Cephalosporin C acylase mutant with substrate specificity and application thereof

Abstract

According to the invention, cephalosporin C acylase is modified by a site-directed mutagenesis technology, so that a mutant which has improved enzyme activity on cephalosporin C and has substrate specificity is obtained. The conversion rate of the obtained mutant to cephalosporin C is 1.2-3 times higher than that of a starting enzyme; the conversion rate of the deacetoxycephalosporin C is 28-20 times lower than that of the starting enzyme; and the conversion rate of the deacetylcephalosporin C is 10-15 times lower than that of the starting enzyme. Therefore, the mutant provided by the invention can efficiently and specifically catalyze the substrate cephalosporin C to generate 7-aminocephalosporanic acid, and has the advantages of mild reaction, simple operation, environmental friendliness and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and relates to a method for synthesizing a cephalosporin mother nucleus by utilizing cephalosporin C acylase to catalyze the synthesis of the cephalosporin mother nucleus. Background Art

[0002] β-lactam antibiotics are a class of antibacterial drugs that account for more than half of the global antibiotic market. Among them, cephalosporins are market leaders in the treatment of a variety of infectious diseases. 7-aminocephalosporanic acid (7-ACA) is the core of the synthesis of β-lactam antibiotics. 7-ACA was originally discovered in filamentous fungi and was obtained by the reaction of cephalosporin C (CPC). CPC is the main metabolic source for the synthesis of 7-ACA. 7-aminocephalosporin acid is the main core compound of current and future semi-synthetic cephalosporin drugs.

[0003] In the early days, 7-ACA was synthesized from CPC through a series of chemical reactions in industry. Not only does it use organic solvents at extremely low temperatures, but this chemical production method also releases toxic chemical waste and phosphate-containing contaminated water, causing serious environmental damage. Therefore, in recent years, enzyme catalysis has gradually become a new direction in industrial production due to its low energy consumption and environmental friendliness. Initially, a two-step enzymatic method was used in industry to synthesize 7-aminocephalosporanic acid from cephalosporin C. CPC was first catalyzed by D-amino acid oxidase (DAAO) to oxidize and decarboxylate to form glutaryl-7-aminocephalosporanic acid (GL-7-ACA), and then hydrolyzed by glutaryl-7-aminocephalosporanic acid acylase (GLA) to form 7-aminocephalosporanic acid. This two-step enzymatic method for producing 7-ACA requires two different bacterial fermentation procedures and a complex enzyme purification-immobilization process, so the manufacturing cost is still high. Later, a one-step enzymatic method was developed, which can directly catalyze cephalosporin C to form 7-aminocephalosporanic acid by cephalosporin C acylase (CCA). In contrast, a one-step enzymatic method to directly convert CPC to 7-ACA is more economically feasible while avoiding the production of hydrogen peroxide.

[0004] The biosynthesis of cephalosporin C starts with three amino acids, namely L-valine, L-cysteine, and L-aminoethanedioic acid. These three amino acids are catalyzed by aminocephalosporanic acid (ACV) synthetase in the cytoplasm to generate the tripeptide ACV, which is then cyclized to generate IPN by isopenicillin N (IPN) synthetase; the generated IPN is isomerized to penicillin N by a two-component outer membrane enzyme located in the peroxisome; penicillin N is converted to deacetyloxycephalosporin C (DAOC) by DAOC synthetase; cytoplasmic hydroxylase promotes the modification of the DAOC side chain to generate deacetylcephalosporin C (DAC). Finally, acetyltransferase adds an acetyl group to DAC to generate CPC, which is then secreted from the fungal cytoplasm into the extracellular space.

[0005] Since cephalosporin C acylase has catalytic activity on DAOC, DAC, and CPC, in order to ensure full utilization of components in the metabolic pathway and more efficient production of 7-ACA, CPC acylase (CCA) with substrate specificity and high enzyme activity is obtained. The present invention has made great efforts in protein engineering of CCA, and obtained an acylase with CPC catalytic activity and inhibition of DAOC and DAC to support industrial applications, and make the synthesis of various core β-lactam antibiotics possible. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to efficiently prepare the cephalosporin mother nucleus 7-ACA by a biocatalytic synthesis method, so as to solve the problem of low catalytic activity and low substrate specificity of the mutant cephalosporin C acylase in the prior art.

[0007] In order to solve the above technical problems, the present invention provides a cephalosporin C acylase mutant, wherein the amino acid sequence of the wild-type acylase is shown in SEQ ID NO:1, which is derived from Bosea sp.OK403, and the acylase mutant is a protein obtained by mutating the wild-type sequence as shown in SEQ ID NO:2, and then mutating at positions R158, M163, R261, and A602 using SEQ ID NO:2 as a template. It is found that the mutant obtained by mutating the A602 site has higher substrate specificity.

[0008] The present invention accordingly provides the encoding nucleic acid molecule of the above-mentioned cephalosporin C acylase mutant, its expression cassette, a recombinant vector containing the encoding gene or its expression cassette, and a recombinant microorganism containing the gene or the expression cassette or the recombinant vector;

[0009] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0010] The expression cassette of the nucleic acid molecule refers to a DNA capable of expressing the mutant cephalosporin C acylase in a host cell, and the DNA may include not only a promoter for initiating transcription of the mutant cephalosporin C acylase encoding gene, but also a terminator for transcription of the mutant cephalosporin C acylase. Furthermore, the expression cassette may also include an enhancer sequence.

[0011] The recombinant vector of the nucleic acid molecule can be a bacterial plasmid (such as an expression vector based on a T7 promoter expressed in bacteria, such as pET-28a, etc.), a bacteriophage, a yeast plasmid (such as a YEp series vector, etc.) or a retroviral packaging plasmid carrying the mutant cephalosporin C acylase encoding gene.

[0012] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.

[0013] The recombinant microorganism containing the nucleic acid molecule encoding the mutant cephalosporin C acylase can be yeast, bacteria, algae or fungi, such as Escherichia coli, carrying a gene encoding cephalosporin or the cephalosporin mutant protein.

[0014] The present invention also provides a method for preparing cephalosporin nucleus 7-ACA, which specifically comprises taking compound I as a substrate and contacting with a catalyst of cephalosporin C acylase as shown in SEQ ID NO: 1 or the cephalosporin mutant to carry out a catalytic reaction to obtain compound II.

[0015]

[0016] Wherein R is acetoxy, hydroxyl or hydrogen.

[0017] In the above method, the recombinant cell can be obtained by introducing a recombinant vector capable of expressing the cephalosporin C acylase mutant into a biological cell.

[0018] The biological cell may be a microorganism. The microorganism may be Escherichia coli or other bacteria. In an embodiment of the present invention, the microorganism is Escherichia coli BL21 (DE3).

[0019] The reaction temperature may be 10-40°C, specifically 25°C; the reaction is carried out in a phosphate buffer with a pH of 6.5-9.5, specifically 8.0. The reaction may generally be 0.5-24h, specifically 1h. The lysate of the recombinant cell may be obtained by lysing the recombinant cell.

[0020] The invention constructs mutants through semi-rational design, obtains mutants with improved enzyme activity and substrate specificity, and the obtained mutants can catalyze cephalosporin derivatives to produce cephalosporin mother nuclei, and has good industrial application prospects in the biocatalytic preparation of cephalosporin mother nuclei. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 . Flowchart of 7-ACA generation by DAOC.

[0022] Figure 2 . Flow chart of the production of 7-ACA from CPC catalyzed by acylase in the present invention.

[0023] Figure 3 .Standard curve diagram of CPC and 7-ACA content in the present invention.

[0024] Figure 4 .HPLC test results of CPC and 7-ACA in the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described below through specific implementation modes to provide a better understanding of the present invention, but does not constitute a limitation of the present invention.

[0026] Example 1 Construction and high-throughput screening of acylase mutants

[0027] 1.1 Construction of starting strain

[0028] According to the sequence of CPC acylase L159S / A419V published in CN 117247925 A, the sequence SEQ ID NO: 2 was constructed, and its encoded amino acid sequence is shown in SEQ ID NO: 1. It was subcloned into the corresponding site of vector pET-28a to obtain plasmid pET-28a-L159S / A419V, and transformed into the expression host Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli expressing the initial CPC acylase.

[0029] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0030] MTTDADRDALEAALPPLSGSLSLSGLEAPVSVRRDAWGIPHIKAVGEADAYRALGFV

[0031] HAQDRLFQMELTRRKALGRAAEWLGAEAAEADILVRRLGMEQACRRDYDALADAAKA

[0032] MLQAYAAGVNAFLESGAPVPCEYTLLGATPETWEPWHSIAVMRRSGLLMGSIWFKLWR

[0033] MLALPIVGAENALKLRYDDGGRDLLCIPPGAQADRFEADLAALAPTVDHLLKAMGGDA

[0034] SDAAGGGSNNWAVGPDRTATGRPILAGDPHRVFEIPGMYAQHHLACDRFDMIGLTVPGVP

[0035] GFPHFAHNGSVAYCVTHAFMDIHDLYLEQFTDEGRAVRFGEGFEPVIHRRDRIAVRGGAD

[0036] REFEIFETRHGPVIAGDPREGVGLSLRSVQFAETDLSFDCLTRMPAASTVSELYEATRGWG

[0037] LIDHNLVVGDTAGAIGHLVRARVPVRSRDNGWLPVPGWTAEHEWQGWIAHEAMPCVID

[0038] PPGGLIVTANNRVVADDHPDYLCTDCHPPYRAERILQRLDSGAPFAVADAAAIHADTQSP

[0039] HLDLFRTRLAVLGLREQADAERLRQDLLAWDGRMDADSTSAAAYNAFRRALTRIVTVRS

[0040] RLEQATAHRFAAVAPGVSPQGQVWWAVPTLLRDDDAGMLNGLTWDDVLSEALSEAAAT

[0041] LTGKTWGEEHQPRFAHPLSAQFPDWAQRLDPPSLPVGGDGDTVLAIGIVPSAGPTATYGA

[0042] LARYVFDVGNWDNSRWAVFHGTSGHPASPHYADQNTPWSACEMVPMLYDWERIAAEA

[0043] KTVQELVPPRA。

[0044] The nucleotide sequence of the coding gene of SEQ ID NO:1 is shown as SEQ ID NO:2:

[0045]

[0046] 2.1 Construction of mutants

[0047] In order to find a cephalosporin C acylase mutant with high activity and substrate specificity, a rational design method was used to site-directedly mutate the amino acid sites using SEQ ID NO:2 as a template. Molecular docking of SEQ ID NO:2 with the substrate CPC was selected. The points R158, M163, R261, and A602 within the range that have a steric hindrance to the binding of the substrate and the enzyme were site-directedly mutated to cysteine ​​and methionine.

[0048] Primers were designed using the above selected sites for site-directed mutagenesis. The primers are shown in Table 1.

[0049] Table 1 Primers for mutation sites

[0050]

[0051] The specific steps for constructing mutants are as follows:

[0052] 2.1.1 PCR for site-directed mutagenesis

[0053] Using SEQ ID NO: 2 as a template, 1.5 μL of each of the upper and lower primers in Table 1 (eg, R158M-F; R158M-R) was added to a 50 μL system to amplify the gene fragment.

[0054] 50μL system: 25μL Taq enzyme, 21μL sterile water, 1μL template, 1.5μL upper and lower primers

[0055] 2.1.2 Construction of genetically engineered strains

[0056] The PCR product was added with 1 μL DpnⅠ at 37°C for 1 hour to digest the template. After digestion, 4 μL of the PCR product was added to the competent E. coli for transformation, and then spread on the LB solid plate containing kanamycin resistance (50 μg / ml). After culturing in a 37°C incubator for 12-14 hours, a single colony grew, which was the acylase mutant after site-directed mutagenesis.

[0057] 2.2 Mutant screening and enzyme activity determination

[0058] The single colonies of the mutant strains were inoculated in 96-well plates, 300 μL of LB liquid medium (containing kanamycin resistance) was added, and the culture was shaken at 37°C and 800 rpm for 12 hours for the normal growth of E. coli. After 12 hours, 700 μL of TB medium (containing a final concentration of 0.1 mM IPTG and 50 μL / mg kanamycin) was taken out and shaken at 25°C and 800 rpm for 14 hours for the expression of acylase. After the culture was completed, the cells were collected by centrifugation, frozen at -80°C for 1 hour, and thawed at room temperature for 30 minutes. 200 μL of phosphate buffer (0.1M PH 8.0) was added to resuspend the cells.

[0059] Different substrates (CPC, DAOC, DAC) were added to 500 μL of lysate and reacted at 25°C, 1000 rpm for 2 h. 100 μL of sample was taken, diluted with methanol and measured by HPLC.

[0060] HPLC conditions for CPC detection: The selected chromatographic column was Agilent ZORBAX SB-C18 (4.6*150mm 4um), the mobile phase was 0.1% formic acid aqueous solution and methanol, the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, the detection time was 15 min, and the column oven was kept constant at 40°C.

[0061] HPLC conditions for detecting DAOC: chromatographic column Agilent ZORBAX SB-C18 (4.6*150mm 4um), mobile phase using 0.1% trifluoroacetic acid solution and methanol, flow rate 1.0 ml / min, detection wavelength 254 nm, detection time 17 min, column oven constant at 40°C.

[0062] HPLC conditions for detecting DAC: chromatographic column Agilent ZORBAX SB-C18 (4.6*150mm 4um), mobile phase using 0.1% trifluoroacetic acid solution and methanol, flow rate 1.0ml / min, detection wavelength 254nm, detection time 7min, column oven constant at 40°C.

[0063] Through screening, it was found that the mutant A602M had improved catalytic activity towards the substrate CPC, but reduced catalytic activity towards DAOC and DAC.

[0064] Table 2 Conversion rates of mutants for different substrates

[0065]

[0066] Example 3 Extraction and reaction of pure enzyme of mutant A602M

[0067] 3.1 Cell culture

[0068] The monoclonal mutant A602M constructed in Example 2 was picked and transferred to a test tube of 5mL LB medium (containing 50μL / ml kanamycin), and cultured overnight at 37°C and 220rpm to obtain the seed solution; the seed solution was then inoculated into 100mL TB medium (50ug / mL kanamycin) at an inoculum size of 1%, and cultured at 37°C and 220rpm. When the OD600 value of the bacterial solution reached 0.6-0.8, 0.1mMIPTG was added, and the culture was continued at 25°C and 220rpm for 16h. The cell bodies were collected by centrifugation at 4°C and 1000rpm for 10min. The bacterial bodies were then washed with phosphate buffer (50mM pH 8.0), and the wet bacteria were weighed after centrifugation. The bacterial suspension was then resuspended in phosphate buffer (10mL / g bacteria). The bacterial suspension was ultrasonically broken for 20min, and the supernatant was taken at 4°C and 6000rpm for 1h to obtain a crude enzyme solution.

[0069] 3.2 Acylase protein purification

[0070] The crude enzyme solution was treated with Ni + Column purification: Use buffer A (100 mM potassium phosphate, 500 mM sodium chloride, 30 mM imidazole, pH 8.0) for balance, then filter the crude enzyme solution collected in 3.1 and add Ni + In the column, buffer B (100 mM potassium phosphate, 500 mM sodium chloride, 250 mM imidazole, pH 8.0) was used to elute and collect the target protein, which was then concentrated by ultrafiltration and desalted to obtain the purified protein, which was then refrigerated at 4°C.

[0071] The concentration of the purified target protein was determined by Bradford method, and the specific enzyme activity was determined using pure enzyme. The target protein was analyzed by SDS-PAGE gel electrophoresis.

[0072] 3.3 Mutant A602M catalyzes CPC to produce 7-ACA

[0073] The CPC conversion product was analyzed by HPLC to obtain its pure enzyme activity. The reaction was 1mL system, 0.1mg / mL pure enzyme, adding substrate CPC, 25℃, 1000rpm, and reacting for 5min. Take 100uL sample, dilute with methanol and measure by HPLC.

[0074] HPLC detection conditions: The selected chromatographic column was Agilent ZORBAX SB-C18 (4.6*150mm 4um), the mobile phase was 0.1% formic acid aqueous solution and methanol, the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, the detection time was 15 min, and the column oven was kept constant at 40°C.

[0075] 3.4 Mutant A602M catalyzes DAOC to produce 7-ADCA

[0076] Deacetoxycephalosporin C (DAOC) is an analog of CPC, which can be generated by a two-step enzymatic method. The structural difference between CPC and DAOC lies in the side chain substituents of the parent six-membered ring, where DAOC is hydrogen (-H) and CPC is acetoxy (-OCOCH3).

[0077]

[0078] The DAOC conversion product was analyzed by HPLC to obtain its pure enzyme activity. The reaction was a 1mL system, 3.2 pure enzyme (0.1mg / ml) was taken, substrate DAOC was added, 25℃, 1000rpm, and the reaction was carried out for 5min. 100uL of sample was taken, diluted with methanol and then measured by HPLC.

[0079] Liquid phase detection conditions: chromatographic column Agilent ZORBAX SB-C18 (4.6*150mm 4um), mobile phase using 0.1% trifluoroacetic acid solution and methanol, flow rate 1.0ml / min, detection wavelength 254nm, detection time 17min, column oven constant at 40°C.

[0080] 3.5 Mutant A602M catalyzes DAC to produce HACA

[0081] Deacetylcephalosporin C (DAC) is a CPC analog. DAC generates CPC under the action of acetyltransferase. The only difference between it and DAC is the side chain substituent of the six-membered ring of the parent nucleus, where DAC is hydroxyl (-OH) and CPC is acetoxy (-OCOCH3).

[0082]

[0083] The DAC conversion product was analyzed by HPLC to obtain its pure enzyme activity. The reaction was a 1mL system, 3.2 pure enzyme (0.1mg / ml) was taken, substrate DAC was added, 25°C, 1000rpm, and the reaction was carried out for 5min. 100uL of sample was taken, diluted with methanol and then measured by HPLC.

[0084] Liquid phase detection conditions: chromatographic column Agilent ZORBAX SB-C18 (4.6*150mm 4um), mobile phase using 0.1% trifluoroacetic acid solution and methanol, flow rate 1.0ml / min, detection wavelength 254nm, detection time 8min, column oven constant at 40°C.

[0085] Table 3 Relative activities of pure enzyme of mutant A602M on different substrates

[0086]

[0087] The mutants obtained by mutating the starting enzyme of SEQ ID NO: 2 have different degrees of improvement or reduction in catalytic activity for different substrates, among which A602M has improved catalytic activity for CPC and reduced conversion rate for deacetyloxycephalosporin C (DAOC) and deacetylcephalosporin C (DAC).

Claims

1. A cephalosporin C acylase mutant, characterized in that: The amino acid sequence is a mutant obtained by substituting amino acid positions of the amino acid sequence described in SEQ ID NO. 2 in the sequence list, including R158, M163, R261, and A602.

2. The cephalosporin C acylase mutant according to claim 1, characterized in that: Compared with the original cephalosporin C acylase, the R158, M163, R261 and A602 positions of its amino acid sequence are replaced by cysteine ​​and methionine; Specifically, R158, R261, and A602 are substituted by cysteine ​​and methionine; and M163 is substituted by cysteine.

3. The biological material related to the cephalosporin C acylase mutant according to any one of claims 1 to 3, which is any one of the following (1) to (4): (1) Nucleic acid molecule encoding the cephalosporin mutant according to claim 1 or 2 (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1), or a vector containing the expression cassette described in (2); (4) A recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3).

4. A method for preparing compound II, characterized in that: The method comprises taking compound I as a substrate and the cephalosporin C acylase mutant according to any one of claims 1 to 2 as a catalyst to carry out a catalytic reaction to obtain compound II: Wherein R is acetoxy, hydroxyl or hydrogen.

5. The method according to claim 4, characterized in that: The recombinant cell is achieved by introducing a recombinant vector capable of expressing the cephalosporin C acylase mutant according to any one of claims 1 to 2 into a biological cell.

6. The method according to claim 5, characterized in that: The cephalosporin C acylase mutant according to any one of claims 1 to 2 is obtained by expression in a recombinant cell, specifically by introducing a recombinant vector capable of expressing the cephalosporin C acylase mutant according to any one of claims 1 to 2 into a biological cell.

7. The method according to claim 5, characterized in that: The cephalosporin C acylase mutant according to any one of claims 1 to 2 catalyzes the reaction in the form of whole cells, crude enzyme solution, crude enzyme powder, immobilized enzyme or pure enzyme.

8. The method according to claim 7, characterized in that: The whole cell is a recombinant microorganism containing a nucleic acid molecule encoding the cephalosporin C acylase mutant according to any one of claims 1 to 2, preferably yeast, bacteria, algae or fungi, such as Escherichia coli; More preferably, the Escherichia coli is BL21 (DE3).

9. The method according to claim 4, characterized in that: The reaction of using the acylase as a biological enzyme to catalyze the substrate to generate the corresponding product is carried out in a phosphate buffer solution with a concentration of 40mmol / L-60mmol / L and a pH of 6.5-9.

5.

10. The method according to claim 9, characterized in that: The temperature of the catalytic reaction may be 20-40° C.; the time of the catalytic reaction may be 0.5-28 h.

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