CN102925423B - Mutated cephalosporin C acylase - Google Patents
Mutated cephalosporin C acylase Download PDFInfo
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- CN102925423B CN102925423B CN201210466978.8A CN201210466978A CN102925423B CN 102925423 B CN102925423 B CN 102925423B CN 201210466978 A CN201210466978 A CN 201210466978A CN 102925423 B CN102925423 B CN 102925423B
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- cpcacy
- cephalosporin
- enzyme
- acrylase
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- HOKIDJSKDBPKTQ-GLXFQSAKSA-N cephalosporin C Chemical compound S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H](N)C(O)=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-N 0.000 title claims abstract description 125
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- HSHGZXNAXBPPDL-HZGVNTEJSA-N 7beta-aminocephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H]([NH3+])[C@@H]12 HSHGZXNAXBPPDL-HZGVNTEJSA-N 0.000 claims abstract description 26
- 238000012217 deletion Methods 0.000 claims abstract description 7
- 230000037430 deletion Effects 0.000 claims abstract description 7
- 230000008859 change Effects 0.000 claims description 28
- 241000588724 Escherichia coli Species 0.000 claims description 16
- 239000013604 expression vector Substances 0.000 claims description 13
- 230000006698 induction Effects 0.000 claims description 4
- 238000005215 recombination Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 150000001413 amino acids Chemical group 0.000 claims 4
- 102000004190 Enzymes Human genes 0.000 abstract description 41
- 108090000790 Enzymes Proteins 0.000 abstract description 40
- 230000035772 mutation Effects 0.000 abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 6
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- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 6
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- FSHURBQASBLAPO-WDSKDSINSA-N Ala-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](C)N FSHURBQASBLAPO-WDSKDSINSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IXUSDMGLUJZNFO-BXUZGUMPSA-N (7R)-7-(4-carboxybutanamido)cephalosporanic acid Chemical compound S1CC(COC(=O)C)=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CCCC(O)=O)[C@@H]12 IXUSDMGLUJZNFO-BXUZGUMPSA-N 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 108091028026 C-DNA Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000317803 Pseudoxya diminuta Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 101150114167 ampC gene Proteins 0.000 description 1
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C([O-])=O)(C)C)=CC=CC=C1 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
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- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
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- 229940049954 penicillin Drugs 0.000 description 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
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- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
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- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a product-resistant inhibitory mutated cephalosporin C acylase, a gene carrier and a transformant of the enzyme, and application of the enzyme in one-step enzymatic production of 7-aminocephalosporanic acid, belonging to the technical field of enzyme engineering and biotechnology industry. The amino acid sequence of the enzyme is obtained by conducting deletion mutation on the amino acid sequence of cephalosporin C acylase coded by the gene sequence SEQ ID NO:1 to obtain enzyme CPCAcy-D2 and CPCAcy-D4. The amino acid sequences are shown as SEQ ID NO:2 and SEQ ID NO:3, respectively. The invention also discloses the gene carrier, the transformant, and the application of the enzyme. The enzyme has high expressing activity, and also the tolerance of the product is improved significantly, so that CPC is catalyzed efficiently to produce 7-ACA.
Description
Technical field
The invention belongs to enzyme engineering and industrial biotechnology field, be specifically related to carrier, the transformant of a kind of resistance to product inhibition type sudden change cephalosporin C acrylase, this enzyme gene and produce the application in 7-amino-cephalosporanic acid at a step enzyme method.
Background technology
The cephalosporin of microorganisms producing (Cephalosporin C, CPC) acylase, can slough side chain generation 7-amino-cephalosporanic acid (7-aminocephalosporanic acid, 7-ACA) by efficient catalytic cephalosporin.Cephalosporins (Cephalosporins) is the same with penicillin, is gang's beta-lactam Broad spectrum antibiotics, by disturbing the destruction of synthesizing and accelerating cell walls of bacteria cell wall to play the effect of sterilization.Utilize that the cephalosporin C acrylase catalytic production 7-amino-cephalosporanic acid of microorganisms has that technique is simple, safety, efficient, pollute the advantages such as little, therefore become gradually the main method that 7-amino-cephalosporanic acid is produced.
The research emphasis of one step enzyme method production 7-amino-cephalosporanic acid is discovery and transformation and the transformation to cephalosporin C acrylase self structure of the bacterial strain of high yield cephalosporin C acrylase.Wherein, aspect the transformation research to CPC acylase, enzyme product inhibition problem low, that substrate conversion efficiency is low and serious alive is that restriction CPC acylase is produced the industrialized principal element of 7-ACA for catalysis CPC.
Aspect the transformation research of living at enzyme; there is scholar to suddenly change in 215,296 and 309 three sites to the CPC acylase that comes from P. diminuta N176; mutant has shown the CPC enzyme higher than GL-7-ACA (Pollegioni L alive; Lorenzi S; Rosini E; et al. Protein Science, 2005,14 (12): 3064 ~ 3076).Korea S scholar has improved enzyme to the activity of substrate CPC and specificity to the CPC acylase from Pseudomonas strain SE83 acy II by multipoint mutation (Val122Ala-Gly140Ser-Phe297Arg-Ile314Thr-Ile415Val-Ser710Cys), and its CPC enzyme work has improved 8～10 times of left and right (WO 2005/014821 A1) with respect to wild mushroom.Tsing-Hua University designs by codon and the synthetic high reactivity CPC acylase that obtained of gene, and pass through the sudden change of substrate ingress related amino acid residue (Ala675Gly), enzyme is lived and further improved 35% (Chinese invention patent, application number: 201110460235.5; Yu H.M. etc., Journal of Bioscience and Bioengineering, 2012,113,36 – 41).Tsing-Hua University is also by decomposing genes involved to composing type host strain E. coli JM105 (match Parkson company) and inducible expression type host strain E. coli JM109 (DE3) (Ding Guo company) substrate: β-lactamase gene ampC and acetyl esterase gene aes knock out; make substrate conversion efficiency significantly improve (Yu H.M. etc.; Journal ofBioscience and Bioengineering; 2012; 113,737 – 741).
Summary of the invention
The object of the present invention is to provide a kind of sudden change cephalosporin C acrylase and gene thereof.
The present invention also aims to provide expression vector and the transformant of said mutation cephalosporin C acrylase.
Object of the present invention is again to provide said mutation cephalosporin C acrylase and gene thereof, contains expression vector and the application of transformant in preparation 7-ACA of sudden change cephalosporin C acrylase gene.
A kind of sudden change cephalosporin (Cephalosporin C; CPC) acylase; it has aminoacid sequence shown in SEQID NO:2; it is to obtain by the 227th Ala of the cephalosporin C acrylase aminoacid sequence of genes encoding shown in SEQ ID NO:1 and the 228th Met are carried out to deletion mutantion, this sudden change cephalosporin C acrylase called after CPCAcy-D2.
Above-mentioned sudden change cephalosporin C acrylase; it has aminoacid sequence shown in SEQ ID NO:3; it is by the 212nd Ala in the aminoacid sequence of genes encoding shown in SEQ ID NO:1, the 213rd Asp, the 214th Leu and the 215th Ala, to carry out deletion mutantion to obtain, this sudden change cephalosporin C acrylase called after CPCAcy-D4.
The gene of coding said mutation cephalosporin C acrylase.
Expression vector containing said mutation cephalosporin C acrylase gene.
The expression vector of the gene of said mutation cephalosporin C acrylase is induction type or constitutive expression carrier, and inducible expression vector is pET or other serial inducible expression vector, as inducible expression vector pET28; Constitutive expression carrier is pMKC constitutive expression carrier or other efficient constitutive expression carrier of maltose binding protein MBP amalgamation and expression.
The transformant of said mutation cephalosporin C acrylase gene, transformant is the recombination bacillus coli of induction type or composing type.
The Host Strains of inducible expression vector is preferably E. coli BL21 (DE3) (Promega company) or JM109 (DE3) (Ding Guo company) or other suitable bacterial strain etc. as recipient bacterium.
The Host Strains of constitutive expression carrier is preferably E. coli TB1 (New England Biolab company) or E. coli JM105 (match Parkson company) or other suitable bacterial strain etc. as recipient bacterium.
The construction process of said mutation CPC acylase gene transformant be conventional Calcium Chloride Method or electroporation conversion method (Sambrook J etc. Molecular Cloning:A Laboratory manual. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press. 1989).
Said mutation CPC acylase, the expression vector that carries said mutation CPC acylase encoding gene or transformant are in the application of preparing on 7-amino-cephalosporanic acid.
First the suddenly change crude enzyme liquid of CPC acylase of the present invention adopts the saturated precipitation of ammonium sulfate to carry out preliminary purification, further adopts resin, silica gel or other being fixed of carrier, can be used for preparing 7-amino-cephalosporanic acid from CPC.
Beneficial effect of the present invention is: the product tolerance of the cephalosporin C acrylase of (1) the present invention improvement significantly promotes, in the 7-ACA of 6g/L concentration solution, enzyme retention rate alive is brought up to respectively 17.5%(CPCAcy-D2 from 7.5%) and 40%(CPCAcy-D4); (2) to improve cephalosporin C acrylase expression activity high in the present invention, i.e. the lifting of stability does not cause the decline of cephalosporin C acrylase activity.Adopt improvement cephalosporin C acrylase provided by the invention, can generate product 7-ACA by efficient catalytic substrate CPC, there is good prospects for commercial application.
Accompanying drawing explanation
Fig. 1 carries the recombinant plasmid pET28-CPCacy of improvement cephalosporin C acrylase gene
mschematic diagram.
The suddenly change protein electrophorese figure of cephalosporin C acrylase and protoenzyme of Fig. 2;
Be with 1 for CPCAcy-D4(D4); Be with 2 for CPCAcy-D2(D2); Be with 3 for protoenzyme contrast CPCacy (C); Be with 4 for protein molecular weight standard.
Fig. 3 improves the product tolerance comparison of cephalosporin C acrylase and protoenzyme
Embodiment
Embodiment 1
The transgenation of sudden change cephalosporin C acrylase CPCacy-D2
From the corresponding gene order of former CPC acylase SEQ ID NO:1 (the SEQ ID NO:4) described in Chinese invention patent ZL200810102219.7; use pUC18-acy(Chinese invention patent ZL 200810102219.7) as template, adopt polymerase chain reaction (PCR) method to build sudden change cephalosporin C acrylase CPCacy
m.According to step described in TaKaRa MutanBEST test kit, with a pair of Up1-Down1 primer (Up1:GGCGGTGATGCCAGCGACGCA; Down1:TCCAGCCGTTGATGCATTACTGAAA) plasmid pUC18-acy is oppositely increased, make it at α subunit C end place, lack these 2 amino-acid residues of 227-228 position Ala-Met.
The reaction system of PCR is: sterilized water 37.7 μ L, 10 * Pyrobest Buffer, 5 μ L; Every kind of dNTP concentration 2.5mM of dNTPs(), 4 μ L; Upstream and downstream primer (concentration 20 μ molL
-1), each 1 μ L; Plasmid template, 1 μ L; Pyrobest archaeal dna polymerase (5 U μ L
-1), 0.3 μ L; Cumulative volume, 50 μ L.
Reaction conditions is: 94 ℃, and 5 min; 94 ℃ of 0.5min, 60 ℃ of 0.5n, 72 ℃ of 5min, circulate 30 times; Last 72 ℃ of 15min.
First adopt method well known to those skilled in the art to carry out end connection; again ligation product is transformed to the competent cell (Ding Guo company) of Host Strains E. coli JM109 (DE3); (substratum consists of: 50ml/300ml shaking flask to adopt penbritin (Amp+) LB solid medium; peptone 10g/L; yeast powder 5g/L; sodium-chlor 10g/L; kantlex; 50mg/L; agar powder; 15g/L, pH 7.0) flat board selects positive colony, obtains the recombinant plasmid pUC18-CPCacy that contains sudden change cephalosporin C acrylase gene segment
m-D2.Submit to Nuo Sai genome company to carry out DNA sequencing recombinant plasmid; the sudden change cephalosporin C acrylase of sequencing result proof gained lacks 227-228 position Ala, Met residue at α subunit C end place; the aminoacid sequence of proteins encoded is SEQ ID NO:2, i.e. saltant type CPCAcy-D2.
Embodiment 2
Sudden change cephalosporin C acrylase CPCacy-D4 transgenation after improvement and transformant thereof build
Other condition is with embodiment 1, but PCR primer used changes Up2-Down2 into, and wherein, the gene order of Up2 is: GCATTACGTCCAGCCGTTGATGCAT; The gene order of Down2 is: AGGCGTGGAAGCGGAGCGTCTGGAG.After pcr amplification, connection, obtain recombinant plasmid pUC18-CPCacy
m-D4, carry the sudden change CPC acylase gene of 212-215 position Ala-Asp-Leu-Ala disappearance.The aminoacid sequence of proteins encoded is SEQ ID NO:3, i.e. saltant type CPCAcy-D4.
Embodiment 3
Expression vector pET28-CPCacy
mand pMKC-CPCacy
mbuild and transformant structure
(1) plasmid pET28-CPCacy
m-D2structure: plasmid vector pUC18-acy that embodiment 1 is obtained
m-D2and pET28a (Novagen company) adopts respectively BamHI/HindIII double digestion.Endonuclease reaction volume 50 μ L, plasmid consumption 17 μ L, each enzyme dosage 1 μ L, damping fluid 5 μ L, adopt sterilized water to complement to 50 μ L.37 ℃ of reactions are spent the night, and obtain the linear plasmid skeleton that enzyme is cut product C PC acylase gene and pET28a.PCR product reclaims test kit (TIANGEN Biotech (Beijing) Co., Ltd.) purifying enzyme and cuts after product; the linear plasmid skeleton of CPC acylase gene and pET28a is carried out to ligation with T4 DNA ligase at 4 ℃ with the concentration ratio of 3:1, and the ligation time is 14 h to 16h.Ligation reaction is transformed to Host Strains E. coli DH5 α competent cell, coating LB dull and stereotyped (kalamycin resistance, Kan
+), select positive colony, carry out shake-flask culture 12h.Harvested cell, adopts conventional test kit to extract in a small amount plasmid and carries out that conventional enzyme is cut and electrophoresis checking, obtains recombinant plasmid pET28-CPCacy
m-D2.
(2) transformant E.coli JM109 (DE3)/pET28-CPCacy
m-D2with E. coli BL21 (DE3)/pET28-CPCacy
m-D2structure: by plasmid pET28-CPCacy
m-D2adopt respectively conventional electroporation conversion method (electroporation: Bole company, voltage 1250V) transform host e. coli E.coli JM109 (DE3) and E. coli BL21 (DE3), cell after conversion adds the SOC liquid nutrient medium (formula is shown in < < molecular cloning guide > >) of 800 μ l, be placed in 37 ℃, 220rpm shaking table is cultivated 30min activation.Get 50 μ l bacterium liquid and coat that to contain that antibiotic LB solid medium of 50 μ g/ml cards dull and stereotyped, put into 37 ℃ of incubators and cultivate 20 hours, obtain respectively gene recombination bacterium JM109 (DE3)/pET28-CPCacy
m-D2and BL21 (DE3)/pET28-CPCacy
m-D2.
(3) plasmid pET28-CPCacy
m-D4structure and transformant thereof build: plasmid vector pUC18-acy that embodiment 2 is obtained
m-D4and pET28a (Novagen company) adopts respectively method as shown in (1), (2) in embodiment 3, acquisition recombinant plasmid pET28-CPCacy
m-D4and transformant JM109 (DE3)/pET28-CPCacy
m-D4and BL21 (DE3)/pET28-CPCacy
m-D4.
Carry the recombinant plasmid pET28-CPCacy of sudden change cephalosporin C acrylase gene
mas shown in Figure 1; Wherein, M represents Ala-Met deletion mutantion or the Ala-Asp-Leu-Ala deletion mutantion of 212-215 position of 227-228 position.
(4) plasmid pMKC-CPCacy
m-D2and pMKC-CPCacy
m-D4structure and transformant thereof build: plasmid pMKC-CPCacy
m-D2and pMKC-CPCacy
m-D4construction process with embodiment 3(1), adopt BamHI/HindIII double digestion, obtain the full gene of sudden change CPC acylase (CPCAcy-D2 or CPCAcy-D4).Adopt simultaneously same procedure digested plasmid carrier pMKC-Acy (Yu HM etc. Journal of Molecular Biocatalysis B:Enzymatic, 2006,43,118-123), obtain the linear plasmid skeleton of pMKC.After purifying with PCR product recovery test kit, the linear plasmid skeleton of CPCAcy-D2 or CPCAcy-D4 gene and pMKC is adopted to T4 at 4 ℃
dNA ligase carries out ligation 14h, builds constitutive expression recombinant plasmid pMKC-CPCacy
m-D2and pMKC-CPCacy
m-D4.Method for transformation is with embodiment 3(2), adopt electroporation conversion method by pMKC-CPCacy
m-D2and pMKC-CPCacy
m-D4transform respectively intestinal bacteria E.coli JM105(TIANGEN Biotech (Beijing) Co., Ltd.), obtain transformant JM 105/pMKC-CPCacy
m-D2and JM 105/pMKC-CPCacy
m-D4.
Embodiment 4
Sudden change cephalosporin C acrylase CPCacy-D2 and the expression of CPCacy-D4 in transformant
The present invention that embodiment 3 is obtained improves inducible expression bacterial strain E.coli JM109 (DE3)/pET28-CPCacy of cephalosporin C acrylase CPCacy-D2 and CPCacy-D4
m-D2and JM109 (DE3)/pET28-CPCacy
m-D4carry out shake-flask culture, and take do not suddenly change CPC acylase recombinant bacterium E.coli JM109 (DE3)/pET28-CPCacy as contrast.First containing (substratum consists of: 10ml/50ml shaking flask, peptone 10g/L, yeast powder 5g/L, sodium-chlor 10g/L, pH 7.0) in the LB substratum of 50 mg/L kantlex, inoculating respectively single bacterium colony, 37 ℃, 200 rpm are cultivated 12h, make kind of a bottle.
From kind of bottle, according to 5% inoculum size, be transferred to that in the fermention medium containing 50mg/L kantlex, (50ml/300ml shaking flask, substratum is: corn steep liquor 50 g/L, yeast extract paste 10 g/L, NH
4cl 2.5 g/L, glycerine 5.0 g/L, KH
2pO
42.3 g/L, K
2hPO
416.4 g/L, lactose 3g/L, pH 7.5), under 28 ℃, 200 rpm conditions, shake-flask culture is 24 hours.
Take CPC as substrate, adopt the enzyme of paradimethy laminobenzaldehyde (PDAB) determination of color CPC acylase to live.First prepare crude enzyme liquid: 5ml bacterium liquid is at 4 ℃, centrifugal 10 min of 10000 rpm, and gained precipitates with being resuspended in 20ml damping fluid (thalline OD after 0.1M PBS damping fluid (pH 8.5) washed twice
600be about 1.0), the broken 10 min(320 W of ice-bath ultrasonic, 7 * 3 * 60 times); At 4 ℃, 12000 rpm are centrifugal, and 5 min obtain clear enzyme solution; 20 μ l substrate (0.1M PBS damping fluids; pH 8.5; prepare 20 mg/ml CPC solution), 20 μ l enzymes, after 37 ℃ of reaction 5 min; add 200 μ l stop buffers (20% acetic acid and 0.05 mol/L NaOH solution 2:1 volume mixture); the centrifugal 3min of 12000rpm, gets 200 μ l supernatant liquors and adds 40 μ l developers (being that 5:95 is made into by PDAB and methanol quality volume ratio), reaction 10min; measure the absorbancy of 415nm, measure the enzyme of CPC acylase and live.Enzyme unit definition alive is that per minute catalysis generates the required enzyme amount of 1 μ mol 7-ACA.
Enzyme activity determination result shows, recombinant bacterium E.coli JM109 (DE3)/pET28-CPCacy, E.coli JM109 (DE3)/pET28-CPCacy
m-D2and JM109 (DE3)/pET28-CPCacy
m-D4the protoenzyme CPCacy expressing and the enzyme work of mutant enzyme CPCacy-D2, CPCacy-D4 are respectively 3219,3380 and 3350 U/L.Collect thalline; after ultrasonication, get supernatant liquor and carry out the expression that conventional sodium laurylsulfonate-polyacrylamide gel electrophoresis of protein (SDS-PAGE) detects enzyme; result shows, the present invention enters to compile CPC acylase and protoenzyme has all obtained a large amount of activated proteins, as shown in Figure 2.
Similarly, to recombinant bacterium E.coli BL21 (DE3)/pET28-CPCacy, BL21 (DE3) (DE3)/pET28-CPCacy
m-D2and BL21 (DE3)/pET28-CPCacy
m-D4carried out shake-flask culture.Result shows, the enzyme further raising approximately 40% alive of protoenzyme CPCacy and mutant enzyme CPCacy-D2, CPCacy-D4.
Equally, to constitutive expression bacterium JM105/pMKC-CPCacy, JM105/pMKC-CPCacy
m-D2with JM105/ pMKC-CPCacy
m-D4carry out parallelly cultivate, plant bottle and shake-flask culture base and cultural method the same, just in shake-flask culture process, do not add inductor lactose, directly shake-flask culture 24 hours under 28 ℃ of the same terms.Result shows, it is suitable with inducible expression that the improvement CPC acylase of constitutive expression and the enzyme of protoenzyme are lived, and the enzyme work that improves acylase CPCacy-D2, CPCacy-D4 improves respectively 5% and 3% a little more than protoenzyme CPCacy().
Embodiment 5
The product inhibition assessment of improvement cephalosporin C acrylase
50ml recombinant bacterium E.coli JM109 (DE3)/pET28-CPCacy that embodiment 4 is cultivated
m-D2and JM109 (DE3)/pET28-CPCacy
m-D4and contrast bacterium JM109 (DE3)/pET28-CPCacy fermented liquid centrifugal cell harvesting respectively, with isopyknic 0.1M phosphate buffered saline buffer PBS(pH 8.5) standby after resuspended, ordinary method ultrasonication.
Respectively get 20 μ l ultrasonication liquid and mix with equal-volume 20 mM CPC solution, add the 7-ACA solution soaking of 6g/L, place the 7-ACA amount of measuring again generation after 5 minutes for 37 ℃.Result shows, is adding under the 7-ACA product rejection condition of 6g/L, and the enzyme of protoenzyme CPCacy retention rate alive is only 7.5%, and the enzyme of modified form CPCAcy-D2 and CPCAcy-D4 retention rate alive brings up to respectively 17.5% and 40%, as shown in Figure 3.Improvement CPCAcy-D2 and the CPCAcy-D4 of constitutive expression have same result.
Embodiment 6
The present invention improves CPC acylated enzyme catalysis CPC and generates 7-ACA test
Induction type transformant JM109 (DE3)/pET28-CPCacy that embodiment 4 is obtained
m-D2and JM109 (DE3)/pET28-CPCacy
m-D4carry out shake-flask culture.Collect 50ml bacterium liquid, with low temperature ultrasonication after 0.1M PBS damping fluid (pH 8.5) washing, the crude enzyme liquid after cytoclasis is directly used in 3%CPC sodium salt to the catalyzed conversion of 7-ACA, shaking table oscillatory reaction 1h at 28 ℃.Sample 200 μ l, with PDAB development process, analyze, result shows, CPC acylase CPCAcy-D2 of the present invention and CPCAcy-D4 can successfully transform substrate CPC mono-step to generate 7-ACA, and production concentration is respectively 2.80 and 2.83g/L.Improvement CPCAcy-D2 and the CPCAcy-D4 of constitutive expression have same result.
Claims (10)
- One kind sudden change cephalosporin C acrylase; it is characterized in that; its aminoacid sequence is as shown in SEQ ID NO:2, and it is to obtain by the 227th Ala of the cephalosporin C acrylase aminoacid sequence of genes encoding shown in SEQ ID NO:1 and the 228th Met are carried out to deletion mutantion.
- 2. a kind of sudden change cephalosporin C acrylase according to claim 1; it is characterized in that; its aminoacid sequence is as shown in SEQ ID NO:3, and it is by the 212nd Ala in the aminoacid sequence of genes encoding shown in SEQ ID NO:1, the 213rd Asp, the 214th Leu and the 215th Ala, to carry out deletion mutantion to obtain.
- 3. the gene of the sudden change cephalosporin C acrylase of any one in coding claim 1-2.
- 4. the expression vector that contains the cephalosporin C acrylase gene that suddenlys change described in claim 3.
- 5. the transformant that contains expression vector described in claim 4.
- 6. transformant according to claim 5, is characterized in that, described transformant is the recombination bacillus coli of induction type or composing type.
- 7. the application of cephalosporin C acrylase in preparing 7-amino-cephalosporanic acid suddenlys change described in claim 1-2 any one.
- 8. the application in preparing 7-amino-cephalosporanic acid of the gene of cephalosporin C acrylase suddenlys change described in claim 3.
- 9. the application of expression vector in preparing 7-amino-cephalosporanic acid described in claim 4.
- 10. the application of transformant in preparing 7-amino-cephalosporanic acid described in claim 5-6 any one.
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| CN109072215B (en) * | 2017-03-15 | 2020-02-18 | 山西新宝源制药有限公司 | Cephalosporin C acylase mutant and application thereof |
| CN111172142B (en) * | 2020-02-14 | 2021-09-28 | 上海陶宇晟生物技术有限责任公司 | Cephalosporin C acylase mutant with high thermal stability |
| KR102405289B1 (en) | 2021-12-24 | 2022-06-07 | 아미코젠주식회사 | Polypeptide having cephalosporin c acylase activity and use thereof |
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