is a protein molecular weight standard; the molecular weight of decarboxylase is about 63.1 kDa;

Fig. 2 is the HPLC collection of illustrative plates of the product generated by decarboxylase catalysis;

Figure 3 is the nuclear magnetic resonance spectrum of the product catalyzed by decarboxylase; (a) (b) are the H-NMR spectrum and C-NMR spectrum of the product 2-furanyl hydroxymethyl ketone standard respectively; (c) (d) are the H-NMR spectrum and C-NMR spectrum of the product 2-furanyl hydroxymethyl ketone catalyzed by the whole cell of decarboxylase respectively.

Detailed ways

The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only for explanation and illustration and do not limit the scope of the present invention in any way.

Main reagents and consumables

pET-28a(+) plasmid is a known E. coli expression vector with a vector size of 5369 bp, T7 promoter, vector tags N-6×His and C-6×His, and vector resistance Kanamycin, purchased from Novogen; E. coli DH5α competent cells (Cat. No. G6016), E. coli Overexpress C43(DE3) competent cells (Cat. No. X17016) and E. coli BL21(DE3) competent cells (Cat. No. G6030) were all purchased from Ang Yu Biotechnology; agar powder (Cat. No. A8190) was purchased from Solarbio; TRYPTONE (Cat. No. LP0042) and YEAST EXTRACT (Cat. No. LP0021) were purchased from OXOID; NaCl (Cat. No. 10019318), K ₂ HPO ₄ ·3H ₂ O (Cat. No. Reagents such as 1H 2 PO 4 (Cat. No. 10017518), KH ₂ PO ₄ (Cat. No. 10017618), and propylene glycol (Cat. No. 10010618) were purchased from Sinopharm Chemical Reagent Co., Ltd.

LB medium

Patent document CN114591938A is referenced here. Each 100mL LB medium contains: 1g TRYPTONE, 0.5g YEAST EXTRACT and 1g NaCl, pH = 7.0-7.2. Preparation method: Dissolve 10g TRYPTONE, 5g YEAST EXTRACT, 10g NaCl in 1L distilled water, and do not adjust the pH value. If preparing solid culture medium, add 1.8g agar powder to 100mL culture medium. Sterilize with high pressure steam at 121℃ for 20min.

TB medium

Patent document CN114591938A is referenced here. Each 100 mL TB medium contains: 1.2 g TRYPTONE, 2.4 g YEAST EXTRACT, 0.4 mL glycerol, 1.6 g K ₂ HPO ₄ ·3H ₂ O and 0.23 g KH ₂ PO ₄ , pH 7.0-7.2. Preparation method: Dissolve 12 g TRYPTONE, 24 g YEAST EXTRACT, 4 mL glycerol, 16 g K ₂ HPO ₄ ·3H ₂ O and 2.3 g KH ₂ PO ₄ in 1 L of distilled water, and do not adjust the pH value. Sterilize with high pressure steam at 121°C for 20 min.

Unless otherwise specified, the reagents used in the following examples are all commercially available reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to relevant experimental manuals or manufacturer instructions.

Example 1 Preparation of decarboxylase

1. Acquisition and synthesis of decarboxylase gene

Through gene mining, a decarboxylase gene with NCBI number WP_103376266.1 was screened from the NCBI database. The open reading frame of the gene is 1668 bp in length. The decarboxylase encoded by the gene consists of 555 amino acids. The amino acid sequence (555aa) is shown in SEQ ID NO: 1 (Sequence Table 1); the nucleotide sequence encoding the decarboxylase is shown in SEQ ID NO: 2 (Sequence Table 2);

The decarboxylase with Genbank ID WP_103376266.1 was fully synthesized, and the synthesized gene fragment was ligated into the pET-28a(+) plasmid. The above gene was synthesized by entrusting Sangon Biotech (Shanghai) Co., Ltd.

2. Heterologous Expression of Decarboxylase 6×His Fusion Protein

The following operation steps 1-3 refer to patent document CN114591938A.

1. Transformation of competent cells for protein expression

Take out E.coli BL21(DE3), E.coli Rosetta(DE3), Overexpress C43(DE3), and E.coli BL21-condon plus(DE3) competent cells from the -80℃ ultra-low temperature freezer and place them on ice. After the cells melt, add 1μL of pET-28a(+) plasmid containing the decarboxylase gene to each competent cell and place them on ice for 30 minutes. Heat shock at 42℃ for 50s, then place on ice for 3min. Add 600μL of sterile LB liquid culture medium and culture at 37℃, 200rpm shaking for 1h. Pipette 200μL of the cultured bacterial solution and spread it on the LB plate culture medium containing Kana resistance (50μg/mL), and invert and culture at 37℃ overnight.

2. Preservation of decarboxylase expression strains

A single colony on the LB plate was picked and inoculated into LB liquid medium containing Kana resistance (50 μg/mL), and cultured overnight at 37°C and 200 rpm in a shaking incubator to be used as a strain expressing decarboxylase. Sterile glycerol was added to the bacterial solution at a final volume concentration of 15%, and stored in a -80°C ultra-low temperature refrigerator for a long term.

3. Protein expression

(a) Seed shake flask culture: 100 μL of decarboxylase protein expression glycerol bacteria were inoculated into 50 mL of sterile TB liquid culture medium, the final concentration of kanamycin was 50 μg/mL, 37°C, 200 rpm culture for 8 h, and the decarboxylase protein expression seed bottle bacterial solution was obtained. (b) Fermentation shake flask culture: 10 mL of decarboxylase protein expression seed bottle bacterial solution was inoculated into 350 mL of sterile TB liquid culture medium, the final concentration of kanamycin was 50 μg/mL, 37°C, 200 rpm; when OD600 = 0.6-0.8, sterile IPTG with a final concentration of 0.3 mM was added, and the induction culture was carried out at 28°C, 200 rpm for 20 h to obtain enzyme solution. (c) The enzyme solution was subjected to ultrasonic cell wall breaking treatment, and the whole bacteria, cell wall breaking supernatant and cell wall breaking precipitate were subjected to SDS-PAGE detection to identify its molecular weight and soluble expression. The results are shown in Figure 1. The results of SDS-PAGE showed that the decarboxylase was successfully expressed in both hosts, with a protein molecular weight of about 61.3 kDa. The expression level of the decarboxylase mutant was the highest in the OverExpress C43 (DE3) host. (d) Centrifuge at 4000 rpm for 12 min to collect the OverExpress C43 (DE3) cells that had expressed a large amount of the decarboxylase mutant.

The OverExpress C43 (DE3) wet bacteria obtained in Example 1 and expressing a large amount of decarboxylase mutants were used, as shown in Examples 2 to 6.

Example 2 Synthesis of 2-furanyl hydroxymethyl ketone using decarboxylase

(1) Synthesis of 2-furanyl hydroxymethyl ketone

The substrate solution was prepared with an aqueous solution of pH 7.0, and a fed-batch reaction was carried out. The final concentration of the reaction substrate was 60 g/L of furfural and formaldehyde, the reaction temperature was 35° C., the amount of the decarboxylase wet bacteria in Example 1 was 20 g/L, and the sample aqueous solution was obtained by centrifugation (8000 rpm for 2 min) after the reaction for 6 h. The aqueous solution was extracted with 2 times of ethyl acetate, and 80 g of 2-furanyl hydroxymethyl ketone product was obtained by rotary evaporation at 60° C. and a vacuum degree of -0.1 MPa to recover ethyl acetate.

(2) Detection of 2-furanyl hydroxymethyl ketone

The sample aqueous solution obtained in step 1 was diluted 10 times to achieve a better HPLC separation effect. The purity of 2-furanylhydroxymethylketone in the sample was detected by HPLC. HPLC instrument model: Shimadzu LC 2030C. Parameter settings: chromatographic column: Inertsil ODS-3V (4.6×250mm); wavelength 274nm, column temperature 30℃; mobile phase: phase A is phosphate buffer, 2.73g of potassium dihydrogen phosphate is dissolved in 1000ml of water, and adjusted to 2.8 with dilute phosphoric acid; phase B is acetonitrile; flow rate 1mL/min. The purity of 2-furanylhydroxymethylketone in the sample is determined according to the peak area. The HPLC results are shown in Figure 2. The retention time of 2-furanylhydroxymethylketone is 4.81min, and the liquid phase purity of the product is 97.26%. The product obtained in step 1 was subjected to nuclear magnetic resonance analysis. From the spectrum analysis of Figure 3, it can be further determined that the product generated by the reaction is the target product 2-furanylhydroxymethylketone.

The test shows that the present invention takes less time and further improves the efficiency compared with the prior art document CN114591938A. The product with a liquid phase purity of 97.26% is obtained after 6 hours of reaction, while the prior art reaction takes 20 hours to reach a liquid phase purity of 97.1%. See Example 7 for a specific comparison.

Example 3

The substrate solution was prepared with an aqueous solution of pH 6.0, and a fed-addition reaction was performed. The final concentration of the reaction substrate was 60 g/L of furfural and formaldehyde, the reaction temperature was 37° C., the amount of the decarboxylase in Example 1 was 20 g/L, and the sample aqueous solution was obtained by centrifugation (8000 rpm for 2 min) after the reaction for 6 h. The purity of 2-furanyl hydroxymethyl ketone in the sample was detected by HPLC, and the product purity was 97.23%.

Example 4

The substrate solution was prepared with an aqueous solution of pH 6.5, and a fed-addition reaction was performed, the final concentration of the reaction substrate was 60 g/L of furfural and formaldehyde, the reaction temperature was 25° C., the amount of the decarboxylase in Example 1 was 20 g/L, and the sample aqueous solution was obtained by centrifugation (8000 rpm for 2 min) after the reaction for 8 h. The purity of 2-furanyl hydroxymethyl ketone in the sample was detected by HPLC, and the product purity was 97.19%.

Example 5

The substrate solution was prepared with an aqueous solution of pH 7.0, and a fed-addition reaction was performed, the final concentration of the reaction substrate was 55 g/L of furfural and formaldehyde, the reaction temperature was 35° C., the amount of the decarboxylase in Example 1 was 20 g/L, and the sample aqueous solution was obtained by centrifugation (8000 rpm for 2 min) after the reaction for 5.5 h. The purity of 2-furanyl hydroxymethyl ketone in the sample was detected by HPLC, and the product purity was 97.58%.

Example 6

The substrate solution was prepared with an aqueous solution of pH 7.0, and a fed-addition reaction was performed, the final concentration of the reaction substrate was 65 g/L of furfural and formaldehyde, the reaction temperature was 40° C., the amount of the decarboxylase in Example 1 was 20 g/L, and the sample aqueous solution was obtained by centrifugation (8000 rpm for 2 min) after the reaction for 10 h. The purity of 2-furanyl hydroxymethyl ketone in the sample was detected by HPLC, and the product purity was 97.09%.

Example 7

The product of the present invention is compared with the product in the prior art, and the specific results are as follows:

Table 1 Comparison of the products in the present invention and the products in the prior art

product Furfural concentration (g/L) purity(%) Catalytic time (h) Embodiment 2 of the present invention 2-Furanyl hydroxymethyl ketone 60 97.26 6 Embodiment 3 of the present invention 2-Furanyl hydroxymethyl ketone 60 97.23 6 Embodiment 4 of the present invention 2-Furanyl hydroxymethyl ketone 60 97.19 8 Embodiment 5 of the present invention 2-Furanyl hydroxymethyl ketone 55 97.58 5.5 Embodiment 6 of the present invention 2-Furanyl hydroxymethyl ketone 65 97.09 10 Prior art embodiment 2 1-(2-Furyl)-2-hydroxyethanone 40 95.5 12 Prior art embodiment 3 1-(2-Furyl)-2-hydroxyethanone 20 95.9 8 Prior art embodiment 4 1-(2-Furyl)-2-hydroxyethanone 60 97.1 20

The product in the prior art refers to the carboxylase mutant disclosed in CN114591938A. From the data in the above table, it can be seen that in the process of catalyzing the same type of product, the purity of the obtained 2-furanyl hydroxymethyl ketone can reach 97.26% after 6 hours of catalysis by the decarboxylase of the present invention, which has a high purity and a short time.

In the prior art, in Examples 2 to 4, after 20 hours of catalysis, the purity of the obtained product 1-(2-furanyl)-2-hydroxyethyl ketone is comparable to that of the present invention; after 8 hours of catalysis, the purity of the product is 95.5, which is about 2 percentage points lower than that of the present invention. It is well known to those skilled in the art that it is very difficult to increase the purity of the product furan ammonium salt precursor, i.e., furanyl hydroxy ketone product, by even 1 percentage point. The present invention achieves a purity of about 2 percentage points through a new decarboxylase catalyst, which is an outstanding and significant improvement over the prior art.

Sequence Listing 1

SEQ ID NO:1

MTTPYTVGRYLLDRLAELGIDRLFGVPGDYNLNFLSQVMEDPVIEWVGNRNELNAAYAADGYARIKGIGALITTFGVGELS AINGVAGSYAERVPVVAITGTPATSVMAEGLLVHHSLGDGEFQHFLHAYQEVTAAQTVLQPDTAMDEIDRVLRICWYEKRPVYIALPSDVSGKPATPPQAPLLHSSGPLAPRDNAGPLVLEQILHRLASAKRPFVLADFEVDRYHAQNLVRTFIERTGLPIATLS MGKGIIDETHPQFVGVYHGALSSNELQEQITGSDCLLLIGVQLIDTITGSFTDNIDLSTVIDIHPHHTLIEGMRYEGIDMLWLLDALTRHASQHPMAHKPNPTTVPPILPADMEGPITQEHFWSRFQQFLAPHDVLIADQGTAYFGATSLVLPPGTRFIGQPLW GSIGFTLPALLGTQLADPKRRNILLIGDGSFQLTLQELSTIIRWRLHPIIILINNDGYTVERAIHGPEEAYNDIPMWSYHDIPRVFGDSTALTRQVATVRDLDEALAAVDLARDRLVFLEVMMNRMDAPDILRRLGRAMAAQNRY

Sequence Listing 2

SEQ ID NO:2

atgacaacaccttacaccgttggccgttatctccttgaccgcttagccgaactcggcattgatcgcctctttggagtgcccggcgattacaatctgaactttct tagccaagtcatggaagaccccgtgattgagtgggtggggaaccgcaatgaactcaatgccgcctatgccgccgatggttacgcccgcatcaaaggtatcggagcccttatcaccacatttggtgtgggggaactcagcgccatcaatggcgtcgccggatcgtatgcagaacgagtgccggtagtggccattaccggaa cacctgctacatcggtgatggccgaaggccttttagttcatcattcgttaggcgatggtgagtttcaacatttcctgcacgcgtatcaagaggtcaccgcggcgcaaaccgtgttacaaccggacaccgcgatggatgaaattgatcgcgtcttgcgaatttgttggtatgagaaacgcccggtatacattgccctcccgtcgga cgtcagtggcaaacccgcaacaccgcctcaagcgccgttacttcattcctctgggccgttggcacctcgtgataatgctggccccctggtgctcgaacagattcttcaccgtctcgcatcagcaaagcgtcccttcgtcctagccgattttgaagttgaccggtatcatgcacaaaatcttgttcggacgttcatcgaacgcactg ggctgcctattgccacattaagcatgggcaagggcattattgatgaaacgcatcctcaatttgttggggtgtatcatggagcactgagcagcaatgaacttcaagaacagatcaccggttcggactgtttgttactcattggcgtgcaactaatcgacaccatcaccggaagctttaccgataacatcgacttaagcaccgtga ttgacattcaccctcaccacaccctcattgaggggatgcgctacgaaggaatcgatatgttgtggctgctagatgccctgacccggcacgcgtcccaacaccctatggcgcataaacccaatccaaccacggtcccccctatattgcccgccgacatggaaggccccattactcaagagcacttttggtcccgatttcaacag tttttagccccccatgatgtgctcattgcagaccaaggcacggcttactttggtgcgacttctctcgtcctgccccctggtacccgcttcattggtcaacctctttggggatcgatcgggtttaccttacccgctctacttgggacgcaacttgctgatcctaagcgtcgcaatatcctactcattggagacgggtcctttcaactgac gttacaagagctttccacgattatccgttggcgtctgcacccgatcatcatcctgatcaacaacgatggctacacggttgaacgcgccatacacggccccgaagaagcctataacgacattcccatgtggagttatcacgacatccctcgtgtctttggcgacagtacagctctcacacgccaagttgccacggtgcgcgacct cgatgaggccttggctgctgtggacctagcgcgtgaccgtctcgtctttttagaagtcatgatgaaccgcatggacgcgcccgatatcttgcgccgtttaggtcgtgctatggccgcgcaaaaccgctattaa.

Claims (1)

1. An application of a THDP-dependent decarboxylase, characterized in that the amino acid sequence of the THDP-dependent decarboxylase is shown in SEQ ID NO: 1, and the application is to use the THDP-dependent decarboxylase as a catalyst, under the conditions of 25-40° C. and pH=6.0-7.0, with furfural and formaldehyde as substrates, to catalyze the synthesis of 2-furanyl hydroxymethyl ketone.