CN112662655A - Cephalosporin C acylase mutant and preparation method and application thereof - Google Patents
Cephalosporin C acylase mutant and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, and particularly relates to a cephalosporin C acylase mutant as well as a preparation method and application thereof. The cephalosporin C acylase mutant has the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. SEQ ID NO: 2 by comparing the amino acid sequence shown in SEQ ID NO:1, and performing substitution mutation on the 245-247 amino acid of the amino acid sequence shown in the specification; wherein, the 245 th tyrosine is mutated into phenylalanine, the 246 th arginine is mutated into methionine, and the 247 th leucine is mutated into serine. Compared with the wild GL-7-ACA acylase SED ID NO:1 enzyme activity is improved by more than 210 times, and the method can be applied to one-step enzyme method production of 7-aminocephalosporanic acid (7-ACA).
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a cephalosporin C acylase mutant as well as a preparation method and application thereof.
Background
7-aminocephalosporanic acid (7-ACA) is a main precursor for synthesis of beta-lactam antibiotics, and is mainly synthesized industrially by a chemical method and an enzymatic method. However, the chemical synthesis method has complex process, serious pollution and high energy consumption, and has been replaced by a biological enzyme method in recent years, the biological enzyme method has simple process and mild reaction condition, and the prepared product has stable quality. The chemical method is mature in process, and compared with the chemical method, the biological enzyme method has a large space for rising. With the gradual increase of the national requirements on green environmental protection, the biological enzyme method has a better future prospect.
At present, biological enzyme methods are mainly divided into a one-step enzyme method and a two-step enzyme method. The two-step enzyme method relates to step-by-step catalysis of D-amino acid oxidase and glutaryl transferase, and has been reported to be applied to industrial production. The one-step enzymatic approach involves the direct catalytic production of 7-ACA using cephalosporin C acylase on the substrate CPC. Compared with a two-step enzyme method, the one-step enzyme method has the advantages of simpler process, low equipment requirement, low production cost and the like. However, compared with GL-7-ACA acylase prepared by a two-step enzyme method, the cephalosporin C acylase prepared by the one-step enzyme method has low catalytic efficiency on CPC, and only catalyzes GL-7-ACA by 2% -4%.
Chinese patent CN 108220276A discloses a cephalosporin C acylase mutant and application thereof in 7-aminocephalosporanic acid production, the cephalosporin C acylase mutant can catalyze cephalosporin C to generate 7-aminocephalosporanic acid, the cephalosporin C acylase mutant has higher catalytic activity compared with wild cephalosporin C acylase, and the enzyme activity is improved by about 8.6 times. Obtaining a gene sequence, recombining and expressing protein, detecting enzyme activity and preparing 7-aminocephalosporanic acid by using the primer A.
Chinese patent CN 110129305A discloses a cephalosporin C acylase mutant for preparing 7-ACA, wherein the amino acid sequence of the cephalosporin C acylase is SEQ ID NO. 4, compared with wild type GL-7-ACA acylase SEQ ID NO. 1 from Pseudomonas GK16(Pseudomonas sp.GK16), the enzyme activity is improved by 52 times, and the cephalosporin C acylase mutant can be used for producing 7-aminocephalosporanic acid (7-ACA) by a one-step enzyme method.
Although the enzyme activity of the cephalosporin C acylase mutants provided by the two patents is respectively improved by 8.6 times and 52 times compared with that of the wild cephalosporin C acylase, the enzyme activity is still lower, and the industrial production efficiency is low.
Disclosure of Invention
The invention aims to provide a cephalosporin C acylase mutant which has high enzyme activity and high industrial production efficiency; the invention also provides a preparation method and application of the cephalosporin C acylase mutant.
The cephalosporin C acylase mutant provided by the invention has the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
SEQ ID NO: 2 by comparing the amino acid sequence shown in SEQ ID NO:1, and performing substitution mutation on the 245-247 amino acid of the amino acid sequence shown in the specification; wherein, the 245 th tyrosine is mutated into phenylalanine, the 246 th arginine is mutated into methionine, and the 247 th leucine is mutated into serine.
SEQ ID NO: 2 is the amino acid sequence shown in SEQ ID NO: 3, and (b) 3.
SEQ ID NO: 3 by comparing the nucleotide sequence shown in SEQ ID NO:4, and the base numbers 215, 232, 239, 250, 252, 268 and 327 of the DNA sequence shown in the specification are subjected to substitution mutation; wherein, the 215 th adenine mutation is thymine, the 232 th adenine mutation is thymine, the 239 th guanine mutation is cytosine, the 250 th cytosine mutation is guanine, the 252 th thymine mutation is adenine, the 268 th adenine mutation is thymine, and the 327 th guanine mutation is cytosine.
The preparation method of the cephalosporin C acylase mutant comprises the following steps:
(1) converting SEQ ID NO:4 to obtain the DNA sequence shown in SEQ ID NO: 2 in sequence (b);
(2) cloning the coding gene into a plasmid vector to obtain a recombinant plasmid;
(3) introducing the recombinant plasmid into an escherichia coli competent cell to obtain a recombinant bacterium;
(4) and performing induction fermentation on the recombinant bacteria to obtain the cephalosporin C acylase mutant.
The double-point mutation step in the step (1) is carried out by using SEQ ID NO:4 as template, adding primer to perform PCR amplification;
the primer sequences are as follows:
the upstream primer F1: GGGAATTCCATATGCT F2: GAGAGTTCTGCACCG the flow of the air in the air conditioner,
the downstream primer R1: CCCGGAATTCTCATGG R2: CTTGAAGTTGAAGTTGAAGG the flow of the air in the air conditioner,
wherein, F1 and R2 contain 20bp homology arms, and F2 and R1 contain 20bp homology arms.
The PCR amplification system is as follows:
the PCR reaction condition is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 5s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.
In the step (2), the plasmid carrier is PET-28a+。
And (4) adding IPTG to perform induction fermentation in the step (4).
The application of the cephalosporin C acylase mutant is that the cephalosporin C acylase mutant is used for preparing 7-aminocephalosporanic acid.
The invention has the following beneficial effects:
in order to overcome the defect of low catalytic efficiency of CPC catalyzed by a one-step enzyme method and obtain CPC acylase with higher enzyme activity efficiency, the invention carries out gene mutation and screening on wild type GL-7-ACA acylase (SED ID NO: 1) from pseudomonas GK16 by using genetic engineering technologies such as point mutation and the like to prepare the cephalosporin C acylase mutant with higher enzyme activity efficiency.
Compared with the wild GL-7-ACA acylase SED ID NO:1 enzyme activity is improved by more than 210 times, and the method can be applied to one-step enzyme method production of 7-aminocephalosporanic acid (7-ACA).
Drawings
FIG. 1 is an electrophoretic verification chart of true positive transformants obtained in example 1.
Detailed Description
The present invention is further described below with reference to examples.
Example 1
(1) Using CPC acylase wild type gene SEQ ID NO:4, cloning target genes by taking the primer sequences as follows: the upstream primer F1: GGGAATTCCATATGCT F2: GAGAGTTCTGCACCG downstream primer R1: CCCGGAATTCTCATGG R2: CTTGAAGTTGAAGTTGAAGG, wherein F1 and R2 contain 20bp homology arms, and F2 and R1 contain 20bp homology arms, thereby carrying out double-point mutation.
(2) The PCR amplification system is as follows:
the PCR reaction condition is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 5s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.
(3) Subsequently, a purification kit is adopted to purify and purify the PCR product, the obtained target gene is subjected to double enzyme digestion by BamHI and HinDIII endonucleases, and the plasmid vector PET-28a is subjected to double enzyme digestion+Carrying out the same double enzyme digestion to obtain the recombinant plasmid PET-28a+-CPCA. Escherichia coli TG1 was made into Escherichia coli competent cells by calcium chloride method, and the obtained recombinant plasmid was introduced into Escherichia coli competent cells to obtain a transformant designated TG1/PET-28a+-CPCA. Mixing TG1/PET-28a+CPCA was spread evenly on LB plates (containing ampicillin resistance), incubated overnight at 37 ℃ and plasmids were extracted from single colonies growing on the plates.
The primer sequence for designing the recombinant plasmid is as follows: f: CGAAATCTTTGTTGGTGGG R: ATGAAGGATACATAAACGGCTAC are provided.
The PCR amplification system is as follows:
the PCR reaction condition is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 5s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.
And (3) carrying out nucleic acid electrophoresis verification on the amplification product, wherein the electrophoresis result is shown in figure 1, and the single colony picked is confirmed to be a true positive transformant.
(4) Selecting the colony of the verified true positive transformant to an LB liquid culture medium containing ampicillin, adding 0.1mM IPTG at 37 ℃ and 200rpm to promote the expression of the target protein, and shaking the bacteria overnight until the OD value is 0.6-0.8 to obtain a bacterial liquid; centrifuging the bacterial liquid at 4000rpm for 10min, discarding the supernatant, retaining the precipitate, freezing at-20 ℃ for 1h, thawing at normal temperature, adding 200ul of 0.9% physiological saline, cracking the bacteria by using an ultrasonic cell crusher, incubating at 37 ℃ for 1h, centrifuging at 4000rpm for 10min to obtain a cephalosporin C acylase mutant, and retaining 20ul of the supernatant for CPC activity determination.
(5) And (4) resuspending the bacterial liquid obtained in the step (4) by using 50ml of buffer solution, carrying out ultrasonic lysis, centrifuging the crushed thallus at 12000rpm at 4 ℃ for 20min, and collecting supernatant. The supernatant was passed through a nickel column at a rate of 1ml/min, the nickel column was washed with 30mM imidazole, the hetero-protein was washed with 300mM imidazole, the target protein was collected until the reaction solution did not turn blue any more, and the peak eluent was collected. Desalting the obtained eluate with 10KD hyperconcentration column, and removing acylase to obtain purified enzyme. And (3) determining the Protein concentration of the purified enzyme by adopting a Protein Assay Kit so as to obtain the specific activity of the purified enzyme. The results of enzyme activity measurement of wild-type CPC acylase (wtCPC) and various CPC acylase mutants are shown in Table 1.
TABLE 1 measurement results of enzyme activities of wild-type CPC acylase and various CPC acylase mutants
As is clear from Table 1, by substituting an amino acid in the wild-type CPC acylase, a mutant strain ED4 (CPC acylase mutant prepared in example 1) having a relative specific activity increased 210.1-fold was obtained.
The invention successfully constructs the CPC acylase mutant, successfully improves the specific activity of wild acylase by more than 210 times, has faster reaction efficiency when being used for producing 7-ACA, and has wide industrialization prospect.
(6) The substrate reaction solution was 0.1M potassium phosphate salt buffer (ph8.0) containing 2 wt.% CPC sodium salt,
terminating the reaction solution: 0.05M NaOH, 20 v/v% glacial acetic acid,
color developing agent: containing 0.5 wt% of PDAB in methanol,
definition of enzyme activity: the amount of enzyme required to catalyze CPC to produce 1uM7-ACA per minute was defined as one unit (U).
Adding 20ul of each of the wild-type CPC acylase and the CPC acylase mutants in the step (5) into a substrate reaction solution, culturing overnight at 37 ℃, adding 200ul of a termination reaction solution, centrifuging at 5000rpm for 10min, adding 40ul of a color developing agent, reacting for 10min, and detecting the yield of 7-ACA under the absorbance of 415nm, wherein the results are shown in Table 2.
TABLE 2 determination of 7-ACA yields of wild-type CPC acylase and various CPC acylase mutants
| Strain numbering | wtCPC | ED1 | ED2 | ED3 | ED4 |
| 7-ACA yield (ug/min) | 0.15 | 0.69 | 17.8 | 23.1 | 32 |
(7) Effect of medium pH on cephalosporin C acylase secretion expression
After the bacterial liquids obtained in the step (4) are respectively cultured in LB culture media with pH values of 7.5, 8.0, 8.5, 9.0 and 9.5 for 24 hours, the activity of cephalosporin C acylase of respective intact cells is determined, and the results are shown in Table 3.
TABLE 3 determination of cephalosporin C acylase Activity
As is clear from Table 3, the enzyme activity was the greatest and the expression level was the highest when cultured in a medium having a pH of 8.0.
Sequence listing
<110> Shandong Kingkory chemical Co., Ltd
<120> cephalosporin C acylase mutant and preparation method and application thereof
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accaccgttg acaaaccgct ggaaatccgt tcttctgttc acggtccggt tttcgaacgt 900
gctgacgcat gccatgcgct gttgctgttg ctgttcgtgt tgctggtctg gaccgtccgg 960
gtatgctgga acagaccttc gacatgatca ccgctgactc tttcgacgac tacgaagctg 1020
ctctggctcg tatgcaggtt ccgaccttca acatcgttta cgctgaccgt gaaggtacca 1080
tcaactactc tttcaacgca caagctccga aacgtgctga aggtgacatc gctttctggc 1140
agggtctggt tccgggtgac tcttctcgtt acctgtggac cgaaacccac ccgctggacg 1200
acctgccgcg tgttaccaac ccgccgttcg ccgagaccga cctttccttc gattgcctga 1260
cgcggatgcc gggcgcatcg accgtggcgc agctttacga cgcgacgcgc ggctggggcc 1320
tgatcgacca taatcgacca taatctcgtc gccggggatg tcgcgggctc gatcggccat 1380
ctggtccgcg cccgcgtccc gtcccgcccg cgcgagaacg gctggctgcc ggtgccgggc 1440
tggtccggcg tggtccggcg agcatgaatg gcgcggctgg attccgcacg aggcgatgcc 1500
gcgcgtcatc gatccgccgg gcggcctcat cgtcacggcg aacaaccgcg tcgtggccga 1560
tgatcatccc gattatctct gtaccgattg ccatccgccc taccgcgccg aacggatcat 1620
ggagcgcctg gtcgccagtc cggctttcgc cgtcgacgat gcggccgcga tccacgccga 1680
tacgctgtcc cccatgtcgg cttgctgcgc gcgaggctcg aagcgctcgg aatccagggc 1740
agtctccctg ccgaagagtt gaggcagacc ctcatcgcct gggacggccg catggatgct 1800
ggctcgcagg cggcttccgc ttataatgcg ttccgcaggg cgctgacgcg gctggtaacg 1860
gcccgcagcg ggctggagca agcgatagcg catcccttcc tgcgcaacga cgatgccggg 1920
atgctgaaag gctggagctg ggacgaggcc ttgtcggagg ccctgtccgt cgcgacgcag 1980
aacctgaccg ggcgcggctg gggcgaggag catcggccgc gtttcacgca cccgctctcc 2040
gcgcagttcc cggcctgggc cgcgctgctg aacccggttt cgcgcccgat cggcggcgat 2100
ggcgacaccg tgctggcgaa cgggctcgtc ccatcggccg gacctgaggc gacctatggc 2160
gccctgtcgc gctacgtctt cgatgtcggc aattgggaca atagccgctg ggtcgtcttc 2220
cacggcgcct cggggcactc ggccagcccc cactatgccg accagaatgc gccatggagc 2280
gactgcgcga tggtgccgat gctctatagc tgggacagga tcgccgcgga ggccgtgacc 2340
tcgcaggaac tcgtcccggc ctga 2364
Claims (10)
1. A cephalosporin C acylase mutant characterized in that it has the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
2. Cephalosporin C acylase mutant according to claim 1 characterized in that the amino acid sequence of SEQ ID NO: 2 by comparing the amino acid sequence shown in SEQ ID NO:1, and performing substitution mutation on the 245-247 amino acid of the amino acid sequence shown in the specification; wherein, the 245 th tyrosine is mutated into phenylalanine, the 246 th arginine is mutated into methionine, and the 247 th leucine is mutated into serine.
3. Cephalosporin C acylase mutant according to claim 1 characterized in that the amino acid sequence of SEQ ID NO: 2 is the amino acid sequence shown in SEQ ID NO: 3, and (b) 3.
4. Cephalosporin C acylase mutant according to claim 3, characterized in that the amino acid sequence of SEQ ID NO: 3 by comparing the nucleotide sequence shown in SEQ ID NO:4, and the base numbers 215, 232, 239, 250, 252, 268 and 327 of the DNA sequence shown in the specification are subjected to substitution mutation; wherein, the 215 th adenine mutation is thymine, the 232 th adenine mutation is thymine, the 239 th guanine mutation is cytosine, the 250 th cytosine mutation is guanine, the 252 th thymine mutation is adenine, the 268 th adenine mutation is thymine, and the 327 th guanine mutation is cytosine.
5. A process for preparing cephalosporin C acylase mutant of any of claims 1 to 4, characterized in that it comprises the following steps:
(1) converting SEQ ID NO:4 to obtain the DNA sequence shown in SEQ ID NO: 2 in sequence (b);
(2) cloning the coding gene into a plasmid vector to obtain a recombinant plasmid;
(3) introducing the recombinant plasmid into an escherichia coli competent cell to obtain a recombinant bacterium;
(4) and performing induction fermentation on the recombinant bacteria to obtain the cephalosporin C acylase mutant.
6. The method for preparing cephalosporin C acylase mutant according to claim 5, characterized in that the step of double-point mutation in step (1) is represented by SEQ ID NO:4 as template, adding primer to perform PCR amplification;
the primer sequences are as follows:
the upstream primer F1: GGGAATTCCATATGCT F2: GAGAGTTCTGCACCG the flow of the air in the air conditioner,
the downstream primer R1: CCCGGAATTCTCATGG R2: CTTGAAGTTGAAGTTGAAGG the flow of the air in the air conditioner,
wherein, F1 and R2 contain 20bp homology arms, and F2 and R1 contain 20bp homology arms.
7. The method for preparing cephalosporin C acylase mutant according to claim 6, characterized in that the PCR amplification system is as follows:
the PCR reaction condition is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 45 ℃ for 5s, extension at 72 ℃ for 3min, and 30 cycles; extension at 72 ℃ for 10 min.
8. The method for preparing cephalosporin C acylase mutant according to claim 5, characterized in that the plasmid carrier in step (2) is PET-28a+。
9. The method for preparing cephalosporin C acylase mutant according to claim 5, characterized in that the induction fermentation in step (4) is an induction fermentation by adding IPTG.
10. Use of a cephalosporin C acylase mutant according to any of claims 1 to 4 for the preparation of 7-aminocephalosporanic acid.
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