Cephalosporin C acylase mutants and their application in cephalosporin nucleus synthesis
By mutating and immobilizing the amino acid sequence of the cephalosporin C acylase from Bosea sp. OK403, the problem of low catalytic efficiency in existing enzymatic methods has been solved, achieving efficient and environmentally friendly preparation of cephalosporin nuclei, which has good prospects for industrial application.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
- Filing Date
- 2022-06-10
- Publication Date
- 2026-08-04
AI Technical Summary
Existing cephalosporin C acylases have low hydrolytic activity, making it difficult to efficiently catalyze the preparation of cephalosporin nuclei. Furthermore, traditional chemical methods suffer from high energy consumption and environmental pollution.
A highly efficient mutant enzyme was constructed by mutating the amino acid sequence of cephalosporin C acylase derived from Bosea sp. OK403. The enzyme was then immobilized using a carrier such as epoxy resin or amino resin to optimize the reaction conditions.
It significantly improves the catalytic efficiency of cephalosporin nuclei, reduces energy consumption and environmental pollution, and provides a biocatalytic method with greater commercial application potential.
Smart Images
Figure QLYQS_1 
Figure QLYQS_2 
Figure BDA0003687462410000031
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biocatalysis technology and relates to a method for synthesizing cephalosporin nuclei using cephalosporin C acylase catalysis. Background Technology
[0002] Cephalosporin antibiotics are among the most widely used β-lactam antibiotics for many years. Their key parent nucleus is 7-aminocephalosporanic acid (7-ACA) and its related derivatives. These antibiotics account for approximately 40% of the global antibiotic market and have significant medical value.
[0003] Early industrial preparation of 7-ACA involved chemical cleavage of cephalosporin C (CPC), followed by removal of the molecular side chains to obtain 7-ACA. This process was demanding, requiring ultra-low temperatures. Chemical methods involved large amounts of toxic and harmful organic solvents, were complex, energy-intensive, and caused environmental pollution. Therefore, in recent years, enzymatic catalysis has gradually become a new direction for industrial production due to its low energy consumption and environmental friendliness. Early industrial production employed a two-step enzymatic method, primarily utilizing D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase. First, CPC was converted to GL-7-ACA via DAAO; then, GL-7-ACA was converted to 7-ACA via GL-7-ACA acylase. However, in the two-step enzymatic catalysis, the first step product hydrogen peroxide reacts with the DAO substrate or product to generate a large number of byproducts. Moreover, the process is long, difficult to control, and costly. Therefore, the one-step enzymatic catalysis of CPC cleavage reaction is currently more commonly used.
[0004] A one-step enzymatic method utilizes CPC acylase to catalyze the hydrolysis of CPC amide bonds. Since the 1980s, strains that secrete CPC acylases have been discovered in nature, such as the acyII gene from Pseudomonas SE83; the CPC acylase gene from Pseudomonas N176; the CPC acylase gene from Pseudomonas V22; and the CPC acylase gene from Pseudomonas P130. However, the hydrolytic activity of cephalosporin C acylase genes urgently needs improvement; compared to GL-7-ACA, its hydrolytic activity is only 2-4%. Furthermore, new cephalosporin C acylases are still to be discovered to develop acylases with greater commercial potential and higher reusability after immobilization. Summary of the Invention
[0005] The technical problem to be solved by the present invention is how to efficiently prepare cephalosporin nucleus 7-ACA using a biocatalytic method.
[0006] To address the above technical problems, this invention first provides an 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. The cephalosporin C acylase mutant is a protein obtained by mutating the wild-type sequence at positions L159, G160, L161, L162, I166, W167, H249, P259, V262, H294, F295, V305, F309, E308, A419, N479, H496, P603, and Y704.
[0007] The protein obtained by mutating the mutant cephalosporin C acylase using any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or all of the following mutations:
[0008] (1) L159 is alanine, replaced by lysine, serine, cysteine, glutamine, and threonine;
[0009] (2) G160 is replaced by phenylalanine, asparagine, and serine;
[0010] (3) L161 is replaced by phenylalanine, methionine, alanine, lysine, serine, and cysteine;
[0011] (4) L162 is replaced by alanine, methionine, and histidine;
[0012] (5) I166 is replaced by tryptophan;
[0013] (6) W167 is replaced by alanine, serine, and asparagine;
[0014] (7) H249 is replaced by alanine and serine;
[0015] (8) P259 is replaced by valine, alanine, lysine, and aspartic acid;
[0016] (9) V262 is replaced by leucine, isoleucine, and serine;
[0017] (10) H294 is replaced by alanine and serine;
[0018] (11) F295 is replaced by valine, asparagine, and leucine;
[0019] (12) V305 is replaced by arginine, lysine, and serine;
[0020] (13) F309 is replaced by aspartic acid and glutamine;
[0021] (14) E318 is replaced by threonine;
[0022] (15) A419 is replaced by valine;
[0023] (16) N479 is replaced by leucine;
[0024] (17) H496 is replaced by phenylalanine;
[0025] (18) P603 is replaced by glutamine or asparagine; or
[0026] (19) Y704 is replaced by serine and cysteine.
[0027] Preferably, the mutant contains the following combination of amino acid substitutions:
[0028] L159A / E318T, L159Q / A419V, L159S / A419V, L159S / G160S, L159Q / G160S, L159S / P60 3Q, L159Q / P603Q, L159S / G160S / P603Q, L159Q / G160S / P603Q, L159S / A419V / G160S, L159S / A419V / P603Q, L159S / A419V / G160S / P603Q, G160S / P603Q, L161K / H249A, L161K / H249S, L161K / V305K, V262S / V305S, L159T / V262S, H294S / V305K or H294S / E318T.
[0029] The present invention correspondingly provides a nucleic acid molecule encoding the above-mentioned mutant cephalosporin C acylase, 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.
[0030] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
[0031] The aforementioned nucleic acid expression cassette refers to DNA capable of expressing the mutant cephalosporin C acylase in host cells. This DNA may include not only a promoter to initiate transcription of the gene encoding the mutant cephalosporin C acylase, but also a terminator to terminate transcription of the mutant cephalosporin C acylase. Furthermore, the expression cassette may also include an enhancer sequence.
[0032] The recombinant vector for the nucleic acid molecule may be a bacterial plasmid (such as an expression vector based on the T7 promoter expressed in bacteria, specifically pET-28a), bacteriophage, yeast plasmid (such as YEp series vectors), or retroviral packaging plasmid carrying the gene encoding the mutant cephalosporin C acylase.
[0033] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.
[0034] The recombinant microorganism containing the nucleic acid molecule encoding the mutant cephalosporin C acylase can be a yeast, bacteria, algae, or fungus carrying a gene encoding cephalosporin or the mutant cephalosporin protein, such as Escherichia coli.
[0035] This invention also provides a method for preparing cephalosporin nuclei, specifically comprising using compound I as a substrate, and conducting a catalytic reaction in contact with a catalyst of cephalosporin C acylase as shown in SEQ ID NO: 1 or the cephalosporin mutant thereon to obtain compound II:
[0036]
[0037] Where R represents acetoxy, hydroxyl, or hydrogen.
[0038] Preferably, the acylase of the present invention is an immobilized enzyme, for example, using epoxy resin, amino resin, or macroporous adsorption resin as a carrier to immobilize the acylase, thereby obtaining an immobilized enzyme with high conversion activity.
[0039] In the above method, the recombinant cells can be obtained by introducing a recombinant vector that can express the mutant cephalosporin C acylase into biological cells.
[0040] The biological cells may be microorganisms. The microorganisms may be *Escherichia coli* or other bacteria. In one embodiment of the present invention, the microorganism is *Escherichia coli* BL21(DE3).
[0041] The reaction temperature can be 10-40℃, specifically 20℃; the reaction is carried out in a phosphate buffer solution with a pH of 6.5-9.5, specifically 8.0. The reaction time is generally 0.5-24 hours, specifically 1 hour. The lysis products of the recombinant cells can be obtained by lysing the recombinant cells.
[0042] Meanwhile, the present invention also provides the application of a cephalosporin C acylase derived from Bosea sp. in catalyzing the hydrolysis or acylation of cephalosporin C or deacetoxycephalosporin C, wherein the amino acid sequence of the cephalosporin C acylase is shown in SEQ ID NO: 1.
[0043] This invention first identifies a cephalosporin C acylase derived from Bosea sp. OK403 as a potential catalyst for the synthesis of cephalosporin nuclei. Furthermore, through a semi-rational design, a mutant library was constructed, resulting in mutants with significantly improved performance. These mutants can catalyze the production of cephalosporin nuclei from cephalosporin derivatives, demonstrating promising industrial applications in the biocatalytic preparation of cephalosporin nuclei. Attached Figure Description
[0044] Figure 1 This is a flowchart of the acylase-catalyzed hydrolysis reaction of cephalosporin C in this invention.
[0045] Figure 2 This is a standard curve of 7-ACA and CPC content in this invention.
[0046] Figure 3 These are the HPLC detection results of 7-ACA and CPC in this invention.
[0047] Figure 4 The CPC acylase E744 in this invention is used for the construction of substrate saturated mutant libraries and related residue sites. Detailed Implementation
[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0050] Example 1: Acylase Sequence Mining and Enzyme Activity Characterization
[0051] 1.1 Acylase Sequence Mining
[0052] The acylase sequence SEQ ID NO.1 (named E744) from Bosea sp. OK403 was obtained through NCBI search.
[0053] 1.2. Induction, expression, and purification of cephalosporin C acylase
[0054] The nucleic acid sequence containing the amino acid sequence of SEQ ID NO.1 was transformed into Escherichia coli BL21(DE3) bacterial culture and evenly spread on kanamycin-resistant (concentration of 50 μg / mL) LB plates. After incubation at 37℃ for 14 h, single colonies grew, which were the transformants containing the cephalosporin C acylase nucleic acid sequence.
[0055] The transformants were transferred to test tubes containing 5 mL of LB medium (kanamycin concentration 50 μg / mL) and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. Then, 1% of the seed culture was inoculated into 100 mL of TB medium (kanamycin concentration 50 μg / mL) and cultured at 37°C with shaking at 220 rpm for 3 hours. The culture was then incubated until the OD500 of the transformed culture was reached. 600 When the pH reaches 0.6-0.8, add IPTG to a final concentration of 0.1 mM and continue incubation at 25°C and 220 rpm with shaking for 16 h; collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 10 min. Wash the cells with phosphate buffer (50 mM, pH 8.0), centrifuge, and then resuspend in phosphate buffer (10 mL / g of bacteria) to obtain a bacterial suspension. After sonication for 15 min, centrifuge for 60 min (4°C, 12000 rpm) and collect the supernatant to obtain the crude enzyme solution.
[0056] 1.3 Purification of Acylase Protein
[0057] Using Ni + Column purification: After pre-equilibration with buffer A (100mM potassium phosphate, 500mM NaCl, 30mM imidazole, pH 8.0), the wild-type crude enzyme solution collected in step 1.2 was filtered and added to Ni. + The column was eluted with buffer B (100mM potassium phosphate, 500mM sodium chloride, 250mM imidazole, pH 8.0) and the target protein was collected. The protein was then concentrated by ultrafiltration, desalted and purified, and stored at 4°C.
[0058] The concentration of the purified target protein was determined using the Bradford method. The target protein was analyzed using SDS-PAGE gel electrophoresis.
[0059] 1.4. Cephalosporin C acylase catalyzes the formation of 7-ACA from CPC.
[0060] Reaction system (1 mL): 40 mM CPC, 0.1 mg acylase, phosphate buffer added to 1 mL. Reaction conditions: 37℃, 1000 rpm for 30 min. Take 100 μL of sample, add 9 times the volume of methanol to terminate the reaction and dilute the system. Detect enzyme activity by HPLC. Analyze the CPC-converted 7-ACA product by HPLC.
[0061] Detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 96% phosphate buffer (pH 7.0, 50mM) with 4% methanol (v / v); flow rate: 0.8 mL / min; detection wavelength: 254 nm; column temperature: constant at 25℃.
[0062] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 micromolar (μmol) 7-ACA per minute from CPC, which is defined as 1 U.
[0063] Table 1 Acylase Activity Test
[0064]
[0065] Example 2: Construction of acylase mutants
[0066] 2.1 Single-point saturation mutation
[0067] Using amino acid E744 of the acyltransferase as a template, SWISS-MODEL was used for modeling. After modeling, an insertion model was established between the protein and the substrate molecule CPC. Docking analysis was performed to determine the binding sites. The results showed that within the 6A region of the substrate molecule, sites L159, L161, L162, I166, W167, P259, V262, H294, F295, V305, F309, I413, and P603 in the acyltransferase significantly affected the binding of the CPC side chain, thus impacting the substrate's activity space within the pocket. Within the 6A region of the substrate molecule, sites G160, M163, G164, H260, R261, Y269, T306, H307, H313, H415, N479, H496, and Y704 played a crucial role in the formation of hydrogen bonds in the structural region, stabilizing the substrate binding pocket. Therefore, these amino acids were selected for single-point saturation mutagenesis.
[0068] Single-point saturation mutagenesis was performed using the selected primers, and the corresponding primers are shown in Table 2.
[0069] Table 2 Primer sequence list
[0070]
[0071]
[0072]
[0073] The specific construction method is as follows:
[0074] 1. First round of PCR
[0075] Using plasmid SEQ ID NO.1 as a template, primers L159-F1, L159-F2, L159-F3, and L159-F4 from Table 2 were mixed in a ratio of 12:6:1:1. 2 μL of this mixture was used as the upstream primer, and KU-R1 from Table 4 was used as the downstream primer to amplify gene fragment L1. The amplification methods for sites G160, L161, L162, M163, G164, I166, and W167 were the same as for gene fragment L1. Gene fragment amplification was performed using KU-R2 as the downstream primer for sites P259, H260, R261, V262, and Y269; KU-R3 as the downstream primer for sites H294, F295, V305, T306, H307, F309, and H313; KU-R4 as the downstream primer for sites I413 and H415; KU-R5 as the downstream primer for sites N479 and H496; KU-R6 as the downstream primer for site P603; and KU-R7 as the downstream primer for site Y704.
[0076] The first round of PCR program was as follows: 28 cycles of incubation at 94℃ for 2 min, followed by incubation at 98℃ for 15 s, 55℃ for 30 s, and 72℃ for 30 s, and then incubation at 72℃ for 5 min.
[0077] 2. Second round of PCR
[0078] Using 2 μL of the first-round PCR products L1, L2, L3, and L4 as primers, and plasmid SEQ ID NO.1 as a template, a second-round PCR was performed to construct a single-site saturated mutant library containing L159, G160, L161, L162, M163, G164, I166, W167, P259, H260, R261, V262, Y269, H294, F295, V305, T306, H307, F309, H313, I413, H415, N479, H496, P603, and Y704. The second-round PCR program was: 28 cycles of 94℃ for 2 min, (98℃ for 15 s, 60℃ for 30 s, and 72℃ for 5 min), followed by a final incubation at 72℃ for 10 min.
[0079] 3. Construction of engineered mutant strains
[0080] The obtained second-round PCR product was subjected to the following operations: 1 μL of Dpn I was added to the second-round PCR product to digest the plasmid template, and the mixture was treated at 37°C for 3 h. 2 μL of the enzyme-digested second-round PCR product was taken and electroporated into *E. coli* BL21(DE3). The electroporated *E. coli* BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. After the growth of a single colony, this was the engineered mutant strain of the acyltransferase SEQ ID NO. 1.
[0081] 2.2 High-throughput screening and activity assay of acylase mutant libraries
[0082] Single colonies from the mutant library were inoculated into 96-well plates and cultured in 300 μL of LB medium (containing 50 μg / mL kanamycin) at 37°C for 8–12 h. A negative control group (empty vector of pET-28a) was also included. Then, 200 μL of seed culture was inoculated into 800 μL of TB medium (containing 0.1 mM IPTG and 50 μg / mL kanamycin). Expression was performed at 25°C for 16 h. Cells were collected by centrifugation, frozen at -80°C for 1 h, and thawed at room temperature for 30 min. 200 μL of potassium phosphate buffer (0.1 M pH 8.0, containing 1 mg / mL lysozyme) was added to resuspend the cells, and incubated at 37°C for 1 h. 200 μL of substrate CPC (20 mM) was added to the cell lysis buffer, and the reaction was carried out at 37°C for 4–6 h. Transfer 40 μL of the reaction solution to a new 96-well plate, add 200 μL of stop reaction solution (20% glacial acetic acid: 0.05M NaOH = 2:1), and centrifuge for 10 min (4℃, 5000 rpm). Take 200 μL of the supernatant, add 40 μL of colorimetric reagent (0.5% p-DAB dissolved in methanol), react at room temperature for 10 min, and then measure the absorbance at 415 nm.
[0083] Table 3. Relative viability test of random mutant strains
[0084]
[0085]
[0086] Example 3: Construction of acylase combinatorial mutants
[0087] Based on the high-activity mutants screened in Example 2, the following mutants were constructed: L159A / E318T, L159Q / A419V, L159S / A419V, L161K / H294A, L161K / H294S, L161K / V305S, V262L / V305S, L159T / V262L, H294S / V305S, H294S / E318T, and L159Q / H294S / A419V. Primers were designed for constructing the mutants, as shown in Table 4.
[0088] Table 4. Primers for site-directed mutagenesis
[0089]
[0090]
[0091] The specific construction method is as follows:
[0092] 1) PCR
[0093] Using plasmid L159A as a template, and taking E318T-F and E318T-R from Table 4 as upstream and downstream primers, the mutant L159A / E318T was amplified; using plasmid L159T as a template, and taking V262L-F and V262L-R from Table 4 as upstream and downstream primers, the mutant L159T / V262L was amplified; using plasmids L159Q and L159S as templates, and taking A419V-F / A419V-R, G160S-F / G160S-R, and P603Q-F / P603Q-R from Table 4 as downstream primers, respectively, the mutants were amplified. Upstream and downstream primers were used to amplify mutants L159Q / A419V, L159S / A419V, L159Q / P603Q, L159S / P603Q, L159Q / G160S, and L159S / G160S. Using plasmid G160S as a template, P603Q-F / P603Q-R from Table 4 were used as upstream and downstream primers to amplify mutant G160S / P603Q. Using plasmid L161K as a template, H294A-F and H294A-R, H294S-F and H294S-R from Table 4 were used respectively. Using plasmid V262L as the upstream and downstream primers, the mutants L161K / H294A, L161K / H294S, and L161K / V305S were amplified. Using plasmid H294S as the template, V305S-F and V305S-R from Table 4 were used as upstream and downstream primers to amplify the mutant V262L / V305S. Using plasmid H294S as the template, V305S-F and V305S-R, and E318T-F and E318T-R from Table 4 were used as upstream and downstream primers to amplify the mutants H294S / V305S and H294S / E. Using plasmid L159S / A419V as a template, and taking G160S-F / G160S-R and P603Q-F / P603Q-R from Table 4 as upstream and downstream primers, respectively, the mutants L159S / A419V / G160S and L159S / A419V / P603Q were amplified; using plasmid L159S / A419V / G160S as a template, and taking P603Q-F / P603Q-R from Table 4 as upstream and downstream primers, the mutants L159S / A419V / G160S / P603Q were amplified.
[0094] PCR program: 28 cycles of incubation at 94℃ for 2 min, (98℃ for 15 s, 55℃ for 30 s, 72℃ for 5 min), followed by incubation at 72℃ for 10 min.
[0095] 2) Construction of engineered mutant strains
[0096] The obtained PCR product was subjected to the following operations: 1 μL of Dpn I was added to the PCR product to digest the plasmid template, and the mixture was treated at 37°C for 3 h. 2 μL of the enzyme-digested PCR product was then electroporated into *E. coli* BL21(DE3). The electroporated *E. coli* BL21(DE3) bacterial culture was evenly spread on kanamycin-resistant (50 μg / mL) LB agar plates and cultured at 37°C for 14 h. After the growth of a single colony, this was identified as the mutant strain of the acylase SEQ ID NO.1.
[0097] Example 4: Whole-cell catalytic synthesis of 7-ACA using cephalosporin C acylase mutant
[0098] 4.1 Cell Culture
[0099] Single clones of the mutants constructed in Example 3 were transferred to test tubes containing 5 mL of LB medium (kanamycin concentration of 50 μg / mL) and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. 1% of this seed culture was then inoculated into 100 mL of TB medium (kanamycin concentration of 50 μg / mL) and cultured at 37°C with shaking at 220 rpm for 3 hours. The culture was then incubated until the OD500 of the bacterial culture was... 600 When the pH reaches 0.6-0.8, add IPTG (0.1 mM) and continue incubation at 25°C and 220 rpm with shaking for 16 h; collect the bacterial cells by centrifugation at 4°C and 4000 rpm for 10 min. Wash the bacterial cells with phosphate buffer (50 mM pH 8.0), centrifuge, weigh the wet bacterial cells, and then resuspend them in phosphate buffer (10 mL / g bacteria) to obtain a bacterial suspension.
[0100] 4.2 Whole-cell response
[0101] The conversion rate of the whole cells obtained in step 4.1 was determined by analyzing the CPC conversion product using HPLC. The reaction was carried out in a 1 mL system. 800 μL of the whole cells obtained in step 4.1 was used for the reaction, and the substrate CPC (40 mM) was added. The reaction was carried out at 37 °C and 1000 rpm for 30 min. 100 μL of the sample was then diluted tenfold with methanol, and the conversion rate was determined by HPLC.
[0102] HPLC detection conditions: The selected column was an Agilent ZORBAX SB-C18 (4.6*150mm 4um), the mobile phase ratio was 96% phosphate buffer (pH 7.0, 50mM), 4% methanol (v / v), the flow rate was 0.8mL / min, the detection wavelength was 254nm, and the column temperature was kept constant at 25℃.
[0103] Table 5. Whole-cell transformation efficiency of the combined mutants to CPC
[0104]
[0105]
[0106] 4.3 Crude enzyme solution catalyzes the synthesis of 7-ACA from CPC
[0107] The crude enzyme solution was prepared according to the method in Example 1, and the conversion rate of the crude enzyme solution from which the mutant was obtained was verified.
[0108] Reaction system (1 mL): substrate CPC (40 mM), 0.1 mL crude acylase solution, and phosphate buffer added to 1 mL. Reaction conditions: 37 °C, 1000 rpm, 30 min. Take 100 μL of sample, dilute it tenfold with methanol, and determine the conversion rate by HPLC.
[0109] Detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 96% phosphate buffer (pH 7.0, 50mM) with 4% methanol (v / v); flow rate: 0.8 mL / min; detection wavelength: 254 nm; column temperature: constant at 25℃.
[0110] Table 6. Conversion rate of crude enzyme to CPC by combined mutants
[0111]
[0112] Example 5: Whole-cell catalytic synthesis of 7-ADCA from cephalosporin C acylase mutant.
[0113] Deacetoxycephalosporin C (DAOC) is an analogue of CPC, the difference in structure being the substituents in the six-membered ring side chain of the parent nucleus. In DAOC, the substituent is hydrogen (-H), while in CPC, it is acetoxy (-OCOCH3).
[0114]
[0115] The conversion efficiency of DAOC was obtained by analyzing the DAOC conversion product using HPLC. The reaction was performed in a 1 mL system. 800 μL of the whole cells obtained in step 3.1 were used for the reaction, and DAOC (1 mg / mL) was added. The reaction was carried out at 37°C and 1000 rpm for 30 min. 100 μL of the sample was then diluted tenfold with acetonitrile before the conversion efficiency was determined by HPLC.
[0116] Liquid chromatography detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 95% ammonium formate, 5% acetonitrile; flow rate: 1 ml / min; detection wavelength: 254 nm; column temperature oven: constant at 25℃.
[0117] Table 7. Conversion rate of DAOC by cephalosporin C acylase mutant.
[0118]
[0119] Example 6: Cephalosporin C acylase mutant catalyzes whole-cell synthesis of deacetylated 7-ACA from DAC.
[0120] Deacetylcephalosporin C (DAC) is an analogue of CPC. The only difference between CPC and DAC and DAOC is the substituent in the side chain of the six-membered ring of the parent nucleus. In DAC, the substituent is hydroxyl (-OH), while in CPC, it is acetoxy (-OCOCH3).
[0121]
[0122] The conversion rate of the whole cells obtained in step 4.1 was determined by analyzing the DAC conversion product using HPLC. A Hypersil BDS C18 column (4.6*200mm, 5µm) was used. The reaction was carried out in 1mL systems. 800µL of the whole cells obtained in step 4.1 was used for the reaction, and 200µL of 40mM DAC substrate was added. The reaction was carried out at 37℃ and 1000rpm for 30min. 100µL of the sample was then diluted tenfold with acetonitrile before the conversion rate was determined by HPLC.
[0123] The HPLC mobile phase was prepared as follows: 96% ammonium acetate buffer (pH 6.2, 50 mM), 4% acetonitrile (v / v), with a flow rate of 1 mL / min, a detection wavelength of 254 nm, and a constant column temperature of 25 °C.
[0124] Table 8. Conversion rate of cephalosporin mutants to GL-7-D-ACA
[0125]
[0126] Example 7: Immobilized cephalosporin C acylase mutant
[0127] (1) Acylase mutants (L159S / A419V / G160S) were immobilized on epoxy resin and macroporous adsorption resin.
[0128] Carrier equilibration: Wash three times repeatedly with immobilization buffer at a carrier / buffer ratio of 1:5 (mass / volume ratio), and then dry after washing.
[0129] Adjustment of the ratio of carrier to acylase: Dissolve the enzyme in immobilization buffer with a ratio of 1:4 (mass / volume ratio). This ratio can be further optimized between 1:1 and 1:4.
[0130] The enzyme-containing buffer and carrier were added to the reactor and reacted at 25°C and 800 rpm for 3 hours, after which stirring was stopped and the mixture was allowed to stand for 2 hours.
[0131] Immobilized enzymes were collected by filtration after immobilization, and the protein content in the supernatant was determined using a 280 nm absorption wavelength. The immobilization rate and yield were calculated. The enzymes were washed with distilled water, and the process was repeated three times. Subsequently, the reactivity of the epoxy resin-immobilized enzymes was detected using HPLC.
[0132] (2) Amino resin immobilized enzyme treatment
[0133] Carrier equilibration: Immobilization buffer, wash repeatedly 3 times with a carrier / phosphate buffer solution (50mM, pH8.0) ratio of 1:5 (mass / volume ratio), filter and dry after washing.
[0134] Prepare a 2% glutaraldehyde solution using immobilization buffer. Add the carrier and glutaraldehyde buffer at a ratio of 1:4 (mass / volume ratio), and stir at 22°C and 180 rpm for 1 hour. Then wash the carrier three times with buffer, filter, and store at 4°C.
[0135] (3) Immobilized enzyme activity test
[0136] Reaction conditions (total volume 4 ml): Immobilized acylase (400 mg) was added to the reaction flask, followed by substrate CPC (40 mM), and buffer was added to a final volume of 4 ml. The reaction was carried out at 37°C and 1000 rpm for 30 min. A 100 μL sample was taken, diluted tenfold with methanol, and the conversion rate was determined by HPLC.
[0137] Detection conditions: Agilent ZORBAX SB-C18 column (4.6*150mm 4um); mobile phase: 96% phosphate buffer (pH 7.0, 50mM) with 4% methanol (v / v); flow rate: 0.8 mL / min; detection wavelength: 254 nm; column temperature: constant at 25℃. The conversion rate reached 67%. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Cephalosporin C acylase mutants and their application in cephalosporin nucleus synthesis <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 774 <212> PRT <213> Arsenic-antimony oxidizing strain (Bosea sp.) <400> 1 MTTDADRDALEAALPPLSGSLSLSGLEAPVSVRRDAWGIPHIKAVGEADAYRALGFVHAQDRLFQMELTRRKALGRAAEWLGAEAAEADILVRRLGMEQACRRDYDALADAAKAMLQAYAAGVNAFLESGAPVPCEYTLLGATPETWEPWHSIAVMRRLGLLMGSIWFKLWRMLALPIVGAENALKLRYDDGGRDLLCIPPGAQADRFEADLAALAPTVDHLLKAMGGDASDAAGGGSNNWAVGPDRTATGRPILAGDPHRVFEIPGMYAQHHLACDRFDMIGLTVPGVPGFPHFAHNGSVAYCVTHAFMDIHDLYLEQFTDEGRAVRFGEGFEPVIHRRDRIAVRGGADREFEIFETRHGPVIAGDPREGVGLSLRSVQFAETDLSFDCLTRMPAASTVSELYEATRGWGLIDHNLVAGDTAGAIGHLVRARVPVRSRDNGWLPVPGWTAEHEWQGWIAHEAMPCVIDPPGGLIVTANNRVVADDHPDYLCTDCHPPYRAERILQRLDSGAPFAVADAAAIHADTQSPHLDLFRTRLAVLGLREQADAERLRQDLLAWDGRMDADSTSAAAYNAFRRALTRIVTVRSRLEQATAHRFAAVAPGVSPQGQVWWAVPTLLRDDDAGMLNGLTWDDVLSEALSEAAATLTGKTWGEEHQPRFAHPLSAQFPDWAQRLDPPSLPVGGDGDTVLAIGIVPSAGPTATYGALARYVFDVGNWDNSRWAVFHGTSGHPASPHYADQNTPWSACEMVPMLYDWERIAAEAKTVQELVPPRA 774
Claims
1. A cephalosporin C acylase mutant, characterized in that: The amino acids were obtained by substituting the following amino acids into the amino acid sequence shown in SEQ ID NO:1: L159A, L159K, L159S, L159C, L159Q, L159T, L159A / E318T, L159Q / A419V, L159S / A419V, L159S / G160S, L159Q / G160S, L159S / P603Q, L159Q / P603Q, L159S / G160S / P603Q. L159Q / G160S / P603Q, L159S / A419V / G160S, L159S / A419V / P603Q, L159S / A419V / G160S / P603Q, or L159T / V262S.
2. A nucleic acid molecule encoding the cephalosporin C acylase mutant according to claim 1.
3. An expression cassette containing the nucleic acid molecule according to claim 2.
4. A recombinant vector containing the nucleic acid molecule according to claim 2.
5. A recombinant microorganism containing the nucleic acid molecule according to claim 2, the expression cassette according to claim 3, and the recombinant vector according to claim 4.
6. A method for preparing compound II, characterized in that, The reaction involves using compound I as a substrate and contacting it with the cephalosporin C acylase mutant as described in claim 1 as a catalyst to catalyze the reaction, yielding compound II: I II Where R represents acetoxy, hydroxyl, or hydrogen.
7. The method according to claim 6, characterized in that: The cephalosporin C acylase mutant as described in claim 1 was obtained through recombinant cell expression.
8. The method as described in claim 7, characterized in that, This is achieved by introducing a recombinant vector into biological cells that can express the cephalosporin C acylase mutant as described in claim 1.
9. The method according to claim 6, characterized in that: The cephalosporin C acylase mutant as described in claim 1 catalyzes enzymes in the form of whole cells, crude enzyme solution, crude enzyme powder, immobilized enzyme, or pure enzyme.
10. The method according to claim 9, characterized in that: The whole cells are recombinant microorganisms containing nucleic acid molecules encoding the cephalosporin C acylase mutant as described in claim 1.
11. The method according to claim 10, characterized in that: The recombinant microorganism is a bacterium or a fungus.
12. The method according to claim 11, characterized in that: The recombinant microorganism is yeast.
13. The method according to claim 11, characterized in that: The recombinant microorganism is Escherichia coli.
14. The method according to claim 13, characterized in that: The Escherichia coli mentioned is BL21(DE3).
15. The method according to claim 6, characterized in that: The catalytic reaction was carried out in phosphate buffer solutions with concentrations of 40 mmol / L to 60 mmol / L and pH values of 6.5 to 9.
5.
16. The method according to claim 15, characterized in that: The temperature of the catalytic reaction is 20–40°C; the time of the catalytic reaction is 0.5–28 h.