A gene encoding penicillin G acylase and the use of the expressed mutated penicillin G acylase in the preparation of cephalexin

Abstract

The invention provides a gene encoding penicillin G acylase and an application of the expressed mutated penicillin G acylase in preparing cephalexin, and relates to the technical fields of gene engineering and enzyme engineering. The nucleotide sequence of the gene encoding penicillin G acylase is shown in SEQ ID NO.1, and the gene encoding penicillin G acylase is expressed to obtain the mutated penicillin G acylase; the mutated penicillin G acylase obtained by expression has high enzyme activity, can further promote the forward progress of the cephalexin synthesis reaction, reduce the hydrolysis (H) function of the expression host strain product, improve the synthesis (S) function of the product, obtain the S / H ratio high expression host strain, compared with the old expression host strain, the S / H ratio is improved by 5 times, the generation amount of hydrolysis byproducts is reduced, the generation amount of the main product cephalexin is increased, the molar ratio of the two reactants is reduced, not only the product quality is improved but also the enterprise cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering and enzyme engineering, and in particular to a gene encoding penicillin G acylase and the application of the expressed mutated penicillin G acylase in the preparation of cephalexin. Background Art

[0002] After years of development, cephalexin (cephalexin, cephalosporin IV) has become a major variety of cephalosporin antibiotics and the most consumed cephalosporin antibiotic in the world.

[0003] At present, cephalosporin antibiotics are the most widely used β-lactam antibiotics, accounting for 40% of the global antibiotic market. Among them, cephalexin is a major variety of cephalosporin antibiotics. It achieves bactericidal effect by inhibiting the synthesis of cell wall. It is a semi-synthetic cephalosporin with large clinical use. Cephalexin is a first-generation cephalosporin oral antibiotic. It was used in clinical practice in the 1960s and is currently the most widely used and most common type of antibiotic. It is suitable for acute tonsillitis, pharyngitis, otitis media, sinusitis, bronchitis, pneumonia and other respiratory tract infections, urinary tract infections and skin and soft tissue infections caused by sensitive bacteria. Its antibacterial spectrum is similar to that of cephalothin, but its antibacterial activity is poorer than that of the latter. Except for Enterococcus and methicillin-resistant Staphylococcus, most strains of Streptococcus pneumoniae, hemolytic Streptococcus, and Staphylococcus aureus that produces or does not produce penicillinase are sensitive to cephalexin. Cephalexin has a good antibacterial effect on Neisseria, but a poor effect on Haemophilus influenzae. It has a certain antibacterial effect on some Escherichia coli, Proteus mirabilis, Salmonella and Shigella. Other Enterobacteriaceae, Acinetobacter, Pseudomonas aeruginosa and Bacteroides fragilis are resistant to cephalexin, while Fusobacterium and Veillonella are generally sensitive, and anaerobic Gram-positive cocci are moderately sensitive.

[0004] There are two methods for the synthesis of cephalexin, namely chemical semi-synthesis (referred to as chemical method, including mixed anhydride method and acyl chloride method) and enzyme semi-synthesis (referred to as enzyme method). The traditional synthesis method of cephalexin is to obtain the side chain from benzaldehyde through chemical synthesis; obtain penicillin G from Penicillium fermentation, obtain the parent nucleus 7-ADCA through chemical ring expansion and cracking, and then condense the parent nucleus and side chain through chemical methods. At present, most of the companies producing cephalexin in the world still use this method. The whole production process requires about 10 chemical reactions, and the final three wastes produced per kilogram of product are as high as 30kg or more. The enzymatic synthesis technology of cephalexin began in the 1970s. Toyo Brewing Company of Japan used penicillin acylase (penicillin acylase, penicillin acyltransferase, penicillin amidase, penicillin acetylase) produced by Bacillus megaterium B-400 and Achromobacterium B-402 as acyltransferase (catalyst) to condense cephalexin, and began industrial research in 1973. The reaction can be carried out in aqueous solution, with very little organic solvent and mild reaction conditions, so the technology has received widespread attention. However, due to the poor performance of the enzyme at that time, the technical problem of separating the reaction product and the excess raw material from the reaction mixture has not been well solved. Therefore, the enzymatic synthesis technology has been in the research and trial production stage for many years, and domestic and foreign literature has introduced it in detail. my country has started research on biotransformation of β-lactam antibiotics since the 1980s and has achieved some results. However, since there has been no substantial breakthrough in the extraction, separation and immobilization technology of acylase and the separation and recovery technology of products and raw materials, it is difficult to form technical and economic advantages, and therefore it is difficult to apply it in production.

[0005] The traditional chemical method generates a large amount of three wastes due to the use of dichloromethane and other solvents, stoichiometric amounts of silanizing agents (or other carboxyl protecting agents, such as DBU, TMG, etc.), side chain protecting agents of Dunn's salts, and activators of acetylation reactions (such as pivaloyl chloride). The wastewater discharged during the enzymatic synthesis of cephalexin contains only some simple inorganic salts, which will not cause harm to the human living environment and is known as the "green route". Therefore, the improvement of the enzyme activity of the enzymatic synthesis of cephalexin and the optimization of the conversion process have become issues that need to be solved urgently. Summary of the invention

[0006] The object of the present invention is to provide a gene encoding penicillin G acylase, wherein the mutated penicillin G acylase expressed has high enzyme activity and can further promote the forward progress of the cephalexin synthesis reaction.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a gene encoding penicillin G acylase, the nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0009] Preferably, the gene is obtained by modifying the initial gene of penicillin G acylase, and the nucleotide sequence of the initial gene of penicillin G acylase is shown in SEQ ID NO.2.

[0010] The present invention also provides a mutated penicillin G acylase obtained by expressing the gene encoding penicillin G acylase.

[0011] The present invention also provides the use of the gene encoding penicillin G acylase or the mutated penicillin G acylase in the preparation of cephalexin.

[0012] The present invention also provides a method for preparing cephalexin using the gene encoding penicillin G acylase, comprising the following steps:

[0013] (1) constructing an expression vector carrying the gene encoding penicillin G acylase, and transforming it into Escherichia coli to obtain a recombinant expression strain and the penicillin G acylase expressed;

[0014] (2) Immobilization of mutated penicillin G acylase;

[0015] (3) The 7-amino-3-deacetoxycephalosporanic acid solution is mixed with immobilized penicillin G acylase at a temperature of 18 to 22° C. to obtain a mixed solution; then the D-phenylglycine methyl ester hydrochloride solution is added dropwise to the mixed solution to react, and the pH is maintained at 7.0 during the reaction process.

[0016] Preferably, the pH of the 7-amino-3-deacetoxycephalosporanic acid solution in step (3) is 7.0.

[0017] Preferably, in step (3), the mass ratio of the immobilized penicillin G acylase to the 7-amino-3-deacetoxycephalosporanic acid in the 7-amino-3-deacetoxycephalosporanic acid solution is 0.4-1.2:1.

[0018] Preferably, in step (3), the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.05-1.10:1.

[0019] Preferably, the dripping time in step (3) is 240 to 260 minutes, and the reaction temperature is 18 to 22°C.

[0020] Technical effects and advantages of the present invention:

[0021] The invention provides a gene encoding penicillin G acylase and an application of the expressed mutated penicillin G acylase in the preparation of cephalexin. The enzymatic synthesis process of cephalexin is based on 7-aminodeacetoxycephalosporanic acid (7-ADCA) as a parent nucleus, phenylglycine methyl ester as an acyl donor, and immobilized penicillin G acylase (PGA) as an acyltransferase, which is used as a catalyst to catalyze the synthesis of cephalexin. The enzymatic synthesis of cephalexin has the advantages of mild reaction conditions, simple process operation, no need for group protection, clean and safe, no pollution, etc. The enzymatic preparation of cephalosporins has great potential in industry, and the synthesis method and route of biological enzymes have certain social and economic benefits. The invention firstly performs gene site mutation on the gene sequence of penicillin G acylase. The hydrolysis (H) function of the expression host strain product is reduced, and the synthesis (S) function of the product is improved, so as to obtain a host strain with a high S / H ratio. Compared with the old expression host strain, the S / H ratio is increased by 5 times, the generation of hydrolysis by-products is reduced, the generation of the main product cephalexin is increased, and the molar ratio of the two reactants is reduced, which not only improves the product quality but also reduces the enterprise cost; secondly, the amount of enzyme added to the reaction, the feed ratio, the feed sequence, the reaction temperature, the pH value, the reaction time, etc. are optimized, so that the 7-ADCA conversion rate reaches more than 99.5%, the purity of the product cephalexin is improved and there is no need to recover the mother liquor, which greatly reduces the enterprise cost, improves the product quality, avoids the pollution caused by the chemical reaction, and greatly reduces the environmental protection pressure of the enterprise. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the liquid chromatogram of Example 1;

[0023] Figure 2 This is the liquid chromatogram of Example 2;

[0024] Figure 3 This is the liquid chromatogram of Comparative Example 1;

[0025] Figure 4 This is the liquid chromatogram of Comparative Example 2;

[0026] Figure 5 This is the liquid chromatogram of Comparative Example 3. DETAILED DESCRIPTION

[0027] The present invention provides a gene encoding penicillin G acylase, the nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0028]

[0029]

[0030] The present invention also provides a mutated penicillin G acylase obtained by expressing the gene encoding penicillin G acylase.

[0031] The present invention also provides the use of the gene encoding penicillin G acylase or the mutated penicillin G acylase in the preparation of cephalexin.

[0032] The present invention also provides a method for preparing cephalexin using the gene encoding penicillin G acylase, comprising the following steps:

[0033] (1) constructing an expression vector carrying the gene encoding penicillin G acylase, and transforming it into Escherichia coli to obtain a recombinant expression strain and express the mutated penicillin G acylase;

[0034] (2) Immobilization of mutated penicillin G acylase;

[0035] (3) The 7-amino-3-deacetoxycephalosporanic acid solution is mixed with the mutated immobilized penicillin G acylase at a temperature of 18 to 22° C. to obtain a mixed solution; then the D-phenylglycine methyl ester hydrochloride solution is added dropwise to the mixed solution to react, and the pH is maintained at 7.0 during the reaction process.

[0036] In the method for preparing cephalexin of the present invention, step (1) constructs an expression vector carrying the gene encoding penicillin G acylase, and transforms it into Escherichia coli to obtain a recombinant expression strain and express the resulting mutated penicillin G acylase; the nucleotide sequence of the gene encoding penicillin G acylase is preferably SEQ ID NO.1, and the blank expression vector is preferably a PET28a vector.

[0037] In the method for preparing cephalexin of the present invention, step (2) is to immobilize penicillin G acylase, centrifuge and concentrate the recombinant expression strain obtained in step (1) and the penicillin G acylase mutant obtained by expression, dilute the concentrate, homogenize the dilution, break the acylase cells and release the acylase into the supernatant; add a flocculant to precipitate the cell fragments of the acylase, and filter; add ammonium sulfate to the filtered clear solution for ammonium sulfate precipitation, dissolve the obtained precipitate, perform secondary ammonium sulfate precipitation, after the secondary ammonium sulfate precipitation is dissolved, pass the dissolved solution through a chromatography resin, perform secondary chromatography on the effluent, and immobilize, cross-link, wash and package the secondary chromatography effluent to obtain the immobilized enzyme to obtain the immobilized penicillin G acylase.

[0038] In the method for preparing cephalexin of the present invention, step (2) of immobilizing penicillin G acylase specifically comprises the following steps:

[0039] a. Centrifugal concentration: the recombinant expression strain obtained in step (1) and the penicillin G acylase mutant obtained by expression are adjusted to pH 8.0 and concentrated by centrifugation;

[0040] b. Homogenization: pressure 800-900 MPa, temperature 4-6°C, pH 7.0-7.5, homogenization treatment;

[0041] c. Filtration, ultrafiltration and concentration: Filtration ensures that the filtrate is clear, and the filtrate is ultrafiltrated and concentrated;

[0042] d. Primary ammonium sulfate precipitation: add 25% ammonium sulfate to the ultrafiltration concentrate, pH 7.2, temperature 8-10°C, time 2h, and stir, filter, and dissolve;

[0043] e. Secondary ammonium sulfate precipitation: add 25% ammonium sulfate to the primary ammonium sulfate precipitation solution, pH 7.2, temperature 8-10°C, time 2h, and stir, filter, and dissolve;

[0044] f. Primary chromatography: feed the dissolved filtrate into the chromatography tank for adsorption, the feed rate is 600-700 L / h, the feed pressure is 0.2-0.4 kg, the temperature is 4-10 ° C, the pH is 6-8, and the effluent is;

[0045] g. Secondary chromatography: the effluent from the first chromatography is continuously fed into the chromatography tank for adsorption, the feed rate is 600-700 L / h, the feed pressure is 0.2-0.4 kg, the temperature is 4-10°C, the pH is 6-8, and the enzyme activity is tested in the effluent;

[0046] h. Immobilization: Resin pretreatment: The resin dosage is 1500U, use 1g wet resin, add activation solution (0.02M phosphate buffer containing 2% glutaraldehyde, pH value is 8.0), use 2mL activation solution for each gram of wet resin, stir at 25Hz / h at room temperature, wash 3 times with 2 volumes of purified water; add enzyme solution and stir evenly, add immobilization solution (0.2M, pH is 8.0 phosphate buffer) to control the solution volume and resin weight ratio to 3:1, stir at 25Hz for 4h; then stir for 10min every 4h, fix for 40-48h and wash 3 times with 2 volumes of purified water; after washing twice with 2 volumes of storage buffer (1.0M, pH is 7.0 phosphate buffer), vacuum filter the immobilized enzyme solution until particles appear on the surface of the immobilized enzyme, stop filtering, and put it into 1 volume of storage buffer.

[0047] In the method for preparing cephalexin of the present invention, the 7-amino-3-deacetoxycephalosporanic acid solution in step (3) is mixed with the mutated immobilized penicillin G acylase at a temperature of 18 to 22° C. to obtain a mixed solution; the mass ratio of the mutated immobilized penicillin G acylase to the 7-amino-3-deacetoxycephalosporanic acid in the 7-amino-3-deacetoxycephalosporanic acid solution is preferably 0.4 to 1.2:1, more preferably 0.6 to 1.0:1; the pH of the 7-amino-3-deacetoxycephalosporanic acid solution is preferably 7.0; and the mixing temperature is 18 to 22° C., more preferably 19 to 21° C.

[0048] In the method for preparing cephalexin of the present invention, in step (3), a D-phenylglycine methyl ester hydrochloride solution is added dropwise to the mixed solution for reaction, and the pH value is maintained at 7.0 during the reaction process; the molar ratio of the D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is preferably 1.05-1.10:1, more preferably 1.06-1.09:1, and further preferably 1.07-1.08:1; the dropping time is preferably 240-260 min, more preferably 245-255 min, and further preferably 248-252 min; the reaction temperature is preferably 18-22° C., and further preferably 19-21° C.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] (1) using pET28a as a blank vector, constructing an expression vector carrying a gene encoding penicillin G acylase with a nucleotide sequence as shown in SEQ ID NO.1, and transforming it into Escherichia coli to obtain a recombinant expression Escherichia coli, which expresses the mutated penicillin G acylase;

[0052] (2) Immobilization of mutated penicillin G acylase;

[0053] (3) the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.10:1, the mutated immobilized penicillin G acylase and 7-amino-3-deacetoxycephalosporanic acid solution are in a mass ratio of 0.6, the reaction temperature is 20° C., D-phenylglycine methyl ester hydrochloride is added dropwise, and the immobilized penicillin G acylase obtained in step (2) is used;

[0054] Take a 200mL beaker, weigh 15g 7-ADCA, add 49.5mL pure water, start stirring, adjust the pH to 7.0 with 3M ammonia water, control the temperature at 20℃, and add 9g mutated immobilized penicillin G acylase; take another beaker and weigh 15.53g D-phenylglycine methyl ester hydrochloride, dissolve it in cold water, and keep it in an ice bath, then slowly add the side chain D-phenylglycine methyl ester hydrochloride dropwise to the mother core 7-ADCA solution, add dropwise for 4h, start the reaction, and use 3M ammonia water to maintain pH 7.0 during the reaction.

[0055] Sampling of the liquid phase began after 4 h of reaction to detect the highest point of 7-ADCA conversion;

[0056] Liquid phase conditions:

[0057] Column model: Inertsil ODS-sp;

[0058] Mobile phase preparation: Add 900 mL of purified water, 11.53 g of phosphoric acid, and 40 mL of acetonitrile to a 1 L beaker, adjust the pH to 3.0 with 15% potassium hydroxide solution, and make up to 1 L;

[0059] Wavelength: 210nm;

[0060] Temperature: 25℃;

[0061] Flow rate: 1mL / min; Results Figure 1 As shown, the reaction conversion rate reached the highest point of 99.74% in 4h10min.

[0062] Example 2

[0063] (1) using pET28a as a blank vector, constructing an expression vector carrying a gene encoding penicillin G acylase with a nucleotide sequence as shown in SEQ ID NO.1, and transforming it into Escherichia coli to obtain a recombinant expression Escherichia coli, which expresses the mutated penicillin G acylase;

[0064] (2) Immobilization of mutated penicillin G acylase;

[0065] (3) the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.05:1, the mutated immobilized penicillin G acylase and 7-amino-3-deacetoxycephalosporanic acid solution are in a mass ratio of 0.6, the reaction temperature is 20° C., D-phenylglycine methyl ester hydrochloride is added dropwise, and the mutated penicillin G acylase obtained in step (2) is used;

[0066] Take a 200mL beaker, weigh 15g 7-ADCA, add 49.5mL pure water, start stirring, adjust the pH to 7.0 with 3M ammonia water, control the temperature at 20℃, and add 9g immobilized penicillin G acylase; take another beaker and weigh 14.82g D-phenylglycine methyl ester hydrochloride to dissolve in cold water, and keep it in an ice bath, then slowly add the side chain D-phenylglycine methyl ester hydrochloride to the mother core 7-ADCA solution, add for 4h, start the reaction, and use 3M ammonia water to maintain pH 7.0 during the reaction.

[0067] Sampling of the liquid phase began after 4 h of reaction to detect the highest point of 7-ADCA conversion;

[0068] Liquid phase conditions:

[0069] Column model: InertsilODS-sp;

[0070] Mobile phase preparation: Add 900 mL of purified water, 11.53 g of phosphoric acid, and 40 mL of acetonitrile to a 1 L beaker, adjust the pH to 3.0 with 15% potassium hydroxide solution, and make up to 1 L;

[0071] Wavelength: 210nm;

[0072] Temperature: 25℃;

[0073] Flow rate: 1mL / min; Results Figure 2 As shown, the reaction conversion rate reached the highest point of 99.48% in 4h20min.

[0074] Comparative Example 1

[0075] (1) using pET28a as a blank vector, constructing an expression vector carrying the initial gene of penicillin G acylase with a nucleotide sequence as shown in SEQ ID NO.2, and transforming it into Escherichia coli to obtain recombinant expression Escherichia coli, thereby expressing penicillin G acylase;

[0076] (2) Immobilization of penicillin G acylase;

[0077] (3) the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.18:1, the mass ratio of immobilized penicillin G acylase to 7-amino-3-deacetoxycephalosporanic acid solution is 0.6:1, the reaction temperature is 20°C, the two substrates react simultaneously, and the immobilized penicillin G acylase adopts the immobilized penicillin G acylase obtained in (2);

[0078] Take a 200mL beaker, weigh 15g 7-ADCA, 16.66g D-phenylglycine methyl ester hydrochloride, add 99mL pure water, start stirring, adjust the pH to 7.0 with 3M ammonia water, control the temperature at 20℃, add 9g immobilized penicillin G acylase, start the reaction, and use 3M ammonia water to maintain pH 7.0 during the reaction.

[0079] Sampling of the liquid phase began after 3.5 h of reaction to detect the highest point of 7-ADCA conversion;

[0080] Liquid phase conditions:

[0081] Column model: InertsilODS-sp;

[0082] Mobile phase preparation: Add 900 mL of purified water, 11.53 g of phosphoric acid, and 40 mL of acetonitrile to a 1 L beaker, adjust the pH to 3.0 with 15% potassium hydroxide solution, and make up to 1 L;

[0083] Wavelength: 210nm;

[0084] Temperature: 25℃;

[0085] Flow rate: 1mL / min; Results Figure 3 As shown, the reaction conversion rate reached the highest point of 93% in 4 h.

[0086] Comparative Example 2

[0087] (1) using pET28a as a blank vector, constructing an expression vector carrying the initial gene of penicillin G acylase with a nucleotide sequence as shown in SEQ ID NO.2, and transforming it into Escherichia coli to obtain recombinant expression Escherichia coli, thereby expressing penicillin G acylase;

[0088] (2) Immobilization of penicillin G acylase;

[0089] (3) the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.18:1, the mass ratio of immobilized penicillin G acylase to 7-amino-3-deacetoxycephalosporanic acid solution is 0.6:1, the reaction temperature is 20°C, D-phenylglycine methyl ester hydrochloride is added dropwise, and the immobilized penicillin G acylase adopts the immobilized penicillin G acylase obtained in (2);

[0090] Take a 200mL beaker, weigh 15g 7-ADCA, add 49.5mL pure water, start stirring, adjust the pH to 7.0 with 3M ammonia water, control the temperature at 20℃, and add 9g immobilized penicillin G acylase; take another beaker and weigh 16.66g D-phenylglycine methyl ester hydrochloride to dissolve in cold water, and keep it in an ice bath, then slowly add the side chain D-phenylglycine methyl ester hydrochloride dropwise to the mother core 7-ADCA solution, add dropwise for 4h, start the reaction, and use 3M ammonia water to maintain pH 7.0 during the reaction.

[0091] Sampling of the liquid phase began after 4 h of reaction to detect the highest point of 7-ADCA conversion;

[0092] Liquid phase conditions:

[0093] Column model: InertsilODS-sp;

[0094] Mobile phase preparation: Add 900 mL of purified water, 11.53 g of phosphoric acid, and 40 mL of acetonitrile to a 1 L beaker, adjust the pH to 3.0 with 15% potassium hydroxide solution, and make up to 1 L;

[0095] Wavelength: 210nm;

[0096] Temperature: 25℃;

[0097] Flow rate: 1mL / min; Results Figure 4 As shown, the reaction conversion rate reached the highest point of 99.48% in 4h10min.

[0098] Comparative Example 3

[0099] (1) using pET28a as a blank vector, constructing an expression vector carrying the initial gene of penicillin G acylase with a nucleotide sequence as shown in SEQ ID NO.2, and transforming it into Escherichia coli to obtain recombinant expression Escherichia coli, thereby expressing penicillin G acylase;

[0100] (2) Immobilization of penicillin G acylase;

[0101] (3) the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.10, the mass ratio of immobilized penicillin G acylase to 7-amino-3-deacetoxycephalosporanic acid solution is 0.6, the reaction temperature is 20°C, D-phenylglycine methyl ester hydrochloride is added dropwise, and the immobilized penicillin G acylase adopts the immobilized penicillin G acylase obtained in (2);

[0102] Take a 200mL beaker, weigh 15g 7-ADCA, add 49.5mL pure water, start stirring, adjust the pH to 7.0 with 3M ammonia water, control the temperature at 20℃, and add 9g immobilized penicillin G acylase; take another beaker and weigh 15.53g D-phenylglycine methyl ester hydrochloride, dissolve it in cold water, and keep it in an ice bath, then slowly add the side chain D-phenylglycine methyl ester hydrochloride dropwise to the mother core 7-ADCA solution, add dropwise for 4h, start the reaction, and use 3M ammonia water to maintain pH 7.0 during the reaction.

[0103] Sampling of the liquid phase began after 4 h of reaction to detect the highest point of 7-ADCA conversion;

[0104] Liquid phase conditions:

[0105] Column model: InertsilODS-sp;

[0106] Mobile phase preparation: Add 900 mL of purified water, 11.53 g of phosphoric acid, and 40 mL of acetonitrile to a 1 L beaker, adjust the pH to 3.0 with 15% potassium hydroxide solution, and make up to 1 L;

[0107] Wavelength: 210nm;

[0108] Temperature: 25℃;

[0109] Flow rate: 1mL / min; Results Figure 5 As shown, the reaction conversion rate reached the highest point of 96.62% in 4h10min.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene encoding penicillin G acylase, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The gene encoding penicillin G acylase according to claim 1, characterized in that The gene is obtained by modifying the original gene of penicillin G acylase, and the nucleotide sequence of the original gene of penicillin G acylase is shown in SEQ ID NO.

2.

3. The mutated penicillin G acylase obtained by expressing the gene encoding penicillin G acylase according to claim 1.

4. Use of the gene encoding penicillin G acylase according to claim 1 or 2 or the mutated penicillin G acylase according to claim 3 in the preparation of cephalexin.

5. The method for preparing cephalexin using the gene encoding penicillin G acylase according to claim 1 or 2, characterized in that: The following steps are involved: (1) constructing an expression vector carrying the gene encoding penicillin G acylase according to claim 1, and transforming it into Escherichia coli to obtain a recombinant expression strain and express the resulting mutated penicillin G acylase; (2) Immobilization of mutated penicillin G acylase; (3) The 7-amino-3-deacetoxycephalosporanic acid solution is mixed with the mutated immobilized penicillin G acylase at a temperature of 18-22°C to obtain a mixed solution; then the D-phenylglycine methyl ester hydrochloride solution is added dropwise to the mixed solution to react, and the pH is maintained at 7.0 during the reaction process.

6. The method for preparing cephalexin according to claim 5, characterized in that: The pH of the 7-amino-3-deacetoxycephalosporanic acid solution in step (3) is 7.

0.

7. The method for preparing cephalexin according to claim 5, characterized in that: The mass ratio of the mutated immobilized penicillin G acylase in step (3) to the 7-amino-3-deacetoxycephalosporanic acid in the 7-amino-3-deacetoxycephalosporanic acid solution is 0.4-1.2:

1.

8. The method for preparing cephalexin according to claim 5, characterized in that: In step (3), the molar ratio of D-phenylglycine methyl ester hydrochloride to 7-amino-3-deacetoxycephalosporanic acid is 1.05-1.10:

1.

9. The method for preparing cephalexin according to claim 5, characterized in that: The dripping time in step (3) is 240-260 min, and the reaction temperature is 18-22°C.

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