Penicillin G expansible ring enzyme mutant, polynucleotide, vector, host cell and application

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a penicillin G expansin mutant, polynucleotide, a vector, a host cell and application. The mutant is a mutant with E150D point mutation on an amino acid sequence as shown in SEQ ID NO.1 in a sequence table. The mutant further comprises point mutation at one or more of the following positions: D28E, E136Q, C155Y, M188I, Q237K, A246V and E290D. The conversion rate of the mutant for catalyzing conversion of penicillin G into G-7-ADCA is obviously improved compared with that of a wild type, and the mutant has a good application prospect; according to the invention, a ring expansor mutant expression library is also constructed, and compared with wild ring expansor, the enzyme activity is greatly improved; meanwhile, the optimal catalysis condition of the expansible enzyme mutant is explored, the conversion rate can be remarkably improved under the condition, and the expansible enzyme mutant has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically relates to a penicillin G expandase mutant and polynucleotide, a vector, a host cell and uses. Background Art

[0002] Cephalosporin antibiotics are widely used antibiotics. Cephalosporin molecules contain semi-synthetic antibiotics called cephem, which belong to the β-lactam antibiotics, similar to penicillin. In the early days, it was believed that the only effect was to inhibit transpeptidase and interfere with the synthesis of bacterial cell wall substances. It has been proven that β-lactam compounds can also bind to certain proteins (β-lactam binding proteins), the essence of which may be some enzymes on the cell membrane. This changes the permeability of bacterial cell membranes, inhibits protein synthesis, and releases autolysins, so it has a lytic effect, or makes it grow into fibers without dividing. It has a strong selective effect on bacteria and is almost toxic to humans. It has the advantages of a wide antibacterial spectrum, strong antibacterial effect, penicillinase resistance, and less allergic reactions than penicillins. Therefore, it is an important antibiotic with high efficiency, low toxicity, and wide clinical application.

[0003] 7-Amino-3-deacetyloxycephalosporanic acid (7-ADCA) is one of the important nuclei for the synthesis of cephalosporin antibiotics. As an important intermediate for semi-synthetic cephalosporin antibiotics, the demand is increasing. At present, the main methods for producing 7-ADCA in industry are chemical enzymatic method and fermentation method. The chemical enzymatic method for preparing 7-ADCA uses penicillin G as raw material, and obtains phenylacetyl-7-amino-3-deacetyloxycephalosporanic acid (G-7-ADCA) through chemical reaction oxidation, ring expansion and rearrangement. Then, the side chain is removed under the action of penicillin acylase to obtain 7-ADCA. This method is costly and environmentally unfriendly. With the development of industrial technology, this method has been gradually abandoned. Secondly, fermentation can also be used to produce 7-ADCA industrially. Studies have found that expandase (DAOCS) can convert the five-membered ring of penicillin G into the six-membered ring of G-7-ADCA. The current method commonly used in industry is to clone the expandase gene into Pseudomonas, Penicillium chrysogenum or Streptomyces clavuligerus, and exogenously add phenylacetic acid, phenylethyl salt or phenylethyl ester as substrates to obtain G-7-ADCA in one step of fermentation. Subsequently, G-7-ADCA removes the phenylacetyl side chain under the catalysis of acyltransferase to obtain 7-ADCA.

[0004] The preparation of 7-ADCA by the whole enzyme method has the advantages of being green, environmentally friendly and low cost. The whole enzyme method starts with cheap penicillin G salt, generates G-7-ADCA through the catalytic ring expansion reaction of penicillin G expandase, and then hydrolyzes and removes the acyl side chain through the action of penicillin acylase to generate 7-ADCA. The reaction route is as follows: .

[0005] Penicillin acylase is a mature enzyme that has been used in industry for many years. 2+ It works in the presence of , O2 and α-ketoglutaric acid, and mainly catalyzes the conversion of the five-membered thiazole ring of penicillin to the six-membered thiazine ring. It is a key step in catalyzing the synthesis of cephalosporin antibiotics by Streptomyces rod-shaped bacteria and other prokaryotes. This reaction does not require the exogenous addition of phenylacetic acid and other easily made-to-drug chemicals as substrates, and is also environmentally friendly. Therefore, DAOCS has important industrial application value. However, in practical applications, it was found that DAOCS had low biological activity in catalyzing penicillin G and could not meet industrial applications, which became the rate-limiting step in the synthesis of 7-ADCA using the whole enzyme method. Therefore, first of all, it is necessary to modify the expandase in order to greatly improve its conversion efficiency to penicillin G; secondly, it is also necessary to explore reaction conditions that are more suitable for the expression of expandase to lay a foundation for industrial applications.

[0006] At present, the crystal structure of scDAOCS from Streptomyces clavuligerus has been obtained, and its amino acid sequence is shown in the sequence list SEQ ID NO.1 (WT-scDAOCS), and the nucleotide sequence is shown in the sequence list SEQ ID NO.2. This makes it possible to transform scDAOCS through protein engineering technology, improve its catalytic activity, and broaden its substrate spectrum, providing broad industrial application prospects for the enzymatic production of 7-ADCA. In actual production, reaction conditions such as buffer concentration, pH, metal ions and the concentration of additives have an important influence on the activity and stability of the enzyme. Therefore, optimizing the reaction conditions is also of great significance for improving the catalytic efficiency of the enzyme. Summary of the invention

[0007] The purpose of the present invention is to provide mutants of penicillin G expandase, which are substituted at one or more positions of the parent polypeptide and have significantly improved activity compared with their parents. The present invention also provides polynucleotides encoding these mutants, nucleic acid constructs, vectors, host cells, and uses of the mutants.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: a penicillin G expandase mutant, which is a mutant having an E150D point mutation in the amino acid sequence shown in SEQ ID NO.1 in the sequence table.

[0009] Preferably, the mutant further comprises point mutations at one or more of the following positions: D28E, E136Q, C155Y, M188I, Q237K, A246V, E290D.

[0010] The present invention also provides a polynucleotide encoding the mutant.

[0011] The present invention further provides an expression vector comprising the polynucleotide, wherein the expression vector is used to encode and produce the one or more mutants in an expression host.

[0012] The present invention further provides a recombinant host cell comprising the expression vector.

[0013] The present invention further provides the use of the mutant, which is used to synthesize phenylacetyl-7-amino-3-deacetoxycephalosporanic acid (G-7-ADCA) using penicillin G as a raw material.

[0014] The present invention further provides a penicillin G ring expansion composition, which contains the mutant, the expression vector or the recombinant host cell.

[0015] The present invention further provides a method for preparing the mutant, which comprises: culturing the recombinant host cell and recovering the mutant.

[0016] The present invention further provides a method for preparing phenylacetyl-7-amino-3-deacetoxycephalosporanic acid using the mutant. The method uses penicillin G as a raw material, adds the mutant, recombinant host cell culture or fermentation liquid into the reaction system, and reacts for 1.5-2 hours.

[0017] Preferably, the reaction system contains phosphate buffer at a concentration of 50-200 mM, FeSO4 at a concentration of 20-80 mg / L, ascorbic acid at a concentration of 0.5-2.0 mM, and α-ketoglutaric acid at a concentration of 30-50 mM; the pH is 6.5-8.0.

[0018] The mutant provided by the present invention catalyzes the conversion rate of penicillin G into G-7-ADCA, which is significantly higher than that of the wild type, and has good application prospects. The present invention also constructs an expandase mutant expression library, and the enzyme activity is greatly improved compared with the wild-type expandase. At the same time, the optimal catalytic conditions of the expandase mutant are explored, under which the conversion rate can be significantly improved, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the plasmid map pET30a-scDAOCS; Figure 2 is the conversion rate of the penicillin G expandase variant ScDAOCS-16 at different phosphate concentrations in the embodiment of the present invention; Figure 3 is the conversion rate of the penicillin G expandase variant ScDAOCS-16 under different pH conditions in the embodiment of the present invention; Figure 4is the conversion rate of the penicillin G expandase variant ScDAOCS-16 at different FeSO4 concentrations in the embodiment of the present invention; Figure 5 is the conversion rate of the penicillin G expandase variant ScDAOCS-16 at different ascorbic acid concentrations in the embodiment of the present invention; Figure 6 Graph (a) shows the conversion rate of the penicillin G expandase variant ScDAOCS-16 at different α-ketoglutarate concentrations in the example of the present invention. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0021] 1. Mutants The present invention relates to isolated penicillin G expandase mutants comprising substitutions at one or more of positions 28, 136, 150, 155, 188, 237, 246, 289 of the polypeptide shown in the sequence table SEQ ID NO.1, and these mutants have the activity of catalyzing the conversion of the five-membered thiazole ring of penicillin into a six-membered thiazine ring.

[0022] The mutants with substitutions or deletions mentioned above should be understood to cover all possible combinations of one or more substitutions or deletions at the specified positions.

[0023] In another aspect, the penicillin G expandase mutant comprises a point mutation at one or more (e.g., several) positions selected from the group consisting of: D28E, E136Q, E150D, C155Y, M188I, Q237K, A246V, E290D.

[0024] When there are two point mutations at the same time, the mutation positions include any one of the following combinations: E150D and D28E; E150D and E136Q; E150D and C155Y; E150D and M188I; E150D and Q237K; E150D and A246V; E150D and E290D.

[0025] When there are three point mutations at the same time, the mutation positions include any one of the following combinations: E150D, D28E and E136Q; E150D, D28E and C155Y; E150D, D28E and M188I; E150D, D28E and Q237K; E150D, D28E and A246V; E150D, D28E and E290D; E150D, E136Q and C155Y; E150D, E136Q and M188I; E150D, E136Q and Q237K; E150D, E136 Q and A246V; E150D, E136Q and E290D; E150D, C155Y and M188I; E150D, C155Y and Q237K; E150D, C155Y and A246V; E150D, C155Y and E290D; E150D, M188I and Q237K; E150D, M188I and A246V; E150D, M188I and E290D; E150D, Q237K and A246V; E150D, Q237K and E290D; E150D, A246V and E290D;.

[0026] When there are 4 point mutations at the same time, the mutation positions include any one of the following combinations: E150D, D28E, E136Q and C155Y; E150D, D28E, E136Q and M188I; E150D, D28E, E136Q and Q237K; E150D, D28E, E136Q and A246V; E150D, D28E, E136Q and E290D; E150D, E136Q, C155Y and M188I; E150D, E136Q, C155Y and Q237K; E150D, E136Q, C155Y and A246V; E150D, E136Q, C155Y and E290D; E150D, C155Y, M188I and Q237K; E150D, C155Y, M188I and A246V; E150D, C155Y, M188I and E290D; E150D, M188I, Q237K and A246V; E150D, M188I, Q237K and E290D; E150D, Q237K, A246V and E290D.

[0027] When there are 5 point mutations at the same time, the mutation positions include any one of the following combinations: E150D, D28E, E136Q, C155Y and M188I; E150D, D28E, E136Q, C155Y and Q237K; E150D, D28E, E136Q, C155Y and A246V; E150D, D28E, E136Q, C155Y and E290D; E150D, E136Q, C155Y, M188I and Q237K; E150D, E136Q, C155Y, M188I and A246V; E150D, E136Q, C155Y, M188I and E290D ; E150D, C155Y, M188I, Q237K and A246V; E150D, C155Y, M188I, Q237K and E290D; E150D, M188I, Q237K, A246V and E290D.

[0028] When there are 6 point mutations at the same time, the mutation positions include any one of the following combinations: E150D, D28E, E136Q, C155Y, M188I and Q237K; E150D, D28E, E136Q, C155Y, M188I and A246V; E150D, D28E, E136Q, C155Y, M188I and E290D; E150D, E136Q, C155Y, M188I, Q237K and A246V; E150D, E136Q, C155Y, M188I, Q237K and E290D; E150D, C155Y, M188I, Q237K, A246V and E290D.

[0029] When there are 7 point mutations at the same time, the mutation positions include any one of the following combinations: E150D, D28E, E136Q, C155Y, M188I, Q237K and A246V; E150D, D28E, E136Q, C155Y, M188I, Q237K and E290D.

[0030] When there are 8 point mutations at the same time, the mutation positions include: E150D, D28E, E136Q, C155Y, M188I, Q237K, A246V and E290D.

[0031] In a further embodiment, except for the specified substitution or deletion, the mutant is selected from the group consisting of: (a) a polypeptide having at least 65% sequence identity with the polypeptide of SEQ ID NO.11 in the sequence listing; (b) a polypeptide encoded by a polynucleotide that hybridizes with the mature polypeptide coding sequence of SEQ ID NO. 11 in the sequence listing, or with the full-length complement of SEQ ID NO. 11 in the sequence listing under low stringency conditions; (c) a polypeptide encoded by a polynucleotide having at least 65% identity with the mature polypeptide coding sequence of SEQ ID NO.11 in the sequence listing; (d) A fragment of the polypeptide of SEQ ID NO. 11 having penicillin G expandase activity.

[0032] These variants may further comprise one or more additional substitutions at one or more (e.g., several) other positions. These amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically smaller deletions of 1-30 amino acids; smaller amino or carboxyl terminal extensions, such as an amino-terminal methionine residue; smaller linker peptides of up to 20-25 residues; or smaller extensions that facilitate purification by changing the net charge or another function, such as polyhistidine sequences (tracts), antigenic epitopes, or binding domains.

[0033] Examples of conservative substitutions are within the scope of the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Alternatively, amino acid changes are essentially changes in the physicochemical properties of the polypeptide. For example, amino acid changes can improve the thermal stability of the polypeptide, change substrate specificity, change pH optimum, etc.

[0034] 2. Preparation of Mutants These mutants can be prepared using any mutagenesis procedure known in the art, such as random mutagenesis, site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, and the like.

[0035] 3. Polynucleotides, nucleic acid constructs and expression vectors The present invention also relates to isolated polynucleotides encoding variants of the present invention. The polynucleotide molecule is specifically a gene sequence encoding the mutant protein described above. A nucleic acid construct that can form a polynucleotide is connected to one or more control sequences of a mutant of the present invention. A recombinant expression vector can be obtained by cloning the polynucleotide of the mutant of the present invention on any vector (e.g., plasmid or virus) that can cause the expression of the polynucleotide. When it is introduced into the host cell, it can be integrated into the genome and replicated together with one or more chromosomes into which it has been integrated. Or it can be isolated from the chromosome and replicate autonomously.

[0036] 4. Recombinant host cells The recombinant host cell provided by the present invention comprises a polynucleotide encoding a mutant, and the polynucleotide is connected to a control sequence to direct the expression of the mutant. The vector or construct can be integrated into the host chromosome or replicated in an extrachromosomal form. The host cell can be a prokaryotic or eukaryotic cell for efficient production of the target mutant.

[0037] 5. Methods for producing mutants The present invention provides a method for producing mutants, comprising culturing recombinant host cells under suitable conditions and recovering mutants. The host cells can be cultured in a medium containing a carbon source, a nitrogen source and an inorganic salt by fermentation in a shake flask or a fermenter. The mutants can be directly recovered from the culture medium or cell lysate and purified by centrifugation, filtration or chromatography. Antibodies or enzyme activity assays can be used to detect mutants, and host cells expressing mutants can be directly used as a source if necessary.

[0038] 6. Application of mutants The present invention also provides a method for producing 7-ADCA by enzymatic method using a penicillin G expandase mutant. The method comprises: using penicillin G as a raw material, adding the penicillin G expandase mutant of the present invention and a culture or fermentation liquid of a recombinant host cell to a reaction system, and reacting for 1.5-2 hours to synthesize 7-ADCA (phenylacetyl-7-amino-3-deacetoxycephalosporanic acid).

[0039] The present invention also provides the optimal reaction conditions of the penicillin G expandase mutant. By respectively screening the phosphate buffer concentration, pH, FeSO4 concentration, ascorbic acid concentration and α-ketoglutaric acid concentration, the reaction conditions that can achieve the highest conversion rate are determined, thereby significantly improving the catalytic efficiency of the mutant.

[0040] The technical solution of the present invention is further described below in conjunction with Examples 1-5 and the accompanying drawings.

[0041] Example 1. Gene acquisition and vector construction Based on the amino acid sequence WT-scDAOCS (SEQ ID NO.1) of the wild-type penicillin G expandase from Streptomyces clavuligerus (GenBank accession number: 1W28_A), the coding gene was optimized with reference to the codon preference of Escherichia coli to obtain the nucleotide sequence (SEQ ID NO. 2). The optimized gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and cloned into the NdeI and XhoI restriction sites of plasmid pET30a to construct the recombinant plasmid pET30a-scDAOCS. The plasmid schematic diagram is shown in Figure 1 Subsequently, the recombinant plasmid was transformed into the Escherichia coli BL21 (DE3) expression host through Ca²⁺-mediated chemical transformation, and the recombinant strain Ec-WT-scDAOCS was successfully constructed.

[0042] Example 2. Chemical mutagenesis to generate expandase mutants Extract the plasmid pET30a-scDAOCS and mix the plasmid with 0.5 M hydroxylamine solution in 0.1 M phosphate buffer, pH 6.0. Incubate at 37°C for 10 hours. Use phenol-chloroform extraction or column purification to remove hydroxylamine and purify the plasmid. Transform the recombinant plasmid into the E. coli BL21 (DE3) expression host and successfully construct a mutant recombinant strain.

[0043] Example 3. Determination of G-7-ADCA synthesis activity of mutants Add 500 μL of substrate solution to each well of the 96-well plate of the induced bacteria. The composition of the substrate solution includes: 50 mM pH 7.4 phosphate buffer, 50 mg / L FeSO4, 0.4 mM ascorbic acid, 10 mM α-ketoglutaric acid and 10 mM penicillin G. After the reaction system was reacted at 30°C for 2 hours, the reaction was terminated by adding 500 μL of methanol. Subsequently, the sample was filtered and analyzed by high-performance liquid chromatography (HPLC), and the content of each component was calculated based on the comparison with the quantitative curve of the standard. Ten strains with significantly improved enzyme activity were selected and commissioned to Suzhou Jinweizhi Company for nucleic acid sequencing. Table 1 shows the transformation efficiency of these mutants.

[0044] Table 1 Conversion rates of expandase mutants generated by chemical mutagenesis .

[0045] A total of 8 mutation sites with significantly improved catalytic activity were obtained, namely D28E, E136Q, E150D, C155Y, M188I, Q237K, A246V and E290D. Among them, the site with the highest mutation efficiency was E150D. This mutation site was further combined with the remaining 7 sites for mutation to construct all mutants containing 2-8 combined mutations. Protein expression purification and catalytic activity determination were performed, and it was found that the catalytic activity of all mutants was higher than that of the wild type. Only the 10 combined mutants with the highest enzyme activity are listed here, named ScDAOCS-16 - ScDAOCS-25, and Table 2 shows the conversion efficiency of these mutants.

[0046] Table 2 Conversion rate of expandase mutant combinations strain Relative conversion rate (%) sequence Mutation location WT-scDAOCS 100 SEQ ID No.1 none ScDAOCS-16 277.52 SEQ ID No.11 D28E, E136Q, E150D, C155Y, M188I, Q237K, A246V, E290D ScDAOCS-17 254.14 SEQ ID No.12 D28E, E136Q, E150D, C155Y, M188I, Q237K, A246V ScDAOCS-18 243.62 SEQ ID No.13 D28E, E136Q, E150D, M188I, Q237K, A246V, E290D ScDAOCS-19 230.28 SEQ ID No.14 D28E, E136Q, E150D, C155Y, M188I, Q237K, E290D ScDAOCS-20 209.35 SEQ ID No.15 E136Q, E150D, C155Y, M188I, A246V, E290D ScDAOCS-21 204.37 SEQ ID No.16 E136Q, E150D, C155Y, M188I, Q237K, A246V ScDAOCS-22 197.42 SEQ ID No.17 E136Q, E150D, C155Y, M188I, Q237K, E290D ScDAOCS-23 184.06 SEQ ID No.18 D28E, E150D, C155Y, Q237K, A246V, E290D ScDAOCS-24 183.28 SEQ ID No.19 D28E, E136Q, E150D, C155Y, M188I, A246V ScDAOCS-25 174.09 SEQ ID No.20 E150D, C155Y, M188I, Q237K, E290D .

[0047] Example 4. Purification and concentration determination of mutants Protein purification for all strains included the following steps: (1) Resuspend the cells in the equilibration solution at a concentration of 15 w / v%, perform ultrasonic disruption, centrifuge for 30 min, and collect the supernatant after disruption; (2) Use 4 column volumes of equilibration solution to equilibrate the His-tagged protein purification column and pass the supernatant through the column; (3) Use 4 column volumes of impurity removal solution to pass through the column to remove impurities. When the Coomassie brilliant blue of the impurity removal solution turns light blue, add the eluent; (4) Elute with 3 column volumes of eluent until Coomassie Brilliant Blue turns light blue; (5) Take the eluted sample with the darkest Coomassie brilliant blue color and load it onto the desalting column. The desalting column is first equilibrated with desalting equilibrium solution, and then the eluted sample is loaded. The absorbance of the eluted liquid is monitored by a UV spectrophotometer. When the absorbance is greater than 0.1, the desalted sample is collected. When the absorbance drops back to 0.1, the desalting is completed. In this way, the variant protein solution is prepared, and the protein concentration is measured by Nanodrop.

[0048] Example 5. Optimization of mutant reaction conditions (1) Screening of phosphate buffer concentration At 30°C, pH 7.4, 50 mg / L FeSO4, 0.4 mM ascorbic acid, 10 mM α-ketoglutaric acid and 10 mM penicillin G were used as the reaction system, and the concentration of phosphate buffer was adjusted to 0-200 mM for 2 hours. After the reaction, 500 μL of methanol was added to terminate the reaction, and the sample was filtered and analyzed by high performance liquid chromatography (HPLC). The content of each component was calculated by comparing with the quantitative curve of the standard. The experimental results are shown in Table 3 and Figure 2shown.

[0049] Table 3 Conversion rate of penicillin G catalyzed by ScDAOCS-16 under different phosphate concentrations Phosphate concentration (mM) Conversion rate (%) 0 42.22 25 53.45 50 61.33 75 75.15 100 74.94 125 73.99 150 72.12 175 71.11 200 68.27 .

[0050] Experimental verification showed that the mutant ScDAOCS-16 had the highest penicillin G conversion rate (75.15%) when the phosphate buffer concentration was 75 mM, so 75 mM phosphate buffer was selected as the subsequent phosphate buffer concentration.

[0051] 2. pH screening At the optimized phosphate buffer concentration (75 mM), the pH was adjusted to 5.0-9.0 with a step length of 0.5. After 2 hours of reaction, 500 μL of methanol was added to terminate the reaction, and the sample was filtered and analyzed by high performance liquid chromatography (HPLC). The content of each component was calculated by comparing with the quantitative curve of the standard. The experimental results are shown in Table 4 and Figure 3 shown.

[0052] Table 4 Conversion rate of penicillin G catalyzed by ScDAOCS-16 at different pH pH Conversion rate (%) 5.0 54.36 5.5 59.21 6.0 66.47 6.5 79.63 7.0 84.34 7.5 80.77 8.0 74.16 8.5 54.74 9.0 30.35 .

[0053] Experimental verification showed that the conversion rate was the highest (84.34%) when the pH was 7.0, so 7.0 was selected as the subsequent pH value.

[0054] 3. Screening of FeSO4 concentration Under the optimized buffer concentration (75 mM) and pH (7.0) conditions, the FeSO4 concentration was adjusted to 0-100 mg / L. After 2 hours of reaction, 500 μL of methanol was added to terminate the reaction, and the sample was filtered and analyzed by high performance liquid chromatography (HPLC). The content of each component was calculated by comparing with the quantitative curve of the standard. The experimental results are shown in Table 5 and Figure 4 shown.

[0055] Table 5 Conversion rate of penicillin G catalyzed by ScDAOCS-16 at different FeSO4 concentrations <![CDATA[FeSO4 concentration (mg / L)]]> Conversion rate (%) 0 60.65 20 74.44 40 85.31 60 89.27 80 73.33 100 50.47 .

[0056] Experimental verification shows that the conversion rate is highest (89.27%) when the FeSO4 concentration is 60 mg / L, so the FeSO4 concentration is optimized to 60 mg / L.

[0057] 4. Screening of Ascorbic Acid Concentration Under the optimized conditions (75 mM phosphate buffer, pH 7.0, 60 mg / L FeSO4), the ascorbic acid concentration was adjusted to 0-2 mM. After 2 hours of reaction, 500 μL of methanol was added to terminate the reaction, and the sample was filtered and analyzed by high performance liquid chromatography (HPLC). The content of each component was calculated by comparing with the quantitative curve of the standard. The experimental results are shown in Table 6 and Figure 5 shown.

[0058] Table 6 Conversion rate of penicillin G from ScDAOCS-16 at different ascorbic acid concentrations Ascorbic acid concentration (mM) Conversion rate (%) 0 32.36 0.3 60.27 0.6 77.36 0.9 85.37 1.2 87.36 1.5 88.31 1.8 80.07 2.1 74.69 2.4 72.54 .

[0059] The experimental results showed that the conversion rate was the highest (88.31%) when the ascorbic acid concentration was 1.5 mM, so 1.5 mM ascorbic acid was optimized as the concentration for subsequent experiments.

[0060] 5. Screening of α-ketoglutarate concentration Under the optimized conditions (75 mM phosphate buffer, pH 7.0, 60 mg / L FeSO4 and 1.5 mM ascorbic acid), the concentration of α-ketoglutaric acid was adjusted to 0-50 mM for conversion. After 2 hours of reaction, 500 μL of methanol was added to terminate the reaction, and the sample was filtered and analyzed by high performance liquid chromatography (HPLC). The content of each component was calculated by comparing with the quantitative curve of the standard. The experimental results are shown in Table 7 and Figure 6 shown.

[0061] Table 7 Conversion rate of penicillin G catalyzed by ScDAOCS-16 at different α-ketoglutarate concentrations α-Ketoglutarate concentration (mM) Conversion rate (%) 0 6.35 10 23.69 20 47.36 30 76.00 40 92.33 50 90.17 .

[0062] Experimental verification showed that the conversion rate was highest when the concentration of α-ketoglutarate was 40 mM, and the conversion rate of penicillin G reached 92.33%.

[0063] In summary, the optimal reaction conditions for the penicillin G expandase mutant are: 75 mM phosphate buffer (pH 7.0), 60 mg / L FeSO4, 1.5 mM ascorbic acid, 40 mM α-ketoglutaric acid, and 10 mM penicillin G. Under these conditions, the conversion rate of the penicillin G expandase mutant was significantly improved, providing an efficient and economical solution for the production of G-7-ADCA.

Claims

1. A penicillin G expandase mutant, characterized in that: The mutant is a mutant having an E150D point mutation in the amino acid sequence shown in SEQ ID NO.1 of the sequence listing.

2. The penicillin G expandase mutant according to claim 1, characterized in that: The mutant also comprises point mutations at one or more of the following positions: D28E, E136Q, C155Y, M188I, Q237K, A246V, E290D.

3. A polynucleotide encoding the mutant according to claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3, characterized in that: The expression vector is used to encode and produce one or more mutants as claimed in claim 1 or 2 in an expression host. A recombinant host cell comprising the expression vector of claim 4.

6. Use of the mutant according to claim 1 or 2, characterized in that: The mutant is used to synthesize phenylacetyl-7-amino-3-deacetoxycephalosporanic acid using penicillin G as raw material.

7. A penicillin G ring expansion composition, characterized in that: The composition comprises the mutant according to claim 1 or 2, the expression vector according to claim 4 or the recombinant host cell according to claim 5.

8. A method for preparing the mutant according to claim 1 or 2, characterized in that: The mutant is recovered after culturing the recombinant host cell according to claim 5.

9. A method for synthesizing phenylacetyl-7-amino-3-deacetoxycephalosporanic acid, characterized in that: The method uses penicillin G as a raw material, adds the mutant described in claim 1 or 2, or the culture or fermentation liquid of the recombinant host cell described in claim 5 into the reaction system, and reacts for 1.5-2 hours.

10. The method for synthesizing phenylacetyl-7-amino-3-deacetoxycephalosporanic acid according to claim 9, characterized in that: The reaction system contains phosphate buffer with a concentration of 50-200 mM, FeSO4 with a concentration of 20-80 mg / L, ascorbic acid with a concentration of 0.5-2.0 mM, and α-ketoglutaric acid with a concentration of 30-50 mM; the pH value is 6.5-8.0.

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