CN102925423A - Mutated cephalosporin C acylase - Google Patents

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

A mutant cephalosporin C acylase

technical field

The invention belongs to the field of enzyme engineering and industrial biotechnology, and in particular relates to a product-inhibition-resistant mutant cephalosporin C acylase, a carrier of the enzyme gene, a transformant and its use in one-step enzymatic production of 7-aminocephalosporanic acid Applications.

Background technique

Cephalosporin C (CPC) acylase produced by microorganisms can efficiently catalyze the removal of the side chain of cephalosporin C to generate 7-aminocephalosporanic acid (7-ACA). Cephalosporins, like penicillin, are a family of β-lactam broad-spectrum antibiotics, which play a bactericidal role by interfering with the synthesis of bacterial cell walls and accelerating the destruction of cell walls. The use of cephalosporin C acylase produced by microorganisms to catalyze the production of 7-aminocephalosporanic acid has the advantages of simple process, safety, high efficiency, and low pollution, so it has gradually become the main method for the production of 7-aminocephalosporanic acid.

The focus of one-step enzymatic production of 7-aminocephalosporanic acid is the discovery and transformation of high-yield cephalosporin C acylase strains and the transformation of the structure of cephalosporin C acylase itself. Among them, in the research on the transformation of CPC acylase, low enzyme activity, low substrate conversion rate and serious product inhibition are the main factors restricting the industrialization of CPC acylase to catalyze the production of 7-ACA from CPC.

In terms of enzyme activity modification research, some scholars mutated the CPC acylase derived from P. diminuta N176 at the 215th, 296th and 309th positions. Higher CPC enzyme activity (Pollegioni L, Lorenzi S, Rosini E, et al. Protein Science, 2005, 14(12): 3064~3076). Korean scholars have improved the activity and specificity of the CPC acylase from Pseudomonas strain SE83 acy II through multiple point mutations (Val122Ala-Gly140Ser-Phe297Arg-Ile314Thr-Ile415Val-Ser710Cys), and its CPC activity is higher than that of wild Bacteria increased by about 8 to 10 times (WO 2005/014821 A1). Tsinghua University has obtained highly active CPC acylase through codon design and gene synthesis, and through the mutation of the relevant amino acid residues at the entrance of the substrate (Ala675Gly), the enzyme activity has been further increased by 35% (Chinese invention patent, application number : 201110460235.5; Yu H.M. et al., Journal of Bioscience and Bioengineering, 2012, 113, 36–41). Tsinghua University also through the compositional host strain E. coli JM105 (Saibaisheng company) and the inducible expression host strain E. coli JM109 (DE3) (Dingguo company) substrate decomposition related genes: β-lactamase gene ampC Knockout of the acetyl esterase gene aes significantly increased the substrate conversion rate (Yu H.M. et al., Journal of Bioscience and Bioengineering, 2012, 113, 737–741).

Contents of the invention

The object of the present invention is to provide a mutant cephalosporin C acylase and its gene.

The object of the present invention is also to provide the expression vector and transformant of the mutant cephalosporin C acylase.

The object of the present invention is to provide the above-mentioned mutant cephalosporin C acylase and its gene, the expression vector containing the mutant cephalosporin C acylase gene and the application of the transformant in the preparation of 7-ACA.

A mutant cephalosporin C (Cephalosporin C, CPC) acylase, which has the amino acid sequence shown in SEQ ID NO: 2, which is the cephalosporin C acylase amino acid encoded by the gene shown in SEQ ID NO: 1 The 227th Ala and the 228th Met of the sequence were deleted and mutated, and the mutant cephalosporin C acylase was named CPCAcy-D2.

The above-mentioned mutant cephalosporin C acylase has the amino acid sequence shown in SEQ ID NO: 3, which is obtained by the 212th Ala and the 213th Asp in the amino acid sequence encoded by the gene shown in SEQ ID NO: 1 , the 214th Leu and the 215th Ala were deleted and mutated, and the mutant cephalosporin C acylase was named CPCAcy-D4.

Gene encoding the mutant cephalosporin C acylase described above.

An expression vector containing the above mutant cephalosporin C acylase gene.

The expression vector of the gene of the above-mentioned mutant cephalosporin C acylase is an inducible or constitutive expression vector, and the inducible expression vector is pET or other series of inducible expression vectors, such as the inducible expression vector pET28; the constitutive expression vector is The pMKC constitutive expression vector or other high-efficiency constitutive expression vectors for fusion expression of maltose binding protein MBP.

The transformant of the mutant cephalosporin C acylase gene is an inducible or constitutive recombinant Escherichia coli.

The host bacterium of the inducible expression vector is preferably E. coli BL21 (DE3) (Promega Company) or JM109 (DE3) (Dingguo Company) or other suitable bacterial strains etc. as the recipient bacterium.

The host bacterium of the constitutive expression vector is preferably E. coli TB1 (New England Biolab Company) or E. coli JM105 (Saibaisheng Company) or other suitable bacterial strains etc. as the recipient bacterium.

The construction method of the above mutant CPC acylase gene transformant is conventional calcium chloride method or electroporation transformation method (Sambrook J et al. Molecular Cloning: A Laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 1989) .

Application of the mutant CPC acylase, the expression vector carrying the gene encoding the mutant CPC acylase or the transformant in the preparation of 7-aminocephalosporanic acid.

The crude enzyme solution of the mutated CPC acylase of the present invention is initially purified by ammonium sulfate saturated precipitation, and further immobilized by resin, silica gel or other carriers, so that it can be used to prepare 7-aminocephalosporanic acid from CPC.

The beneficial effects of the present invention are: (1) The product tolerance of the improved cephalosporin C acylase of the present invention is significantly improved, and in the 7-ACA solution with a concentration of 6g/L, the enzyme activity retention rate is increased from 7.5% respectively to 17.5% (CPCAcy-D2) and 40% (CPCAcy-D4); (2) The improved cephalosporin C acylase expression activity of the present invention is high, that is, the improvement of stability does not lead to cephalosporin C acylase activity decline. The improved cephalosporin C acylase provided by the invention can efficiently catalyze the substrate CPC to generate the product 7-ACA, and has good industrial application prospect.

Description of drawings

Figure 1 Schematic diagram of the recombinant plasmid pET28-CPCacy ^M carrying the improved cephalosporin C acylase gene.

Fig. 2 Protein electrophoresis pattern of mutant cephalosporin C acylase and proenzyme;

Lane 1 is CPCAcy-D4 (D4); Lane 2 is CPCAcy-D2 (D2); Lane 3 is the proenzyme control CPCacy (C); Lane 4 is the protein molecular weight standard.

Figure 3 Comparison of product tolerance between improved cephalosporin C acylase and original enzyme

Detailed ways

Example 1

Gene mutation of mutant cephalosporin C acylase CPCacy-D2

Starting from the gene sequence corresponding to the original CPC acylase SEQ ID NO: 1 (SEQ ID NO: 4 described in Chinese invention patent ZL200810102219.7), using pUC18-acy (Chinese invention patent ZL 200810102219.7) as a template, using Mutant cephalosporin C acylase CPCacy ^M was constructed by polymerase chain reaction (PCR). According to the steps described in the TaKaRa MutanBEST kit, a pair of Up1-Down1 primers (Up1: GGCGGTGATGCCAGCGACGCA; Down1: TCCAGCCGTTGATGCATTACTGAAA) were used to reversely amplify the plasmid pUC18-acy to delete 227-228 at the C-terminus of the α subunit The two amino acid residues of Ala-Met.

The PCR reaction system is: sterile water 37.7 μL, 10 × Pyrobest Buffer, 5 μL; dNTPs (each dNTP concentration 2.5 mM), 4 μL; upstream and downstream primers (concentration 20 μmolL ^-1 ), each 1 μL; plasmid template, 1 μL; Pyrobest DNA polymerase (5 UμL ^-1 ), 0.3 μL; total volume, 50 μL.

The reaction conditions are: 94°C, 5 min; 94°C 0.5min, 60°C 0.5n, 72°C 5min, cycle 30 times; finally 72°C 15min.

Firstly, the method well known to those skilled in the art is used for terminal ligation, and then the ligation reaction product is transformed into competent cells of the host bacterium E. coli JM109 (DE3) (Dingguo Company), and ampicillin (Amp+) LB solid medium (cultivation The basic composition is: 50ml/300ml shake flask, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, kanamycin, 50mg/L, agar powder, 15g/L, pH 7.0) plate selection positive Cloning, the recombinant plasmid pUC18-CPCacy ^M-D2 containing the mutant cephalosporin C acylase gene fragment was obtained. The recombinant plasmid was submitted to Nuosai Genetics Co., Ltd. for DNA sequencing, and the sequencing results proved that the obtained mutant cephalosporin C acylase lacked 227-228 Ala and Met residues at the C-terminus of the α subunit, and the amino acid sequence of the encoded protein was SEQ ID NO: 2, ie mutant CPCAcy-D2.

Example 2

Mutation of the improved mutant cephalosporin C acylase CPCacy-D4 gene and construction of its transformant

Other conditions are the same as in Example 1, but the PCR primers used are changed to Up2-Down2, wherein the gene sequence of Up2 is: GCATTACGTCCAGCCGTTGATGCAT; the gene sequence of Down2 is: AGGCGTGGAAGCGGAGCGTCTGGAG. After PCR amplification and ligation, the recombinant plasmid pUC18-CPCacy ^M-D4 was obtained, which carried the mutant CPC acylase gene with Ala-Asp-Leu-Ala deletion at positions 212-215. The amino acid sequence of the encoded protein is SEQ ID NO: 3, which is the mutant CPCAcy-D4.

Example 3

Expression vector pET28-CPCacy ^M and pMKC-CPCacy ^M construction and transformant construction

(1) Construction of plasmid pET28-CPCacy ^M-D2 : The plasmid vectors pUC18-acy ^M-D2 and pET28a (Novagen) obtained in Example 1 were digested with BamHI/HindIII double enzymes respectively. The volume of enzyme digestion reaction is 50 μL, the amount of plasmid is 17 μL, the amount of each enzyme is 1 μL, buffer solution is 5 μL, and it is made up to 50 μL with sterile water. React overnight at 37°C to obtain the restriction product CPC acylase gene and the linear plasmid backbone of pET28a. After the PCR product recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to purify the digested product, the CPC acylase gene and the linearized plasmid backbone of pET28a were digested with T4 DNA ligase at a concentration ratio of 3:1 at 4°C. Ligation reaction, the connection reaction time is 14 h to 16 h. The ligation reaction was transformed into competent cells of the host strain E. coli DH5α, spread on LB plates (kanamycin resistance, Kan ⁺ ), selected positive clones, and cultured in shake flasks for 12 hours. The cells were harvested, and a small amount of plasmids were extracted using conventional kits for routine enzyme digestion and electrophoresis verification to obtain the recombinant plasmid pET28-CPCacy ^M-D2 .

(2) Construction of transformants E.coli JM109(DE3)/pET28-CPCacy ^M-D2 and E.coli BL21(DE3)/pET28-CPCacy ^M-D2 : The plasmid pET28-CPCacy ^M-D2 was electroporated separately Transformation method (electrotransfer apparatus: Bio-Rad Company, voltage 1250V) to transform host E. coli JM109(DE3) and E. coli BL21(DE3), and add 800 μl of SOC liquid medium to the transformed cells (recipe see Molecular Cloning Guide 》), placed in 37℃, 220rpm shaker for 30min to activate. Take 50 μl of the bacterial liquid and spread it on the LB solid medium plate containing 50 μg/ml kana antibiotics, put it in a 37°C incubator and cultivate it for 20 hours, and obtain the genetically recombinant bacteria JM109(DE3)/pET28-CPCacy ^M-D2 and BL21 respectively (DE3)/pET28-CPCacy ^M-D2 .

(3) Construction of plasmid pET28-CPCacy ^M-D4 and its transformant construction: The plasmid vectors pUC18-acy ^M-D4 and pET28a (Novagen) obtained in Example 2 were respectively used as in Example 3 (1), ( 2) According to the method shown, the recombinant plasmid pET28-CPCacy ^M-D4 and the transformants JM109(DE3)/pET28-CPCacy ^M-D4 and BL21(DE3)/pET28-CPCacy ^M-D4 were obtained.

The recombinant plasmid pET28-CPCacy ^M carrying the mutant cephalosporin C acylase gene is shown in Figure 1; where, M represents the Ala-Met deletion mutation at 227-228 or the Ala-Asp-Leu-Ala deletion at 212-215 mutation.

(4) Construction of plasmids pMKC-CPCacy ^M-D2 and pMKC-CPCacy ^M-D4 and construction of transformants: The construction methods of plasmids pMKC-CPCacy ^M-D2 and pMKC-CPCacy ^M-D4 are the same as in Example 3 (1), The whole gene of mutant CPC acylase (CPCAcy-D2 or CPCAcy-D4) was obtained by double digestion with BamHI/HindIII. At the same time, the plasmid vector pMKC-Acy (Yu HM et al. Journal of Molecular Biocatalysis B: Enzymatic, 2006, 43, 118-123) was digested with the same method to obtain the linear plasmid backbone of pMKC. After purification with the PCR product recovery kit, the CPCAcy-D2 or CPCAcy-D4 gene and the linearized plasmid backbone of pMKC were incubated at 4°C in T4 DNA ligase was used for ligation reaction for 14 hours to construct constitutive expression recombinant plasmids pMKC-CPCacy ^M-D2 and pMKC-CPCacy ^M-D4 . The transformation method was the same as that in Example 3 (2). The electroporation transformation method was used to transform pMKC-CPCacy ^M-D2 and pMKC-CPCacy ^M-D4 into Escherichia coli E.coli JM105 (Tiangen Biochemical Technology (Beijing) Co., Ltd.), respectively, and obtained Transformants JM 105/pMKC-CPCacy ^M-D2 and JM 105/pMKC-CPCacy ^M-D4 .

Example 4

Expression of mutant cephalosporin C acylases CPCacy-D2 and CPCacy-D4 in transformants

The inducible expression strain E.coli JM109(DE3)/pET28-CPCacy ^M-D2 and JM109(DE3)/pET28- CPCacy ^M-D4 was cultured in shake flasks, and the non-mutated CPC acylase recombinant strain E.coli JM109(DE3)/pET28-CPCacy was used as a control. Firstly, in LB medium containing 50 mg/L kanamycin (medium composition: 10ml/50ml shake flask, peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, pH 7.0) respectively Inoculate a single colony, incubate at 37 °C, 200 rpm for 12 hours, and make a seed bottle.

According to the 5% inoculation amount, transfer from the seed bottle to the fermentation medium containing 50mg/L kanamycin (50ml/300ml shaking flask, the medium is: corn steep liquor 50 g/L, yeast extract 10 g/L, NH ₄ Cl 2.5 g/L, glycerin 5.0 g/L, KH ₂ PO ₄ 2.3 g/L, K ₂ HPO ₄ 16.4 g/L, lactose 3 g/L, pH 7.5), shake the flask at 28°C and 200 rpm Incubate for 24 hours.

With CPC as substrate, the enzyme activity of CPC acylase was determined by p-dimethylaminobenzaldehyde (PDAB) chromogenic method. First prepare the crude enzyme solution: 5ml of the bacterial solution was centrifuged at 4°C and 10,000 rpm for 10 minutes, the resulting pellet was washed twice with 0.1M PBS buffer (pH 8.5) and then resuspended in 20ml of buffer (the OD ₆₀₀ of the bacteria was about 1.0) , sonicated in an ice bath for 10 min (320 W, 7×3×60 times); at 4°C, centrifuged at 12,000 rpm for 5 min to obtain the supernatant enzyme solution; 20 μl substrate (0.1M PBS buffer, pH 8.5, prepared 20 mg/ml CPC solution), 20 μl enzyme, react at 37°C for 5 min, add 200 μl stop solution (20% acetic acid mixed with 0.05 mol/L NaOH solution 2:1 volume), centrifuge at 12000 rpm for 3 min, take 200 μl supernatant and add 40μl of chromogenic reagent (made according to the mass volume ratio of PDAB and methanol is 5:95), reacted for 10min, measured the absorbance at 415nm, and measured the enzyme activity of CPC acylase. The enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol 7-ACA per minute.

The results of enzyme activity assay showed that the proenzymes expressed by recombinant bacteria E.coli JM109(DE3)/pET28-CPCacy, E.coli JM109(DE3)/pET28-CPCacy ^M-D2 and JM109(DE3)/pET28-CPCacy ^M-D4 The enzyme activities of CPCacy and mutant enzymes CPCacy-D2 and CPCacy-D4 were 3219, 3380 and 3350 U/L, respectively. The bacteria were collected, and supernatant was taken after ultrasonic crushing for conventional sodium dodecylsulfonate-polyacrylamide protein gel electrophoresis (SDS-PAGE) to detect the expression of the enzyme. The results showed that the invention included CPC acylase and The original enzymes all obtained a large amount of active protein, as shown in Figure 2.

Similarly, shake flask cultures were performed on recombinant strains E.coli BL21(DE3)/pET28-CPCacy, BL21(DE3)(DE3)/pET28-CPCacy ^M-D2 and BL21(DE3)/pET28-CPCacy ^M-D4 . The results showed that the enzyme activities of the original enzyme CPCacy and the mutant enzymes CPCacy-D2 and CPCacy-D4 were further increased by about 40%.

Similarly, the constitutive expression bacteria JM105/pMKC-CPCacy, JM105/pMKC-CPCacy ^M-D2 and JM105/pMKC-CPCacy ^M-D4 were cultured in parallel. The inducer lactose was not added during the flask culture process, and the shake flask culture was directly carried out at 28°C for 24 hours under the same conditions. The results showed that the enzyme activities of the improved CPC acylase and the original enzyme with constitutive expression were equivalent to those of the inducible expression, and the enzyme activities of the improved CPCacy-D2 and CPCacy-D4 were slightly higher than those of the original enzyme CPCacy (increased by 5% respectively and 3%).

Example 5

Product Inhibition Evaluation of Improved Cephalosporin C Acylase

The 50ml recombinant bacteria E.coli JM109(DE3)/pET28-CPCacy ^M-D2 and JM109(DE3)/pET28-CPCacy ^M-D4 and the control bacteria JM109(DE3)/pET28-CPCacy fermentation broth cultivated in Example 4 were centrifuged respectively Cells were harvested, resuspended with an equal volume of 0.1M phosphate buffered saline PBS (pH 8.5), ultrasonically disrupted by conventional methods, and then used for later use.

Take 20 μl of sonication solution and mix it with an equal volume of 20 mM CPC solution, add 6g/L 7-ACA solution for soaking, and measure the amount of 7-ACA generated after standing at 37°C for 5 minutes. The results showed that under the inhibitory condition of adding 6g/L 7-ACA products, the enzyme activity retention rate of the original enzyme CPCacy was only 7.5%, while the enzyme activity retention rates of the improved CPCAcy-D2 and CPCAcy-D4 increased to 17.5% respectively and 40%, as shown in Figure 3. Constitutively expressed modified CPCAcy-D2 and CPCAcy-D4 had the same results.

Example 6

The improved CPC acylase catalyzed by the present invention generates 7-ACA test from CPC

The inducible transformants JM109(DE3)/pET28-CPCacy ^M-D2 and JM109(DE3)/pET28-CPCacy ^M-D4 obtained in Example 4 were cultured in shake flasks. Collect 50ml of bacterial liquid, wash with 0.1M PBS buffer solution (pH 8.5) and then sonicate at low temperature. The crude enzyme solution after cell crushing is directly used for the catalytic conversion of 3% CPC sodium salt to 7-ACA, shaking at 28°C Reaction 1h. 200 μl was sampled and analyzed by the PDAB chromogenic method. The results showed that the CPC acylases CPCAcy-D2 and CPCAcy-D4 of the present invention can successfully convert the substrate CPC into 7-ACA in one step, and the product concentrations are 2.80 and 2.83 g/L respectively. . Constitutively expressed modified CPCAcy-D2 and CPCAcy-D4 had the same results.

Claims (7)

1. A mutant cephalosporin C acylase, characterized in that it has the amino acid sequence shown in SEQ ID NO: 2, and it is by the cephalosporin C acylase encoded by the gene shown in SEQ ID NO: 1 The 227th Ala and the 228th Met of the amino acid sequence are obtained by deletion mutation. 2. a kind of mutant cephalosporin C acylase according to claim 1, is characterized in that, it has the aminoacid sequence shown in SEQ ID NO:3, and it is by the gene coding shown in SEQ ID NO:1 The 212th Ala, the 213th Asp, the 214th Leu and the 215th Ala in the amino acid sequence are obtained by performing deletion mutations. 3. A gene encoding a mutant cephalosporin C acylase according to any one of claims 1-2. 4. An expression vector containing the mutant cephalosporin C acylase gene according to claim 3. 5. The transformant of the mutant cephalosporin C acylase gene according to claim 3. 6. The transformant according to claim 5, characterized in that, the transformant is an inducible or constitutive recombinant Escherichia coli. 7. The application of any one of claims 1-6 in the preparation of 7-aminocephalosporanic acid.

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