CN107653236B - Cephalosporin C acylase mutant and preparation and application thereof - Google Patents
Cephalosporin C acylase mutant and preparation and application thereof Download PDFInfo
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- C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)
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Abstract
The invention discloses a cephalosporin C acylase mutant and preparation and application thereof, belonging to the technical field of enzyme preparation and comprising the following steps: obtaining a gene sequence, recombining and expressing protein, detecting enzyme activity and preparing 7-aminocephalosporanic acid by using the primer A. The cephalosporin C acylase mutant with enhanced enzyme activity is obtained by directed evolution, the enzyme activity is improved by more than 5 times compared with wild cephalosporin C acylase, the substrate conversion rate in three hours reaches 95-98%, and the byproduct D-7ACA is less than 0.8%, which is superior to the industrial standard.
Description
Technical Field
The invention relates to a cephalosporin C acylase mutant, in particular to a cephalosporin C acylase mutant and preparation and application thereof, belonging to the technical field of enzyme preparation.
Background
At present, 7-aminocephalosporanic acid (7-ACA) is an important intermediate for producing semi-synthetic cephalosporin medicines in the pharmaceutical industry, and the cephalosporin C sodium (zinc) salt is produced by removing a side chain of the cephalosporin C sodium (zinc) salt through a chemical method in industry at home and abroad, but the chemical method has the problems of complex process, high cost and the like and can also seriously pollute the environment; compared to chemical methods, enzymatic cleavage can greatly simplify the production process, for example: the cephalosporin C obtained by fermentation can be used for enzymolysis without crystallization, toxic solvents are not used in the production process, the steps of adding protective agents, removing protective agents and the like can be omitted, the product can achieve high yield and high quality, and meanwhile, the cost is reduced and the pollution is reduced. Therefore, in recent years, research on enzymatic production of 7-ACA has been pursued.
Currently, the two-step enzymatic method for preparing 7-ACA is mostly studied, first, cephalosporin C is catalyzed by D-amino acid oxidase (DAAO) under the condition of oxygen gas introduction to produce keto group-containing intermediate (keto-7-ACA) and H2O2This intermediate is less stable and readily co-produced as H2O2Chemical oxidation decarboxylation to convert into glutaryl-7-aminocephalosporanic acid (GL-7-ACA), and then removing the side chain of GL-7-ACA under the action of GL-7-ACA acylase to generate 7-ACA.
At present, most manufacturers of domestic 7-ACA convert the production line of 7-ACA from chemical method to enzymatic method, and 7-ACA D-amino acid oxidase and GL-7-ACA acylase are also produced in large scale in domestic. Although the two-step enzymatic method for preparing 7-ACA is advantageous in terms of production cost and environmental protection, the conversion rate of cephalosporin C to 7-ACA is low compared with the chemical method, DAAO catalytic reaction is difficult to control, cephalosporin C acylase (CPC acylase) can directly convert cephalosporin C to 7-ACA (without intermediate products such as GL-7-ACA and the like, so that the conversion rate is equivalent to the chemical method, and 7-ACA with higher quality can be obtained.
At present, the one-step enzyme method for producing 7-ACA by using CPC acylase is a brand-new 7-ACA enzyme method engineering, has the advantages of high conversion rate and purity, high economy and environmental protection, and has the advantages of simple production process, low equipment requirement, low production cost and the like. However, the naturally occurring cephalosporin C acylase has low catalytic activity on cephalosporin C, which is only 2-4% of GL-7-ACA acylase, and industrialization is difficult to realize, so that development of cephalosporin C acylase mutants with high enzyme activity for industrial production is urgently needed.
The promotion of domestic enterprises on the process for producing 7-ACA by cephalosporin C acylase mainly comprises the following aspects: increasing the activity of cephalosporin C acylase by site-directed mutagenesis; performing gene knockout on escherichia coli so as to eliminate protein expression of decomposed substrates and improve the yield and purity of 7-ACA; the immobilization research of cephalosporin C acylase can reduce the production cost, etc. Although the enzyme activity is improved to a certain extent by the methods, the industrial requirements cannot be met, and the obtained mutant cephalosporin C acylase is easily inhibited by a 7-ACA product, so that the yield of the 7-ACA is low, the conversion rate is low, and the enzyme activity is greatly influenced by the outside.
Disclosure of Invention
The invention mainly aims to provide a cephalosporin C acylase mutant and preparation and application thereof.
The purpose of the invention can be achieved by adopting the following technical scheme:
a cephalosporin C acylase mutant can catalyze cephalosporin C to generate 7-aminocephalosporanic acid, and the cephalosporin C acylase mutant has higher catalytic activity and the enzyme activity is improved by about 6.4 times compared with wild cephalosporin C acylase.
Further, the cephalosporin C acylase mutant is selected from a sequence SEQ ID NO. 1.
Further, the cephalosporin C acylase mutant selected from the sequence SEQ ID NO.1 has higher substrate catalytic activity than the wild-type cephalosporin C acylase.
Further, the detection of enzyme activity comprises the following steps:
step 11: defining enzyme activity as enzyme activity for hydrolyzing 1 mu mol CPC-Na per minute as a unit U, and detecting the enzyme activity by adopting a sodium hydroxide titration method;
step 12: preparing a phosphate buffer solution with the pH value of 8.0;
step 13: preparing a CPC-Na salt solution;
step 14: preheating a three-neck flask in 25 ℃ water bath in advance, sucking 2% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask, adding an appropriate volume of enzyme solution, uniformly mixing a reaction system by using magnetic stirring, controlling the reaction temperature to be 25 ℃, titrating with 0.1mol/L sodium hydroxide titration solution, keeping the pH of the reaction solution at 8.0, and simultaneously recording the consumption milliliter number of the sodium hydroxide titration solution within about 6-8 min of reaction;
step 15: calculating enzyme activity;
step 16: and (4) HPLC analysis.
A polynucleotide encoding a polypeptide which is recombinant by a cephalosporin C acylase mutant selected from the sequence SEQ ID No. 1.
Further, the polynucleotide is selected from the sequence SEQ ID NO. 2.
A method for producing 7-aminocephalosporanic acid, which produces 7-aminocephalosporanic acid by using cephalosporin C acylase mutant selected from SEQ ID NO. 1.
Further, the production method comprises the following steps:
step 21: preparing a phosphate buffer solution with the pH value of 8.0;
step 22: preparing a CPC-Na salt solution;
step 23: placing the three-neck flask in a water bath at 25 ℃ for preheating in advance, and sucking 10% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask;
sampling 500 μ l before adding enzyme solution, and immediately adding 500 μ l stop solution;
after uniform mixing, 12000g of the mixture is centrifuged at high speed for 10min, and then the supernatant is taken for storage;
adding an appropriate volume of enzyme solution, uniformly mixing the reaction system by magnetic stirring, controlling the reaction temperature to be 25 ℃, adjusting the pH value by using 0.1mol/L sodium hydroxide, and keeping the pH value of the reaction solution to be 8.0.
Step 24: sampling 500 μ l after adding enzyme solution for 100min, 200min, 250min, 300min, respectively, adding 500 μ l stop solution, mixing, centrifuging at 12000g for 10min, and collecting supernatant for HPLC detection.
Further, the recombinant expression protein in the process of producing 7-aminocephalosporanic acid by the cephalosporin C acylase mutant comprises the following steps:
step 31: selecting single colony of Escherichia coli containing SEQ ID NO.2 sequence, inoculating in 10ml of culture medium sterilized under high pressure, and culturing at 30 deg.C and 250rpm overnight;
step 32: inoculating 1L triangular flask into 100ml of autoclaved culture medium at an inoculation ratio of 1:100 the next day, and culturing at 30 ℃ until thallus OD 5-6;
step 33: immediately placing the triangular flask in a shaker at 25 ℃ and culturing for 1 hour at 250 rpm;
step 34: IPTG was added to a final concentration of 0.1mM and the culture was continued at 25 ℃ and 250rpm for 16 hours;
step 35: after the culture is finished, centrifuging the culture medium at 4 ℃ and 12000g for 20min to collect wet thalli;
step 36: washing the thallus precipitate twice with distilled water, collecting thallus, and storing at-70 deg.C;
step 37: a small amount of the cells were subjected to SDS-PAGE.
Further, in step 32, the following components are included in each liter of the culture medium:
tryptone: 10g of a mixture;
yeast extract (B): 5g of the total weight of the mixture;
disodium hydrogen phosphate: 3.55 g;
potassium dihydrogen phosphate: 3.4 g;
ammonium chloride: 2.68 g;
sodium sulfate: 0.71 g;
magnesium sulfate heptahydrate: 0.493 g;
ferric chloride hexahydrate: 0.027 g;
glycerol: 5g of the total weight of the mixture;
glucose: 0.8 g;
kanamycin: 50 mg.
The invention has the beneficial technical effects that: according to the cephalosporin C acylase mutant and the preparation and the application thereof, the cephalosporin C acylase mutant with enhanced enzyme activity and the preparation and the application thereof are obtained through directed evolution, the enzyme activity is improved by more than 5 times compared with wild cephalosporin C acylase, the three-hour substrate conversion rate reaches 95 to 98 percent, the byproduct D-7ACA is less than 0.8 percent and is superior to the industrial standard, the protein mutant can be used for effectively manufacturing the important intermediate of semi-synthetic cephalosporin medicaments, the process is simple, the yield is high, the transplantable technology is strong, the cephalosporin C acylase mutant can be put into production only in a general fermentation workshop, such as an amino acid and vitamin production workshop, special equipment does not need to be purchased, and the cephalosporin C acylase mutant is easy to popularize and apply, and has strong practicability and popularization value.
Drawings
FIG. 1 is SDS-PAGE patterns of cephalosporin C acylase mutant protein supernatant and inclusion body according to the invention;
FIG. 2 is an HPLC map of the gene sequence of sample 7-aminocephalosporanic acid in the example according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention more clear and definite for those skilled in the art, the present invention is further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
The cephalosporin C acylase mutant provided by the embodiment can catalyze cephalosporin C to generate 7-aminocephalosporanic acid, and compared with wild cephalosporin C acylase, the cephalosporin C acylase mutant has higher catalytic activity and the enzyme activity is improved by about 6.4 times.
Further, in this example, the cephalosporin C acylase mutant is selected from the sequence SEQ ID NO. 1.
Further, in this example, the cephalosporin C acylase mutant selected from the sequences SEQ ID NO.1 has a higher substrate catalytic activity than the wild-type cephalosporin C acylase.
Further, in this embodiment, the detection of enzyme activity includes the following steps:
step 11: defining enzyme activity as enzyme activity for hydrolyzing 1 mu mol CPC-Na per minute as a unit U, and detecting the enzyme activity by adopting a sodium hydroxide titration method;
step 12: preparing a phosphate buffer solution with the pH value of 8.0;
step 13: preparing a CPC-Na salt solution;
step 14: preheating a three-neck flask in 25 ℃ water bath in advance, sucking 2% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask, adding an appropriate volume of enzyme solution, uniformly mixing a reaction system by using magnetic stirring, controlling the reaction temperature to be 25 ℃, titrating with 0.1mol/L sodium hydroxide titration solution, keeping the pH of the reaction solution at 8.0, and simultaneously recording the consumption milliliter number of the sodium hydroxide titration solution within about 6-8 min of reaction;
step 15: calculating enzyme activity;
step 16: and (4) HPLC analysis.
This example provides a polynucleotide encoding a polypeptide recombinant by a cephalosporin C acylase mutant selected from the group consisting of SEQ ID NO. 1.
Further, in this embodiment, the polynucleotide is selected from the sequence of SEQ ID NO. 2.
This example provides a method for producing 7-aminocephalosporanic acid, which comprises producing 7-aminocephalosporanic acid by using cephalosporin C acylase mutant selected from SEQ ID NO. 1.
Further, in this embodiment, the production method comprises the following steps:
step 21: preparing a phosphate buffer solution with the pH value of 8.0;
step 22: preparing a CPC-Na salt solution;
step 23: placing the three-neck flask in a water bath at 25 ℃ for preheating in advance, and sucking 10% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask;
sampling 500 μ l before adding enzyme solution, and immediately adding 500 μ l stop solution;
after uniform mixing, 12000g of the mixture is centrifuged at high speed for 10min, and then the supernatant is taken for storage;
adding an appropriate volume of enzyme solution, uniformly mixing the reaction system by magnetic stirring, controlling the reaction temperature to be 25 ℃, adjusting the pH value by using 0.1mol/L sodium hydroxide, and keeping the pH value of the reaction solution to be 8.0.
Step 24: sampling 500 μ l after adding enzyme solution for 100min, 200min, 250min, 300min, respectively, adding 500 μ l stop solution, mixing, centrifuging at 12000g for 10min, and collecting supernatant for HPLC detection.
Further, in this example, the recombinant expression of the protein in the production of 7-aminocephalosporanic acid by the cephalosporin C acylase mutant comprises the following steps:
step 31: selecting single colony of Escherichia coli containing SEQ ID NO.2 sequence, inoculating in 10ml of culture medium sterilized under high pressure, and culturing at 30 deg.C and 250rpm overnight;
step 32: inoculating 1L triangular flask into 100ml of autoclaved culture medium at an inoculation ratio of 1:100 the next day, and culturing at 30 ℃ until thallus OD 5-6;
step 33: immediately placing the triangular flask in a shaker at 25 ℃ and culturing for 1 hour at 250 rpm;
step 34: IPTG was added to a final concentration of 0.1mM and the culture was continued at 25 ℃ and 250rpm for 16 hours;
step 35: after the culture is finished, centrifuging the culture medium at 4 ℃ and 12000g for 20min to collect wet thalli;
step 36: washing the thallus precipitate twice with distilled water, collecting thallus, and storing at-70 deg.C;
step 37: a small amount of the cells were subjected to SDS-PAGE.
Further, in this embodiment, in step 32, the following components are included in each liter of the culture medium:
tryptone: 10g of a mixture;
yeast extract (B): 5g of the total weight of the mixture;
disodium hydrogen phosphate: 3.55 g;
potassium dihydrogen phosphate: 3.4 g;
ammonium chloride: 2.68 g;
sodium sulfate: 0.71 g;
magnesium sulfate heptahydrate: 0.493 g;
ferric chloride hexahydrate: 0.027 g;
glycerol: 5g of the total weight of the mixture;
glucose: 0.8 g;
kanamycin: 50 mg.
Further, in this example, FIG. 1 shows the SDS-PAGE patterns of the cephalosporin C acylase mutant protein supernatant and inclusion body; FIG. 2 is an HPLC chromatogram of the gene sequence of sample 7-aminocephalosporanic acid in the example of this example.
The cephalosporin C acylase mutant and the application thereof provided by the embodiment comprise the following steps:
step 1: obtaining a gene sequence through a primer;
step 2: recombinant expression of the protein;
and step 3: detecting enzyme activity;
and 4, step 4: preparing 7-aminocephalosporanic acid.
Further, in this embodiment, in the step 1, acquiring a gene sequence by a primer includes the following steps:
primer Premier (http:// Primer3.ut. ee /) was used for Primer design, the design principle is as follows: ensuring that the Tm value difference is controlled within 3 ℃; and the need to reduce the generation of repetitive sequences and eliminate potential hairpin structures, making secondary structures more transcribable; simultaneously eliminating codons with the frequency of being less than 10 percent in a host transcriptome; and strictly controlling the length of the primer to be within 50 bases to obtain the primer:
the above primers were synthesized, and the obtained primers were dissolved in double distilled water and added to the following reaction system so that the final concentration of each primer was 30nM and the final concentration of the head and tail primers was 0.6. mu.M.
Each 50. mu.l of PCR reaction system contained the following components:
2mM dNTP mix：5μl；
10×Pfu buffer：5μl；
Pfu DNA polymerase(10U/μl)：0.5μl；
ddH2o: and (4) the balance.
The prepared PCR reaction system is placed in a Bori XP cycler gene amplification instrument and amplified according to the following procedures: 30s at 98 ℃, 45s at 55 ℃, 180s at 72 ℃ and 35 x. The DNA fragment obtained by PCR was purified by gel cutting and cloned into the NdeI/XhoI site of pET30a by homologous recombination. And (4) selecting a single clone for sequencing confirmation to obtain a correct sequence.
Further, in this embodiment, in the step 2, the recombinant expression of the protein comprises the following steps:
the single colony of Escherichia coli containing the sequence of SEQ ID NO.2 was selected and inoculated into 10ml of autoclaved medium:
the culture medium comprises the following components per liter:
tryptone: 10g of a mixture;
yeast extract (B): 5g of the total weight of the mixture;
disodium hydrogen phosphate: 3.55 g;
potassium dihydrogen phosphate: 3.4 g;
ammonium chloride: 2.68 g;
sodium sulfate: 0.71 g;
magnesium sulfate heptahydrate: 0.493 g;
ferric chloride hexahydrate: 0.027 g;
glycerol: 5g of the total weight of the mixture;
glucose: 0.8 g;
kanamycin: 50 mg;
30 ℃, 250rpm overnight culture, taking 1L triangular flask the next day, and mixing according to the weight ratio of 1: inoculating 100ml of the culture medium after autoclaving into 100ml of the culture medium;
culturing at 30 deg.C until thallus OD5-6, immediately placing triangular flask in 25 deg.C shaking table, and culturing at 250rpm for 1 hr;
IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;
after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli;
washing the thallus precipitate twice with distilled water, collecting thallus, and storing at-70 deg.C;
a small amount of the cells were subjected to SDS-PAGE.
Further, in this embodiment, in the step 3, detecting the enzyme activity includes the following steps:
defining enzyme activity as enzyme activity for hydrolyzing 1 mu mol CPC-Na per minute as a unit (U), and adopting a sodium hydroxide titration method for detecting the enzyme activity;
preparing a phosphate buffer solution with pH 8.0: weighing KH2PO40.68g of the extract is dissolved in 250ml of purified water to obtain a solution 1; weighing K2HPO4·3H2O2.28 g is put into 500ml of purified water to obtain solution 2; adjusting the pH value of the solution 2 to 8.0 by using the solution 1;
preparing a CPC-Na salt solution: weighing 2.0g of CPC-Na salt, dissolving in about 100ml of the above phosphate buffer solution, and adjusting the pH of the solution to 8.0 by using sodium hydroxide;
preheating a three-neck flask in 25 ℃ water bath in advance, sucking 2% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask, adding an appropriate volume of enzyme solution, uniformly mixing a reaction system by using magnetic stirring, controlling the reaction temperature to be 25 ℃, titrating with 0.1mol/L sodium hydroxide titration solution, keeping the pH of the reaction solution to be 8.0, and simultaneously recording the consumption milliliter number of the sodium hydroxide titration solution within about 6-8 minutes of reaction;
calculating enzyme activity: taking the ml consumed by the sodium hydroxide titration solution within 10 minutes of the test, calculating the ml per minute, and calculating the enzyme activity by the following formula:
V1-determining the volume, ml, of sodium hydroxide titration solution consumed over time;
c is the concentration of sodium hydroxide titration solution, mol/L;
1000-fold micromolar concentration conversion coefficient;
t-measuring the enzyme reaction time, min;
V2-volume of enzyme solution, mL;
500ul of reaction solution was taken, 500. mu.l of stop solution (20% glacial acetic acid and 50mM NaOH mixed at a ratio of 2: 1) was added to stop the reaction, and HPLC analysis (phenomenex C18 column, 150X4.6 mM; mobile phase: 15% methanol, 7.5% acetonitrile, 1% acetic acid; 254nm absorbance detection) was performed, and the results indicated that the mutant was able to produce 7-aminocephalosporanic acid.
Further, in this embodiment, in the step 4, the preparation of 7-aminocephalosporanic acid comprises the following steps:
preparing a phosphate buffer solution with pH 8.0: weighing KH2PO40.68g of the extract is dissolved in 250ml of purified water to obtain a solution 1; weighing K2HPO4·3H2O2.28 g is put into 500ml of purified water to obtain solution 2; adjusting the pH value of the solution 2 to 8.0 by using the solution 1;
preparing a CPC-Na salt solution: weighing 20.0g of CPC-Na salt, dissolving in about 200ml of the phosphate buffer solution, and adjusting the pH value of the solution to 8.0 by using sodium hydroxide;
placing the three-neck flask in a water bath at 25 ℃ for preheating in advance, sucking 10% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask, sampling 500 mu l before adding the enzyme solution, immediately adding 500 mu l of stop solution (20% glacial acetic acid and 50mM NaOH are mixed according to a ratio of 2: 1), uniformly mixing, centrifuging at high speed at 12000g for 10 minutes, and taking the supernatant for storage; adding an appropriate volume of enzyme solution, uniformly mixing the reaction system by magnetic stirring, controlling the reaction temperature to be 25 ℃, adjusting the pH value by using 0.1mol/L sodium hydroxide, and keeping the pH value of the reaction solution to be 8.0;
sampling 500 μ l at 100min, 200min, 250min and 300min after adding enzyme solution, adding 500 μ l stop solution (20% glacial acetic acid and 50mM NaOH mixed at 2: 1), mixing, centrifuging at 12000g at high speed for 10min, and collecting supernatant for HPLC detection.
HPLC results showed a final conversion of 96.95% and approximately 0.77% D-7-aminocephalosporanic acid.
The enzyme activity detection result shows that CPC acylase mutant with 6.4 times higher activity than wild-type acylase is obtained by directed evolution of CPC acylase, and the enzyme activity comparison result is shown in Table 1.
TABLE 1 comparison of enzyme activities
| Species of | Enzyme activity |
| Wild-type acylase | 10.46U/ml |
| Seq ID NO 1 muteins | 67.5U/ml |
The protein mutant of the invention can effectively produce important intermediates of semi-synthetic cephalosporin drugs, has simple process, high yield and strong technical portability, can be put into production only in a common fermentation workshop (such as an amino acid and vitamin production workshop), does not need to purchase special equipment, and is easy to popularize and apply.
In summary, in this example, according to the cephalosporin C acylase mutant and its preparation and application in this example, the cephalosporin C acylase mutant and its preparation and application provided in this example, the cephalosporin C acylase mutant with enhanced enzyme activity is obtained by directed evolution, the enzyme activity is improved by more than 5 times compared with wild cephalosporin C acylase, the substrate conversion rate in three hours reaches 95 to 98 percent, the byproduct D-7ACA is less than 0.8 percent and is superior to the industrial standard, the protein mutant of the invention can effectively produce the important intermediate of semi-synthetic cephalosporin drugs, has simple process, high yield and strong technical portability, can be put into production only in a common fermentation workshop, such as an amino acid and vitamin production workshop, does not need to purchase special equipment, is easy to popularize and apply, and has stronger practicability and popularization value.
The above description is only for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention, and any person skilled in the art can substitute or change the technical solution of the present invention and its conception within the scope of the present invention.
Sequence listing
<110> Nanjing Langen Biotech Ltd
<120> cephalosporin C acylase mutant and preparation and application thereof
<130>2017
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<170>SIPOSequenceListing 1.0
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<213> cephalosporin C acylase mutant (SEQ ID NO.1 Artificial sequence)
<400>1
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Arg Ser His
50 55 60
Gly Asp Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Ile Ser Pro
130 135 140
Glu Val Arg Gln Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Glu Val Tyr Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Val Ile Arg Val Ala Phe Asn Gln Lys Val Ala Tyr
290 295 300
Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu Gln
305 310 315 320
Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu Pro
325 330 335
Phe Glu Arg Arg Gln Ala Ser Tyr Arg Leu Arg Gly Gly Ala Asp Arg
340 345 350
Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly Asp
355 360 365
Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala Glu
370 375 380
Thr Asp Met Leu Glu Gln Thr Phe Asp Met Ile Thr Gly Ala Ser Thr
385 390 395 400
Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp His
405 410 415
Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val Arg
420 425 430
Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val Pro
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Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu Ala
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Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala Asn
465 470 475 480
Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp Cys
485 490 495
His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala Ser
500 505 510
Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr Leu
515 520 525
Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly Ile
530 535 540
Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala Trp
545 550 555 560
Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn Ala
565 570 575
Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu Glu
580 585 590
Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Val Ser Thr Pro
595 600 605
Tyr Gly Leu Arg Asp Pro Lys Ala Ala Val Asp Gln Leu Arg Ser Trp
610 615 620
Asp Glu Ala Leu Ser Glu Ala Leu Ser Val Ala Thr Gln Asn Leu Thr
625 630 635 640
Gly Arg Gly Trp Gly Glu Glu His Arg Pro Arg Phe Thr His Pro Leu
645 650 655
Ser Ala Gln Phe Pro Ala Trp Ala Ala Leu Leu Asn Pro Val Ser Arg
660 665 670
Pro Ile Gly Gly Asp Gly Asp Thr Val Leu Ala Asn Gly Leu Val Pro
675 680 685
Ser Ala Gly Pro Glu Ala Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe
690 695 700
Asp Val Gly Asn Trp Asp Asn Ser Arg Trp Val Val Phe His Gly Ala
705 710 715 720
Ser Gly His Pro Ala Ser Pro His Tyr Ala Asp Gln Asn Ala Pro Trp
725 730 735
Ser Asp Cys Ala Met Val Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala
740 745 750
Ala Glu Ala Val Thr Ser Gln Glu Leu Val Pro Ala
755 760
<210>2
<211>2298
<212>DNA
<213> Polynucleotide (SEQ ID NO.2 Artificial sequence)
<400>2
atgactatgg cggctaaaac tgatcgtgaa gcgctccaag cggcgctccc gccgctctcc 60
ggttctctct ctatcccggg tctctctgcg ccagtgcgtg ttcaacgtga tggttggggc 120
atcccgcaca tcaaggcttc tggcgaagcg gacgcgtatc gtgcgctggg ctttgtgcat 180
gcgcgttctc atggcgatca aatggaactc actcgtcgca aagctctggg tcgtgcggcg 240
gagtggctcg gtgcggaggc tgcggaagct gatatcctgg ttcgtcgtct gggcatggaa 300
aaggtgtgtc gccgcgactt tgaagctctc ggcgctgaag cgaaggatat gctgcgtgct 360
tacgttgcgg gtgttaacgc ttttctcgcg tctggtgcgc cactgccgat cgagtacggt 420
ctgatttccc cagaagttcg tcagtgggaa ccgtggcact ccattgcggt tatgcgccgt 480
ctcggcctgc tgatgggttc tgtttggttc aagctgtggc gtatgctggc tctcccagtg 540
gtgggtgctg ctaatgcgct caagctccgt tacgacgacg gtggccagga cctcctctgt 600
attccaccag gcgtggaagc ggaacgtctc gaggcggacc tcgctgcgct gcgtccagcg 660
gttgacgcgc tgctcaaggc gatgggtggc gacgcttccg acgcggctgg tggtggctct 720
aacaattggg cggttgctcc gggtcgtact gctaccggtc gcccgatcct ggctggcgac 780
ccacaccgtg ttttcgagat tccaggtatg tacgcgcaac atcacctggc ttgtgaccgt 840
ttcgaagttt acggcctcac tgtgccgggc gtgccagtta tccgcgtggc gttcaatcag 900
aaggttgcgt attgtgttac ccatgcgttc atggacatcc acgacctcta cctcgagcag 960
tttgctgaag atggccgcac tgcgcgcttc ggcaatgaat tcgaaccgtt tgaacgtcgt 1020
caggcgtcct atcgcctgcg cggcggtgct gatcgcgagt tcgacatcgt tgaaacccgt 1080
catggcccgg ttattgcggg cgatccactc gaaggtgcgg ctctcaccct ccgttccgtt 1140
cagttcgcgg agaccgacat gctcgaacag acctttgata tgattaccgg cgcgtctact 1200
gtggctcaac tgtacgatgc gacccgtggc tggggcctca ttgaccacaa tctcgttgct 1260
ggtgacgtgg cgggttctat cggccacctc gtgcgtgcgc gtgtgccgtc ccgtccacgt 1320
gaaaacggct ggctgccggt gccaggctgg tccggtgagc acgaatggcg tggttggatc 1380
ccacacgaag cgatgccacg tgtgattgac ccaccgggtg gtctcatcgt taccgctaac 1440
aaccgtgtgg ttgctgacga tcatccagac tacctgtgca ctgattgcca tccaccatat 1500
cgcgcggagc gtatcatgga acgcctcgtg gcttctccgg cgtttgcggt ggacgacgct 1560
gcggctatcc atgcggacac cctgtcccca catgttggtc tcctccgtgc tcgcctggag 1620
gcgctcggta tccagggttc cctgccagcg gaagaactgc gtcaaaccct catcgcgtgg 1680
gatggtcgta tggacgcggg ctcccaggcg gcgtctgcgt acaatgcgtt tcgtcgcgct 1740
ctgactcgtc tcgtgaccgc tcgttctggc ctggaacagg ctattgcgca tccattcgct 1800
gctgtgccac cagtgtccac cccgtatggc ctgcgtgatc cgaaagcggc tgtggatcag 1860
ctgcgttctt gggacgaggc tctgtccgag gcgctgtccg ttgctaccca gaacctgact 1920
ggtcgcggtt ggggcgagga gcatcgtccg cgctttactc acccgctgtc tgctcagttc 1980
ccagcgtggg ctgctctcct gaacccagtt tctcgtccga ttggcggtga cggcgatact 2040
gttctcgcta atggtctggt tccgtctgct ggcccagagg cgacttacgg tgctctctcc 2100
cgctatgtgt tcgatgttgg caactgggat aattcccgtt gggttgtttt ccacggtgcg 2160
tccggtcacc cggcttcccc gcattacgct gaccaaaacg ctccatggtc cgactgtgct 2220
atggtgccaa tgctgtactc ctgggatcgt attgctgctg aggcggttac ttctcaggaa 2280
ctcgttccgg cgtaataa 2298
Claims (9)
1. A cephalosporin C acylase mutant is characterized in that 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 6.4 times; the sequence of the cephalosporin C acylase mutant is SEQ ID NO. 1.
2. The cephalosporin C acylase mutant of claim 1, wherein the cephalosporin C acylase mutant of SEQ ID No.1 has higher substrate catalytic activity than the wild-type cephalosporin C acylase.
3. The cephalosporin C acylase mutant as claimed in claim 1, characterized in that the detection of the enzyme activity comprises the following steps:
step 11: defining enzyme activity as enzyme activity for hydrolyzing 1 mu mol CPC-Na per minute as a unit U, and detecting the enzyme activity by adopting a sodium hydroxide titration method;
step 12: preparing a phosphate buffer solution with the pH value of 8.0;
step 13: preparing a CPC-Na salt solution;
step 14: preheating a three-neck flask in 25 ℃ water bath in advance, sucking 2% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask, adding an appropriate volume of enzyme solution, uniformly mixing a reaction system by using magnetic stirring, controlling the reaction temperature to be 25 ℃, titrating with 0.1mol/L sodium hydroxide titration solution, keeping the pH of the reaction solution at 8.0, and simultaneously recording the consumption milliliter number of the sodium hydroxide titration solution within about 6-8 min of reaction;
step 15: calculating enzyme activity;
step 16: and (4) HPLC analysis.
4. A polynucleotide encoding a polypeptide which is recombined by a cephalosporin C acylase mutant of sequence SEQ ID No. 1.
5. A polynucleotide as claimed in claim 4 wherein the polynucleotide sequence is SEQ ID No. 2.
6. A method for producing 7-aminocephalosporanic acid, characterized in that 7-aminocephalosporanic acid is produced by a cephalosporin C acylase mutant having a sequence of SEQ ID No. 1.
7. The method for producing 7-aminocephalosporanic acid according to claim 6, wherein the steps of the method are as follows:
step 21: preparing a phosphate buffer solution with the pH value of 8.0;
step 22: preparing a CPC-Na salt solution;
step 23: placing the three-neck flask in a water bath at 25 ℃ for preheating in advance, and sucking 10% CPC-Na salt solution preheated to 25 ℃ into the three-neck flask;
sampling 500 μ l before adding enzyme solution, and immediately adding 500 μ l stop solution;
after uniform mixing, 12000g of the mixture is centrifuged at high speed for 10min, and then the supernatant is taken for storage;
adding an appropriate volume of enzyme solution, uniformly mixing the reaction system by magnetic stirring, controlling the reaction temperature to be 25 ℃, adjusting the pH value by using 0.1mol/L sodium hydroxide, and keeping the pH value of the reaction solution to be 8.0;
step 24: sampling 500 μ l after adding enzyme solution for 100min, 200min, 250min, 300min, respectively, adding 500 μ l stop solution, mixing, centrifuging at 12000g for 10min, and collecting supernatant for HPLC detection.
8. The method for producing 7-aminocephalosporanic acid according to claim 6, wherein the cephalosporin C acylase mutant recombinant expression protein is produced by the following steps:
step 31: selecting single colony of Escherichia coli containing SEQ ID NO.2 sequence, inoculating in 10ml of culture medium sterilized under high pressure, and culturing at 30 deg.C and 250rpm overnight;
step 32: inoculating 1L triangular flask into 100ml of autoclaved culture medium at an inoculation ratio of 1:100 the next day, and culturing at 30 ℃ until thallus OD 5-6;
step 33: immediately placing the triangular flask in a shaker at 25 ℃ and culturing for 1 hour at 250 rpm;
step 34: IPTG was added to a final concentration of 0.1mM and the culture was continued at 25 ℃ and 250rpm for 16 hours;
step 35: after the culture is finished, centrifuging the culture medium at 4 ℃ and 12000g for 20min to collect wet thalli;
step 36: washing the thallus precipitate twice with distilled water, collecting thallus, and storing at-70 deg.C;
step 37: a small amount of the cells were subjected to SDS-PAGE.
9. The method for producing 7-aminocephalosporanic acid according to claim 8, wherein the culture medium in step 32 comprises the following components per liter:
tryptone: 10g of a mixture;
yeast extract (B): 5g of the total weight of the mixture;
disodium hydrogen phosphate: 3.55 g;
potassium dihydrogen phosphate: 3.4 g;
ammonium chloride: 2.68 g;
sodium sulfate: 0.71 g;
magnesium sulfate heptahydrate: 0.493 g;
ferric chloride hexahydrate: 0.027 g;
glycerol: 5g of the total weight of the mixture;
glucose: 0.8 g;
kanamycin: 50 mg.
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