Cephalosporin C acylase mutant

Abstract

According to the invention, cephalosporin C acylase is modified by a point mutation method to obtain a mutant with relatively high activity for CPC sodium salt, and the mutant is obtained by substitution mutation at one or more sites of G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603 and L707 in an amino acid sequence as shown in SEQ ID NO: 1. The cephalosporin C acylase mutant disclosed by the invention has higher enzyme activity in production of 7-aminocephalosporanic acid by a one-step enzyme method, which is 2.4-3 times of that of starting cephalosporin C acylase, and has practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and relates to a cephalosporin C acylase mutant and an application thereof in producing 7-aminocephalosporanic acid by a one-step enzymatic method. Background Art

[0002] Cephalosporin antibiotics are widely used β-lactam antibiotics, accounting for about 40% of the global market. The preparation of its key intermediate 7-aminocephalosporanic acid (7-ACA) mainly relies on chemical synthesis and bioenzymatic methods. Early production mostly used chemical synthesis, but due to its complex process, high energy consumption and the generation of a large number of pollutants, it was gradually replaced by the more environmentally friendly bioenzymatic method. The bioenzymatic method is divided into a two-step enzymatic method and a one-step enzymatic method. The two-step enzymatic method first uses D-amino acid oxidase (DAAO) to convert cephalosporin C (CPC) into glutaryl-7-aminocephalosporanic acid (GL-7-ACA), and then converts it into 7-ACA through acylase. However, the process cost is high, and the byproduct hydrogen peroxide (H2O2) has a degradative effect on CPC, which increases the complexity of production.

[0003] In order to simplify the process and reduce costs, a one-step enzymatic method was developed to directly convert CPC into 7-ACA using CPC acylase. In recent years, some genetic engineering studies have attempted to improve the enzymatic activity of CPC acylase on CPC. By modifying CPC acylase from Pseudomonas sp. SE83, mutants with enzyme activity several dozen times higher than that of the wild-type enzyme were obtained, but such enzymes still have strong 7-ACA product inhibition and cannot meet the requirements of industrial production. CPC acylase mutant I215V / F228V / V240F / A306T / Y323T / F347L / V552L / R553L / H623T (CN105543201B) from Pseudomonas sp. GK16 has an activity of 150 times higher than that of the wild-type enzyme in hydrolyzing CPC to generate 7-ACA, while improving substrate specificity and reducing product inhibition. In addition, the CPC acylase mutant 130-ED2 from Pseudomonas sp. P130: G25D / I215V / F228V / V240F / Y323T / F347L / H623T (CN 109072215 B) was 54 times higher than the wild-type enzyme, and on this basis, the mutant 130-V34 was further developed, the enzyme activity was 2.9 times higher than that of 130-ED2, and the stability of the enzyme was improved (CN 111172142 B).

[0004] According to the description of CN 117247925 A, the inventors developed a series of mutants with higher enzyme activity by genetically engineering CPC acylase from Bosea sp. OK403, which significantly improved its catalytic efficiency for cephalosporin C sodium salt. However, in order to meet the high standards of industrial production, the activity of the enzyme still needs to be further improved. Therefore, the present invention is committed to developing a CPC acylase mutant with higher enzyme activity to show greater application potential in industrial production. Summary of the invention

[0005] In order to improve the enzymatic activity of CPC acylase, the present invention transforms and screens the existing CPC acylase through rational design and directed evolution and other technologies to construct CPC acylase mutants with high enzymatic activity.

[0006] The L159S / A419V mutant in CN 117247925 A (amino acid sequence as shown in SE Q ID NO: 1) was further modified by site-directed mutagenesis to obtain a CPC acylase mutant with high enzyme activity using CPC as a substrate. Specifically, the present invention includes the following technical solutions.

[0007] A cephalosporin C acylase (CPC acylase) mutant, which is a protein obtained by substitution mutation at positions G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603, and L707 of the amino acid sequence shown in SEQ ID NO: 1.

[0008] The mutant cephalosporin C acylase is selected from the protein obtained by mutating any one of the above 20 sites to cysteine ​​or methionine:

[0009] (1) G160 is replaced by cysteine ​​or methionine;

[0010] (2) L161 was replaced by cysteine ​​and methionine;

[0011] (3) L162 is substituted by cysteine ​​and methionine;

[0012] (4) G164 was replaced by cysteine ​​and methionine;

[0013] (5) W167 was substituted by cysteine ​​and methionine;

[0014] (6) W171 was substituted by cysteine ​​and methionine;

[0015] (7) L174 is substituted by cysteine ​​and methionine;

[0016] (8) S238 is substituted by cysteine ​​or methionine;

[0017] (9) D258 is substituted by cysteine ​​or methionine;

[0018] (10) P259 is substituted by cysteine ​​or methionine;

[0019] (11) H260 is replaced by cysteine ​​or methionine;

[0020] (12) V262 is substituted by cysteine ​​and methionine;

[0021] (13) T306 is substituted by cysteine ​​and methionine;

[0022] (14) H307 is replaced by cysteine ​​and methionine;

[0023] (15) F309 is substituted by cysteine ​​and methionine;

[0024] (16) I312 is substituted by cysteine ​​or methionine;

[0025] (17) L376 is substituted by cysteine ​​and methionine;

[0026] (18) H415 is substituted by cysteine ​​and methionine;

[0027] (19) P603 is substituted by cysteine ​​and methionine;

[0028] (20) L707 is substituted by cysteine ​​and methionine;

[0029] In one embodiment, the above changes refer to that the 171st W is replaced by M (ie, W171M), the 174th L is replaced by M (ie, L174M), the 312th I is replaced by C (ie, I312C), and the 707th L is replaced by C (ie, L707C).

[0030] The second aspect of the present invention is to provide a gene encoding the above CPC acylase mutant.

[0031] The third aspect of the present invention is to provide a plasmid containing the above gene. The plasmid is a PET series vector, such as pET-28a, but is not limited thereto.

[0032] The fourth aspect of the present invention is to provide a microorganism expressing the above encoding gene, which is transformed with the above encoding gene or plasmid.

[0033] Preferably, the microorganism is selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Cephalosporium acremonium, and Penicillium chrysogenum, preferably Escherichia coli and Cephalosporium acremonium, and more preferably Escherichia coli BL21 (DE3).

[0034] The fifth aspect of the present invention is to provide use of the CPC acylase mutant or microorganism in the preparation of 7-ACA.

[0035] The cephalosporin C acylase mutant or microorganism can be used to produce 7-ACA, especially a one-step enzymatic reaction of producing 7-ACA using cephalosporin C (ie CPC) as a substrate and the cephalosporin C acylase mutant or microorganism as a catalyst.

[0036] Conventional process conditions can be used to produce 7-ACA. For example, the reaction temperature can be selected from 20 to 40°C, preferably 25°C; the pH of the phosphate buffer can be selected from 6.5 to 9.5, preferably 8.0; and the concentration of cephalosporin C can be selected from 5 to 80 mM, preferably 20 mM.

[0037] Compared with the initial enzyme with the amino acid sequence shown in SEQ ID NO: 1, the enzymatic activity of the CPC acylase mutant of the present invention is significantly improved, with the enzyme activity being increased by up to 2.4-3 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a standard curve diagram of HPLC liquid chromatography detection of 7-ACA and CPC standards in the present invention.

[0039] Figure 2 The HPLC test results of 7-ACA and CPC standards in the present invention are shown in FIG.

[0040] Figure 3 It is the residue site interacting with CPC in CPC acylase SEQ ID NO: 1 used for site-directed mutagenesis in the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments. The following examples are only for illustrating the present invention, but are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0042] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0043] For the sake of brevity, the amino acid abbreviations herein may use either single letters or three letters, which are well known to those skilled in the art. These abbreviations are listed in the following table:

[0044] Table 1. Chinese and English correspondence and abbreviations of amino acids

[0045] Alanine Alanine A Ala Phenylalanine Phenylalanine F Phe Cysteine Cysteine C Cys Aspartic acid Aspartic acid / Aspartate D Asp Asparagine Asparagine N Asn Glutamate Glutamic acid / Glutamate E Glu Glutamine Glutamine Q Glu Glycine Glycine G Gly Histidine Histidine H His Leucine Leucine L Leu Isoleucine Isoleucine I Ile Lysine Lysine K Lys Methionine Methionine M Met Proline Proline P Pro Arginine Arginine R Arg Serine Serine S Ser Threonine Threonine T Thr Valine Valine V Val Tryptophan Tryptophan W Trp Tyrosine Tyrosine Y Tyr

[0046] The basic template used to construct the cephalosporin C acylase mutant is the CPC acylase L159S / A419V in CN 117247925 A, i.e., SEQ ID NO: 1:

[0047] MTTDADRDALEAALPPLSGSLSLSGLEAPVSVRRDAWGIPHIKAVGEADAYRALGFVHAQDRLFQMELTRRKALGRAAEWLGAEAAEADILVRRLGMEQACRRDYDALADAAKAMLQAYAAGVNAFLESGAPVPCEYTLLGATPETWEPWHSIAVMRRSGLLMGSIWFKLWRMLALPIVGAENALKLRYDDGGRDLLCIPPGAQADRFEADLAALAPTVDHLLKAMGGDASDAAGGGSNNWAVGPDRTATGRPILAGDPHRVFEIPGMYAQHHLACDRFDMIGLTVPGVPGFPHFAHNGSVAYCVTHAFMDIHDLYLEQFTDEGRAVRFGEGFEPVIHRRDRIAVRGGADREFEIFETRHGPVIAGDPREGVGLSLRSVQFAETDLSFDCLTRMPAASTVSELYEATRGWGLIDHNLVVGDTAGAIGHLVRARVPVRSRDNGWLPVPGWTAEHEWQGWIAHEAMPCVIDPPGGLIVTANNRVVADDHPDYLCTDCHPPYRAERILQRLDSGAPFAVADAAAIHADTQSPHLDLFRTRLAVLGLREQADAERLRQDLLAWDGRMDADSTSAAAYNAFRRALTRIVTVRSRLEQATAHRFAAVAPGVSPQGQVWWAVPTLLRDDDAGMLNGLTWDDVLSEALSEAAATLTGKTWGEEHQPRFAHPLSAQFPDWAQRLDPPSLPVGGDGDTVLAIGIVPSAGPTATYGALARYVFDVGNWDNSRWAVFHGTSGHPASPHYADQNTPWSACEMVPMLYDWERIAAEAKTVQELVPPRA(SEQ ID NO:1).

[0048] The coding gene of SEQ ID NO:1 is SEQ ID NO:2:

[0049]

[0050] In order to obtain CPC acylase mutants with higher enzyme activity, the present invention further performs site-directed mutagenesis on the gene sequence SEQ ID NO: 2 encoding CPC acylase L159S / A419V, i.e. SEQ ID NO: 1. By replacing any one of the sites G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603, and L707 with C or M, four mutants A14-W171M, A14-L174M, A14-I312C, and A14-L707C with improved CPC acylase activity are obtained.

[0051] The number of amino acids in the CPC acylase mutant of the present invention is 774. According to the previous modeling and molecular docking results, a total of 20 sites were selected after molecular dynamics simulation: G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603, and L707. Among them, L161, L162, W167, W171, L174, V262, F309, I312, L376, P603, and L707 are the sites where CPC acylase interacts with the CPC nucleus; G160, G164, S238, D258, P259, H260, T306, H307, and H415 are the sites where CPC acylase interacts with the CPC side chain.

[0052] Example

[0053] The primer synthesis and sequencing in the examples were commissioned to Qingke Company.

[0054] PCR amplification experiments are performed according to the reaction conditions or instructions provided by the DNA polymerase supplier and can be adjusted through simple experiments if necessary.

[0055] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride (solid medium plus 20 g / L agar powder).

[0056] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4, 2.31 g / L KH2PO4, 4 mL / L glycerol.

[0057] Example 1. Construction of the initial CPC acylase L159S / A419V expression strain

[0058] According to the sequence of CPC acylase L159S / A419V published in CN 117247925 A, the sequence SEQ ID NO: 2 in this article was constructed and subcloned into the corresponding site of the vector pET-28a to obtain the plasmid pET-28a-L159S / A419V, which was transformed into the expression host Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli expressing the initial CPC acylase, which was numbered A14.

[0059] Example 2: Construction and screening of acylase mutant library

[0060] 2.1 Site-directed mutagenesis to construct a directed evolution mutation library

[0061] Using SEQ ID NO:2 as a template, a single point mutation library was constructed using site-directed mutagenesis technology. Based on molecular docking, molecular dynamics, rational analysis and other methods of A14, a total of 20 sites were designed for rational design according to the interaction between the substrate binding pocket and the substrate molecule. The present invention selects 20 sites in the amino acid sequence of the acylase variant A14 from Bosea sp.OK403 for transformation, which are G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603, and L707, respectively, and primers are designed for each site and a C or M single point mutant library is constructed.

[0062] To obtain C or M single point mutants, the following experiments were performed:

[0063] 50uL PCR amplification reaction system: 1×KOD-ONE DNA polymerase 25μL, template A141μL, forward primer (10μM) 1.5μL, reverse primer (10μM) 1.5μL, ddH2O 21μL, the specific sequences of forward and reverse primers are shown in Table 2.

[0064] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 15 s, 55°C: 30 s, 72°C: 20 s) 30 cycles, 72°C: 5 min.

[0065] The obtained PCR product was operated as follows: 1 μL of Dpn I enzyme was added to 20 μL of PCR product to digest the plasmid template, and the plasmid template was treated at 37°C for 2 hours. 4 μL of the digested PCR product was transferred to 100 μL of E. coli BL21 (DE3) competent cells, and the transferred E. coli BL21 (DE3) bacterial solution was evenly coated on a kanamycin-resistant (concentration of 50 μg / mL) LB plate, and a single colony was grown after culturing at 37°C for 14 hours, which was the mutant engineering strain of acylase A14. The recombinant plasmid with the target nucleotide subjected to directed mutation contained in the strain was the expression vector for expressing the CPC acylase gene mutant.

[0066] 2.2 High-throughput screening of single-point mutant libraries

[0067] Transformants from the mutant library were selected and inoculated into 96-well deep-well culture plates containing 300uL (containing 50uL kanamycin) LB medium. A blank control, a negative control (pET-28a empty load) and a positive control (A14) were set up. After sealing, the plates were shaken and cultured at 37°C and 800rpm for 12h.

[0068] 700 μL of TB medium (containing 0.1 mM IPTG and 50 ug / mL kanamycin) was added to a 96-well deep-well culture plate and cultured with shaking at 800 rpm at 25°C for 16 h to induce protein expression.

[0069] After 16 hours of culture, the 96-well deep-well culture plate was centrifuged at 4000rpm for 15 minutes, the cells were collected, frozen at -80℃ for 1 hour, thawed at room temperature for 30 minutes, and 200uL (0.1M pH8.0, containing 1mg / mL lysozyme) potassium phosphate buffer was added to resuspend the cells, and incubated at 37℃ for 1 hour. 200uL substrate CPC (20mM) was added to the cell disruption solution, and reacted at 37℃ for 2-4 hours. 40uL of the reaction solution was taken to a new 96-well shallow-well plate, 200uL of the termination reaction solution (20% glacial acetic acid: 0.05M NaOH = 2:1) was added, and centrifuged for 10 minutes (4℃, 4000rpm). 200uL of the supernatant was taken, 40uL of the color developer (0.5% p-DAB dissolved in methanol) was added, and the absorbance at 415nm was detected after reacting at room temperature for 10 minutes.

[0070] Through preliminary screening, four relatively active mutants, A14-W171M, A14-L174M, A14-I312C, and A14-L707C, were obtained. Their conversion rates for substrate CPC were improved compared with A14. The results are shown in Table 2.

[0071] Table 2. Relative activity test of site-directed mutant strains

[0072]

[0073] Example 3: Strain culture, extraction and pure enzyme specific activity determination

[0074] 3.1 Strain culture

[0075] Single colonies of A14 and A14-W171M, A14-L174M, A14-I312C, and A14-L707C in Example 2 were picked up respectively, inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) test tubes, and cultured overnight at 37°C 220 rpm to obtain seed solution; 1% of the inoculum was inoculated into 100 mL of TB medium (containing 50 μg / mL kanamycin), and cultured at 37°C 220 rpm for 3 h. When the bacterial solution OD 600 When the pH reaches 0.6-0.8, add IPTG (0.1 mM) and continue shaking culture at 25°C and 220 rpm for 16 h. Centrifuge at 4°C and 4000 rpm for 10 min to collect the bacteria.

[0076] 3.2 Enzyme extraction

[0077] The bacteria were washed with 50 mL of equilibrium buffer (50 mM potassium phosphate buffer, pH 8.0), and the wet bacteria were weighed after centrifugation. Then, phosphate buffer (10 mL / g bacteria) was added to resuspend the bacteria to obtain a bacterial suspension. Then, the broken bacteria were ultrasonically broken, and the broken bacteria were centrifuged at 4 ° C, 7000 rpm for 40 min, and the supernatant was collected. The supernatant was added to an affinity chromatography column containing 5 mL Ni-NAT matrix at a rate of 2 mL / min, and then the column material was washed with an equilibrium buffer containing 30 mM imidazole to elute impurities. Finally, the target protein was washed and eluted with an equilibrium buffer containing 250 mM imidazole, and the peak eluate was collected.

[0078] The eluate was desalted by an ultrafiltration tube with a molecular weight cutoff of 10 kDa to obtain pure enzyme.

[0079] 3.3 Determination of pure enzyme specific activity

[0080] The CPC conversion product was analyzed by HPLC to determine the activity of the pure enzyme obtained in 3.2. The reaction was 1 mL system, 0.1 mg / mL pure enzyme (diluted with 50 mM potassium phosphate buffer, pH 8.0), substrate CPC (40 mM) was added, 25 ° C, 1000 rpm, and the reaction was carried out for 5 min. Take 100 uL sample, dilute 5 times with methanol, and then use HPLC to determine the product 7-ACA to calculate the activity of the pure enzyme.

[0081] HPLC detection conditions: The selected chromatographic column was Agilent ZORBAX SB-C18 (4.6*150mm 4um), the mobile phase ratio was: 0.1% formic acid water: methanol = 90:10 (v / v), the flow rate was 1.0 mL / min, the detection wavelength was 254 nm, and the column oven was kept constant at 30°C.

[0082] The desalted solution in step 3.2 was used to determine the protein concentration of the pure enzyme using the BCA Protein Assay Kit from Thermo Scientific, thereby obtaining the specific activity of the pure enzyme.

[0083] Table 3. Comparison of enzyme activity at 25°C

[0084]

[0085] Note: All relative activities refer to the percentage of enzyme activity relative to the initial enzyme A14.

[0086] As can be seen from Table 3, the enzyme activities of the cephalosporin C acylase mutants W171M, L174M, I312C and L707C of the present invention are 2.4-3 times that of the initial CPC acylase SEQ ID NO:1.

[0087] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A cephalosporin C acylase mutant, characterized in that: There is a substitution mutation at one or more of G160, L161, L162, G164, W167, W171, L174, S238, D258, P259, H260, V262, T306, H307, F309, I312, L376, H415, P603, and L707 in the amino acid sequence shown in SEQ ID NO:

1.

2. The cephalosporin C acylase mutant according to claim 1, characterized in that: The substitution mutation is a substitution with cysteine ​​or methionine; it is any one of the following substitution mutations: G160 was replaced by cysteine ​​and methionine; L161 was substituted by cysteine ​​and methionine; L162 was substituted by cysteine ​​and methionine; G164 was replaced by cysteine ​​and methionine; W167 was substituted by cysteine ​​and methionine; W171 was substituted by cysteine ​​and methionine; L174 was substituted by cysteine ​​and methionine; S238 was replaced by cysteine ​​and methionine; D258 is substituted by cysteine ​​and methionine; P259 was substituted by cysteine ​​and methionine; H260 is replaced by cysteine ​​and methionine; V262 was substituted by cysteine ​​and methionine; T306 was substituted by cysteine ​​and methionine; H307 was replaced by cysteine ​​and methionine; F309 was substituted by cysteine ​​and methionine; I312 is substituted by cysteine ​​and methionine; L376 was substituted by cysteine ​​and methionine; H415 was replaced by cysteine ​​and methionine; P603 was substituted by cysteine ​​and methionine; L707 was substituted by cysteine ​​and methionine; Preferably, the substitution mutation is W171M, L174M, I312C or L707C.

3. A gene encoding the cephalosporin C acylase mutant according to any one of claims 1 or 2.

4. A recombinant vector containing the coding gene as claimed in claim 3.

5. A recombinant strain containing the coding gene as claimed in claim 3 or the recombinant vector as claimed in claim 4.

6. The recombinant strain according to claim 5, characterized in that The starting bacteria are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Cephalosporium acremonium, and Penicillium chrysogenum, preferably Escherichia coli and Cephalosporium acremonium, and more preferably E. coli BL21(DE3).

7. Use of the cephalosporin C acylase mutant according to claim 1 or 2, the encoding gene according to claim 3, the recombinant vector according to claim 4, or the recombinant strain according to claim 5 or 6 in the preparation of 7-aminocephalosporanic acid.

8. A method for preparing 7-aminocephalosporanic acid, characterized in that: Cephalosporin C is used as a substrate and catalyzed by the cephalosporin C acylase mutant according to any one of claims 1 or 2 or the recombinant strain according to claim 5 or 6 to produce 7-aminocephalosporanic acid.

9. The method according to claim 8, characterized in that The cephalosporin C acylase mutant according to claim 1 or 2 is used in the form of pure enzyme or whole cell to catalyze the preparation of 7-ACA.

10. The method according to claim 8, characterized in that: The concentration of the cephalosporin C mutant can be selected from 5 to 80 mM, preferably 20 mM; the pH of the phosphate buffer can be selected from 6.5 to 9.5, preferably 8.0; Preferably, the temperature of the catalytic reaction may be 20-40° C.; the time of the catalytic reaction may be 5-90 min.

Images