A mutant of penicillin g acylase and its preparation method and application

Abstract

The present application relates to the technical field of biological enzyme, in particular to a penicillin G acylase mutant, a preparation method and application thereof. The penicillin G acylase mutant is based on the penicillin G acylase, and one or two of the following mutations are carried out: the phenylalanine at the 274th site is mutated to alanine, and the tryptophan at the 404th site is mutated to glycine; the amino acid sequence of the penicillin G acylase is shown as SEQ ID NO. 1. It is verified through experiments that, compared with the wild enzyme, the penicillin G acylase mutant provided by the present application significantly improves the efficiency of catalyzing hydrolysis of antibiotics, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioenzyme technology, specifically to a penicillin G acylase mutant, its preparation method, and its application. Background Technology

[0002] Penicillin G acylase (PGA) is an important enzyme in the field of biocatalysis, widely used in the degradation of β-lactam antibiotics and the green synthesis of novel semi-synthetic β-lactam antibiotics. As a biocatalyst with reversible catalytic properties, PGA has become a core tool enzyme in antibiotic pollution control and green chemical synthesis due to its specific catalytic ability for the hydrolysis and synthesis of β-lactam antibiotics.

[0003] In the hydrolysis reaction, PGA can specifically recognize and catalyze the hydrolysis of penicillin G and the side-chain acyl groups of β-lactam antibiotics, efficiently converting them into 6-aminopenicillanic acid (6-APA). This product is a key intermediate in the synthesis of various clinically commonly used semi-synthetic penicillins such as ampicillin and amoxicillin, laying the core material foundation for the large-scale production of novel and highly efficient semi-synthetic antibiotics.

[0004] Under specific reaction conditions, PGA can catalyze the reverse of the above reaction: using 6-APA as the parent nucleus, it undergoes a condensation reaction with the corresponding acyl donor to achieve the targeted synthesis of novel β-lactam antibiotics with specific antibacterial activity requirements.

[0005] However, natural penicillin acylases have poor activity and low catalytic efficiency. In order to improve the activity and catalytic performance of PGA, this invention mutates specific sites of PGA to increase the affinity of PGA for substrates, thereby improving the catalytic performance of PGA. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a penicillin G acylase mutant, its preparation method and application, wherein the penicillin G acylase mutant significantly improves the catalytic hydrolysis efficiency of antibiotics compared with wild-type penicillin G acylase.

[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0008] Technical Topic 1

[0009] A penicillin G acylase mutant, the key being that, based on penicillin G acylase, one or two of the following mutations are performed: mutating phenylalanine at position 274 to alanine, and mutating tryptophan at position 404 to glycine.

[0010] The amino acid sequence of the penicillin G acylase is shown in SEQ ID NO.1.

[0011] As a further improvement of the present invention, the nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase is shown in SEQ ID NO.2.

[0012] As a further improvement of the present invention, the penicillin G acylase is derived from Paracoccus sp. KDSPL-02.

[0013] As a further improvement of the present invention, the penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO.3; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.4.

[0014] As a further improvement of the present invention, the penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO.5; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.6.

[0015] As a further improvement of the present invention, the penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO.7; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.8.

[0016] Technical Theme Two

[0017] An expression vector containing a nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant as described in Technical Subject 1.

[0018] As a further improvement of the present invention, the expression vector is pGEX-6p-1.

[0019] Technical Theme 3

[0020] A host cell, the key feature of which is that the host cell contains an expression vector as described in Technical Subject 2.

[0021] As a further improvement of the present invention, the key feature is that the host cell is E. coli BL21(DE3).

[0022] Technical Theme 4

[0023] A method for preparing a penicillin G acylase mutant as described in Technical Subject 1, the key of which is that it includes the following steps:

[0024] Design and synthesize the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase;

[0025] Design the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant as described in Technical Theme 1;

[0026] Primer sets were designed and synthesized based on the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase, the expression vector, and the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant.

[0027] The target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase was amplified using the primer set via overlap extension PCR technology to obtain the amplification product.

[0028] The expression vector and amplification product were double-digested, and the digested products were ligated to obtain the recombinant plasmid.

[0029] The recombinant plasmid was introduced into competent cells to obtain recombinant cells;

[0030] The recombinant cells were cultured to express the penicillin G acylase mutant.

[0031] The primer set includes a set of original amplification primers for amplifying the nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase. The original amplification primer set includes an original upstream primer and an original downstream primer. The original upstream primer has a nucleic acid sequence as shown in SEQ ID NO. 9, and the original downstream primer has a nucleic acid sequence as shown in SEQ ID NO. 10.

[0032] As a further improvement of the present invention, the primer set includes an upstream primer and a downstream primer for amplifying the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase at position 274, wherein the upstream primer has the nucleic acid sequence shown in SEQ ID NO. 11, and the downstream primer has the nucleic acid sequence shown in SEQ ID NO. 12; and / or

[0033] The primer set also includes an upstream primer and a downstream primer for amplifying the target nucleic acid sequence at site 404, the upstream primer having the nucleic acid sequence shown in SEQ ID NO. 13, and the downstream primer having the nucleic acid sequence shown in SEQ ID NO. 14.

[0034] Technology Theme 5

[0035] Application of a penicillin G acylase mutant as described in Technical Subject 1 in the catalytic hydrolysis of antibiotics.

[0036] The beneficial effects of adopting the above technical solution are as follows:

[0037] This invention provides a penicillin G acylase mutant with one or two mutation sites. Compared to the wild-type enzyme, the penicillin G acylase mutant provided by this invention significantly improves the efficiency of catalytic hydrolysis of antibiotics and has broad application prospects. Attached Figure Description

[0038] Figure 1 This is a schematic flowchart of a method for preparing a penicillin G acylase mutant provided in an embodiment of the present invention;

[0039] Figure 2 Map of pGEX-6p-1 plasmid;

[0040] Figure 3 Agarose gel electrophoresis image to verify enzyme digestion of the F274A mutant plasmid;

[0041] Figure 4 Sequencing results for the F274A mutant plasmid;

[0042] Figure 5 Agarose gel electrophoresis image to verify the restriction enzyme digestion of the W404G mutant plasmid;

[0043] Figure 6 These are the sequencing results for the W404G mutant plasmid;

[0044] Figure 7 Agarose gel electrophoresis image to verify the enzyme digestion of the F274A / W404G mutant plasmid;

[0045] Figure 8 Sequencing results for the F274G / W404G mutant plasmid;

[0046] Figure 9 Figure 1 shows the results of the conversion of potassium penicillin V catalyzed by wild-type penicillin G acylase and its mutant F274A / W404G;

[0047] Figure 10 Figure showing the conversion rate of amoxicillin catalyzed by wild-type penicillin G acylase and its mutant F274A / W404G. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0049] The preparation method of the penicillin G acylase mutant provided by this invention is as follows: Figure 1 As shown;

[0050] The method includes:

[0051] Design and synthesize the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase;

[0052] Design the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant;

[0053] Primer sets were designed and synthesized based on the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase, the expression vector, and the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant.

[0054] The target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase was amplified using the primer set via overlap extension PCR technology to obtain the amplification product.

[0055] The expression vector and amplification product were double-digested, and the digested products were ligated to obtain the recombinant plasmid.

[0056] The recombinant plasmid was introduced into competent cells to obtain recombinant cells;

[0057] The recombinant cells were cultured to express the penicillin G acylase mutant.

[0058] This method is for preparing the above-mentioned penicillin G acylase mutant. Specific information about the penicillin G acylase mutant can be found in the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0059] The PBS buffer used in the embodiments and preparation examples of this invention was purchased from Sangon Biotech (Shanghai) Co., Ltd., item number E607020.

[0060] The penicillin G acylase in this invention is derived from the penicillin G acylase of Paracoccus sp. KDSPL-02, and its amino acid sequence is shown in SEQ ID NO.1;

[0061] SEQ ID NO.1

[0062] MLRSSLGRLLLSAGLSALLACPALADTKVEIIRDSWGVPHVYADDVHGLYAGFGYSVAQDRLFQMEMARRSVLGEVAEVLGIERLPFDIQTRAMFDHAEIRRQIEALAPEERDILRGYAAGFNLWVDRVLAEPAGLMPKQFNDFGFKPRRWTEFDVAMIYVGTMAGRFSGYSSELANARTLAELEAQFGAEQARALFDQMFWDEDPLAPTTVPEGEQYQRKAALTPPSASRFAALLNAPDLPGDDGRPRASNLWIAGPQKTTDGSTILINGPQ F GNFNPSYVFSIGLHGAGFDLTGNTPFAVPNVLFGTNGTIAWGATAGPRDVNDYYRLELNPENPRQYRAGDGWKEMTARRETFRVKGQPEVETTVWESDYGVVGQIDPETATAYAFNRAWKGQEISTLMAWVNSTKAQNYQEWLDQAERVATTINWYYADRDGNIGYVSPGHVPVRAEGHDQRLPAQPGKDDWQGVRPFADAPKAYNPSQGWIANWNNRSAKGDAANFEATPWGSADRVVEITARLDAKDKLTPEEVWNINREISFLDINARYFLPFILDAAEGVDPQSQRAELVEILRNWDGQQIRAEDGRATTPAVGLFQTWLDVMVEDVLRDDSPAIPPAILYNRISRAAQMLHNALLAERAGVPQTHDFFNGADEAGRKEIIL

[0063] The nucleic acid sequence corresponding to the amino acid sequence is shown as SEQ ID NO.2;

[0064] SEQ ID NO.2

[0065]

[0066] Example 1

[0067] The preparation method of penicillin G acylase mutant is as follows:

[0068] (1) Based on penicillin G acylase, perform one or two of the following mutations: mutate phenylalanine at position 274 to alanine, or mutate tryptophan at position 404 to glycine;

[0069] ① When the mutated amino acid at position 274 is alanine, the amino acid sequence of the PGA mutant is shown in SEQ ID NO.3:

[0070] SEQ ID NO.3

[0071] MLRSSLGRLLLSAGLSALLACPALADTKVEIIRDSWGVPHVYADDVHGLYAGFGYSVAQDRLFQMEMARRSVLGEVAEVLGIERLPFDIQTRAMFDHAEIRRQIEALAPEERDILRGYAAGFNLWVDRVLAEPAGL MPKQFNDFGFKPRRWTEFDVAMIYVGTMAGRFSGYSSELANARTLAELEAQFGAEQARALFDQMFWDEDPLAPTTVPEGEQYQRKAALTPPSASRFAALLNAPDLPGDDGRPRASNLWIAGPQKTTDGSTILINGPQ A GNFNPSYVFSIGLHGAGFDLTGNTPFAVPNVLFGTNGTIAWGATAGPRDVNDYYRLELNPENPRQYRAGDGWKEMTARRETFRVKGQPEVETTVWESDYGVVGQIDPETATAYAFNRAWKGQEISTLMAWVNSTKAQNYQEWLDQAERVATTINWYYADRDGNIGYVSPGHVPVRAEGHDQRLPAQPGKDDWQ GVRPFADAPKAYNPSQGWIANWNNRSAKGDAANFEATPWGSADRVVEITARLDAKDKLTPEEVWNINREISFLDINARYFLPFILDAAEGVDPQSQRAELVEILRNWDGQQIRAEDGRATTPAVGLFQTWLDVMVEDVLRDDSPAIPPAILYNRISRAAQMLHNALLAERAGVPQTHDFFNGADEAGRKEIIL

[0072] The nucleic acid sequence designed according to SEQ ID NO.3 is shown as SEQ ID NO.4;

[0073] SEQ ID NO.4

[0074] ATGCTCAGGTCTTCGCTTGGCCGCCTGCTGCTGTCGGCTGGCCTTTCTGCCCTTCTGGCCTGCCCGGCGCTGGCCGACACCAAGGTCGAGATCATCCGCGACAGCTGGGGCGTGCCGCATGTCTATGCCGATGACGTGCATGGGCTTTACGCGGGCTTCGGCTACAGCGTGGCGCAGGACCGGCTGTTCCAGATGGAGATGGCGCGGCGCTCGGTCCTGGGCGAGGTGGCCGAGGTTCTGGGCATCGAGCGGCTGCCCTTCGACATCCAGACCCGCGCGATGTTCGACCATGCCGAGATCCGGCGCCAGATCGAGGCGCTGGCGCCGGAAGAGCGCGACATCCTGCGTGGCTATGCGGCGGGCTTCAACCTGTGGGTGGACCGGGTTCTGGCCGAACCGGCCGGGCTGATGCCCAAGCAGTTCAACGATTTCGGCTTCAAGCCCCGCCGCTGGACCGAATTCGACGTGGCGATGATCTATGTCGGGACCATGGCCGGCCGCTTTTCGGGCTACAGCTCCGAACTGGCCAATGCCAGGACGCTGGCCGAGCTGGAGGCGCAATTCGGCGCCGAACAGGCACGCGCGCTTTTCGACCAGATGTTCTGGGACGAAGACCCGCTGGCCCCGACCACCGTGCCCGAGGGCGAGCAATACCAGCGCAAGGCCGCCCTGACCCCGCCTTCGGCCAGCCGCTTTGCCGCGCTGCTGAACGCGCCGGACCTGCCCGGCGACGATGGGCGTCCGCGCGCCAGCAACCTGTGGATCGCCGGGCCGCAAAAGACCACCGACGGCAGCACCATCCTGATCAACGGCCCGCAA GCC

[0075] ② When the mutated amino acid at position 404 is glycine, the amino acid sequence of the PGA mutant is shown in SEQ ID NO.5:

[0076] SEQ ID NO.5

[0077] MLRSSLGRLLLSAGLSALLACPALADTKVEIIRDSWGVPHVYADDVHGLYAGFGYSVAQDRLFQMEMARRSVLGEVAEVLGIERLPFDIQTRAMFDHAEIRRQIEALAPEERDILRGYAAGFNLWVDRVLAEPAGLMPKQFNDFGFKPRRWTEFDVAMIYVGTMAGRFSGYSSELANARTLAELEAQFGAEQARALFDQMF WDEDPLAPTTVPEGEQYQRKAALTPPSASRFAALLNAPDLPGDDGRPRASNLWIAGPQKTTDGSTILINGPQFGNFNPSYVFSIGLHGAGFDLTGNTPFAVPNVLFGTNGTIAWGATAGPRDVNDYYRLELNPENPRQYRAGDGWKEMTARRETFRVKGQPEVETTVWESDYGVVGQIDPETATAYAFNRAWKGQEISTLMA G VNSTKAQNYQEWLDQAERVATTINWYYADRDGNIGYVSPGHVPVRAEGHDQRLPAQPGKDDWQGVRPFADAPKAYNPSQGWIANWNNRSAKGDAANFEATPWGSADRVVEITARLDAKDKLTPEEVWN INREISFLDINARYFLPFILDAAEGVDPQSQRAELVEILRNWDGQQIRAEDGRATTPAVGLFQTWLDVMVEDVLRDDSPAIPPAILYNRISRAAQMLHNALLAERAGVPQTHDFFNGADEAGRKEIIL

[0078] The nucleic acid sequence designed based on SEQ ID NO.5 is shown in SEQ ID NO.6;

[0079] SEQ ID NO.6

[0080] GGG

[0081] ③ When the mutated amino acid at position 274 is alanine and the mutated amino acid at position 404 is glycine, the amino acid sequence of the PGA mutant is shown in SEQ ID NO.7:

[0082] SEQ ID NO.7

[0083] MLRSSLGRLLLSAGLSALLACPALADTKVEIIRDSWGVPHVYADDVHGLYAGFGYSVAQDRLFQMEMARRSVLGEVAEVLGIERLPFDIQTRAMFDHAEIRRQIEALAPEERDILRGYAAGFNLWVDRVLAEPAGL MPKQFNDFGFKPRRWTEFDVAMIYVGTMAGRFSGYSSELANARTLAELEAQFGAEQARALFDQMFWDEDPLAPTTVPEGEQYQRKAALTPPSASRFAALLNAPDLPGDDGRPRASNLWIAGPQKTTDGSTILINGPQ A GNFNPSYVFSIGLHGAGFDLTGNTPFAVPNVLFGTNGTIAWGATAGPRDVNDYYRLELNPENPRQYRAGDGWKEMTARRETFRVKGQPEVETTVWESDYGVVGQIDPETATAYAFNRAWKGQEISTLMA G VNSTKAQNYQEWLDQAERVATTINWYYADRDGNIGYVSPGHVPVRAEGHDQRLPAQPGKDDWQGVRPFADAPKAYNPSQGWIANWNNRSAKGDAANFEATPWGSADRVVEITARLDAKDKLTPEEVWN INREISFLDINARYFLPFILDAAEGVDPQSQRAELVEILRNWDGQQIRAEDGRATTPAVGLFQTWLDVMVEDVLRDDSPAIPPAILYNRISRAAQMLHNALLAERAGVPQTHDFFNGADEAGRKEIIL

[0084] The nucleic acid sequence designed based on SEQ ID NO.7 is shown in SEQ ID NO.8;

[0085] SEQ ID NO.8

[0086] ATGCTCAGGTCTTCGCTTGGCCGCCTGCTGCTGTCGGCTGGCCTTTCTGCCCTTCTGGCCTGCCCGGCGCTGGCCGACACCAAGGTCGAGATCATCCGCGACAGCTGGGGCGTGCCGCATGTCTATGCCGATGACGTGCATGGGCTTTACGCGGGCTTCGGCTACAGCGTGGCGCAGGACCGGCTGTTCCAGATGGAGATGGCGCGGCGCTCGGTCCTGGGCGAGGTGGCCGAGGTTCTGGGCATCGAGCGGCTGCCCTTCGACATCCAGACCCGCGCGATGTTCGACCATGCCGAGATCCGGCGCCAGATCGAGGCGCTGGCGCCGGAAGAGCGCGACATCCTGCGTGGCTATGCGGCGGGCTTCAACCTGTGGGTGGACCGGGTTCTGGCCGAACCGGCCGGGCTGATGCCCAAGCAGTTCAACGATTTCGGCTTCAAGCCCCGCCGCTGGACCGAATTCGACGTGGCGATGATCTATGTCGGGACCATGGCCGGCCGCTTTTCGGGCTACAGCTCCGAACTGGCCAATGCCAGGACGCTGGCCGAGCTGGAGGCGCAATTCGGCGCCGAACAGGCACGCGCGCTTTTCGACCAGATGTTCTGGGACGAAGACCCGCTGGCCCCGACCACCGTGCCCGAGGGCGAGCAATACCAGCGCAAGGCCGCCCTGACCCCGCCTTCGGCCAGCCGCTTTGCCGCGCTGCTGAACGCGCCGGACCTGCCCGGCGACGATGGGCGTCCGCGCGCCAGCAACCTGTGGATCGCCGGGCCGCAAAAGACCACCGACGGCAGCACCATCCTGATCAACGGCCCGCAA GCCGGCAATTTCAACCCGTCCTATGTCTTCAGCATCGGCCTGCATGGCGCCGGGTTCGACCTGACCGGCAACACGCCCTTTGCGGTTCCCAATGTGCTGTTCGGCACCAATGGCACCATCGCCTGGGGCGCCACCGCCGGGCCGCGCGACGTGAACGACTATTACCGGCTGGAGCTGAACCCCGAGAACCCGCGGCAATACCGCGCCGGCGACGGCTGGAAGGAGATGACGGCGCGCCGCGAGACCTTCCGCGTCAAGGGCCAGCCCGAGGTCGAGACCACGGTCTGGGAAAGCGATTACGGCGTGGTCGGCCAGATCGACCCCGAGACGGCGACCGCCTATGCCTTCAACCGCGCCTGGAAGGGGCAGGAGATCTCGACGCTCATGGCC GGG

[0087] (2) Based on the nucleic acid sequence designed in step (1), the corresponding target gene is synthesized using the nucleic acid sequence of penicillin G acylase shown in SEQ ID NO.2.

[0088] Based on the known penicillin G acylase gene sequence, double restriction sites (BamHI and XhoI sites) on the pGEX-6p-1 plasmid (purchased from Miaoling Biotechnology Co., Ltd.) were selected, and primers were designed based on the principle of overlap extension PCR technology.

[0089] Design a set of original amplification primers for the known unmutated original nucleic acid sequence.

[0090] upstream primer of the original amplification primer set:

[0091] SEQ ID NO.9

[0092] CGGGATCCATGCTCAGGTCTTCGCTTGGCCG

[0093] Downstream primers of the original amplification primer set:

[0094] SEQ ID NO.10

[0095] CCGCTCGAGCTAGCGCGGAACGGCGATGG

[0096] For the case where the mutated amino acid at position 274 is alanine, a primer set was designed.

[0097] Upstream primer:

[0098] SEQ ID NO.11

[0099] TCCTGATCAACGGCCCGCAA GCC GGCAATTTCAACCCGTCCTA

[0100] Downstream primer:

[0101] SEQ ID NO.12

[0102] TAGGACGGGTTGAAATTGCC GGC TTGCGGGCCGTTGATCAAGA

[0103] For the case where the mutated amino acid at site 404 is glycine, a primer set was designed.

[0104] Upstream primer:

[0105] SEQ ID NO.13

[0106] AGATCTCGACGCTCATGGCC GGG GTGAACTCGACCAAGGCGCA

[0107] Downstream primer:

[0108] SEQ ID NO.14

[0109] TGCGCCTTGGTCGAGTTCAC CCC GGCCATGAGCGTAGAGATCT

[0110] (3) The target gene was amplified using the designed amplification primer set to obtain the amplification product:

[0111] Using the different primers designed above, and targeting different combinations of mutation sites, PCR amplification was performed using the corresponding upstream and downstream primers and templates, following the amplification system shown in Table 1 and the amplification program shown in Table 2, to obtain amplification products for different combinations of mutation sites. The initial template was the nucleic acid sequence shown in SEQ ID NO. 2.

[0112] Table 1 PCR amplification reaction system

[0113]

[0114] Table 2 Overlap Extension PCR Reaction Procedure

[0115]

[0116] (4) The recovered and sequence-verified mutant penicillin G acylase gene fragment and the pGEX-6p-1 empty plasmid were double-digested using BamHI and XhoI restriction endonucleases, respectively. 10.0 μL of the amplification product and 2 μL of the expression vector were then double-digested at 37°C for 30 min using 1 μL of BamHI restriction endonuclease and 1 μL of XhoI restriction endonuclease, respectively, to obtain the digested vector and digested product.

[0117] (5) Mix 10 μL of the enzyme digestion product and 2 μL of the enzyme digestion vector, and then add 1 μL of T4 DNA ligase to ligate and obtain the recombinant plasmid vector.

[0118] (6) The above ligation product was transferred into E. coli DH5α(DE3) competent cells by heat shock. After single colonies grew on LB solid plates containing 0.1% kanamycin sulfate (1g peptone, 1g NaCl, 0.5g yeast extract, 100mL ultrapure water, 2g agar powder), several single colonies were randomly picked and inoculated into LB liquid medium containing 0.1% kanamycin sulfate (1g peptone, 1g NaCl, 0.5g yeast extract, 100mL ultrapure water). After overnight incubation until the solution became turbid, plasmids were extracted. The extracted plasmids were then double-digested with BamHI and XhoI restriction endonucleases for verification, and detected by agarose gel electrophoresis. If two bands were present in a lane, the plasmid corresponding to that lane was the recombinant plasmid. A portion of the plasmid solution was sequenced for verification. If the sequencing results showed the presence of the target mutant penicillin G acylase gene sequence, and only a normal mutation occurred compared to the target gene sequence, then the recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, thus completing the construction of the penicillin G acylase mutant engineered bacteria. The agarose gel electrophoresis results of the recombinant cells of the F274A, W404G, and F274A / W404G double mutant engineered bacteria are shown below. Figure 3 , Figure 5 , Figure 7 As shown, the sequencing results of the recombinant plasmids are as follows: Figure 4 , Figure 6 , Figure 8 As shown in the figure, the results demonstrate the successful preparation of the engineered bacteria.

[0119] (7) The preserved penicillin G acylase engineered bacterial suspension and the bacterial suspensions of each mutant penicillin G acylase engineered bacterial suspension were inoculated at a 2% inoculum into small test tubes containing 0.1% kanamycin sulfate in LB liquid medium. The tubes were incubated overnight at 37°C and 200 rpm until the solution became turbid. The next day, the bacterial suspensions in the small test tubes were inoculated at a 1% inoculum into TB medium (6 g peptone, 12 g yeast extract, 2 mL glycerol, 450 mL ultrapure water) containing 0.1% kanamycin sulfate. The tubes were incubated at 37°C and 200 rpm for expansion culture. After about 3 hours of culture, when the OD600 reached 0.6 to 0.8, protein expression could be induced. IPTG was used as an inducer, with a final concentration of 0.1 μmol / mL. The tubes were then incubated at 20°C and 120 rpm for 18 hours to obtain the fermentation broth.

[0120] After induction, the fermentation broth was placed in centrifuge tubes and centrifuged at 7000 rpm for 4 minutes at 4°C. The supernatant was discarded, and the centrifuged cells were retained. 15 mL of PBS buffer solution was added to each centrifuge tube, and the mixture was vortexed to ensure that the cells settled at the bottom were fully mixed with the PBS buffer solution before centrifugation. This process was repeated twice, and then PBS buffer was added again to obtain whole cells of wild-type penicillin G acylase, mutant enzyme F274A, mutant enzyme W404G, and double mutant enzyme F274A / W404G.

[0121] Centrifuge tubes containing suspended bacterial cells were placed in small beakers filled with crushed ice and subjected to ultrasonic disruption using an ultrasonic homogenizer. The parameters were set as follows: 2 seconds of sonication followed by a 3-second interval, with a power ratio of 33% (specifically, 50W). The disruption process lasted 15 minutes to rupture the bacterial cells. After disruption, the lysate was centrifuged at 12000 rpm for 8 minutes in a high-speed refrigerated centrifuge. The supernatant obtained was the crude enzyme solution containing wild-type penicillin G acylase, mutant enzyme F274A, mutant enzyme W404G, and the double mutant enzyme F274A / W404G.

[0122] To obtain pure enzyme solutions of wild-type and mutant penicillin G acylase, protein purification was required. Both expressed proteins were expressed using the pGEX-6p-1 plasmid, which contains a GST tag. Therefore, GST4FF (GST-Tag) agarose resin purchased from Sangon Biotech was used for protein purification.

[0123] First, prepare the relevant solutions. The buffer formula required for protein purification is as follows:

[0124] (1) Equilibrium solution: Binding Buffer (150mM sodium chloride), pH 7.3-7.5;

[0125] (2) Elution buffer: Elution Buffer (10mM reduced glutathione), pH 7.9-8.1.

[0126] After preparation, the prepared equilibration solution, elution solution, and supernatant (containing crude enzyme solution of wild-type penicillin G acylase or mutant penicillin G acylase) are filtered through a 0.22 μm aqueous filter membrane for later use.

[0127] The protein purification steps are as follows:

[0128] (1) Rinse the column with 30 mL of ultrapure water (add 5 mL each time);

[0129] (2) Rinse the column with 25 mL of equilibration buffer (add 5 mL each time);

[0130] (3) Add the broken supernatant to the column (2 mL each time), and repeat this step twice to ensure that the broken supernatant is fully combined with the GST resin;

[0131] (4) Rinse the column with 40 mL of equilibration buffer (add 5 mL each time);

[0132] (5) Rinse the column with 10 mL of elution buffer and collect it with a collection tube for subsequent use (add 5 mL each time).

[0133] (6) Rinse the column with 15 mL of equilibration buffer (add 5 mL each time);

[0134] (7) Rinse the column with 25 mL of ultrapure water (add 5 mL each time);

[0135] (8) Rinse the column with 25 mL of 20% ethanol (add 5 mL each time);

[0136] (9) Add 10 mL of 20% ethanol to the column and then store it in a refrigerator at 4°C.

[0137] After purification, the collected eluents were placed in dialysis bags and then in 100 mM sodium phosphate buffer solution (pH 6.5) for overnight dialysis at 4°C to remove impurities such as glutathione. After dialysis, pure enzyme solutions of wild-type penicillin G acylase, mutant enzymes F274A, W404G, and the double mutant enzyme F274A / W404G were obtained. These solutions were stored at 4°C for later use.

[0138] Preparation Example 1: Preparation of mutant K687G

[0139] The preparation method of the penicillin G acylase mutant strain K687G is the same as in Example 1, except that the primer set designed for the case where the mutated amino acid at position 687 is glycine is as follows:

[0140] Upstream primer:

[0141] SEQ ID NO.15

[0142] CGGACTGGCGCATCGAGATCGGGCAGCATGTGTTCGAGACCAG

[0143] Downstream primer:

[0144] SEQ ID NO.16

[0145] CTGGTCTCGAACACATGCTGCCCGATCTCGATGCGCCAGTCCG

[0146] Example 2

[0147] The Paracoccus sp. KDSPL-02 used in this embodiment was obtained through screening. Those skilled in the art can screen it using conventional technical means or purchase it through conventional channels.

[0148] 1. The hydrolytic activity of whole cells of penicillin G acylase, mutant enzyme F274A, mutant enzyme W404G, and double mutant enzyme F274A / W404G prepared in Example 1, as well as the whole cell of mutant K687G enzyme prepared in Preparation Example 1, was investigated.

[0149] Wild-type and mutant penicillin acylases were subjected to the same amide drug in whole cells, and their hydrolysis performance was detected by high-performance liquid chromatography. This study investigated the changes in the catalytic activity of wild-type and mutant penicillin acylases for different substrates.

[0150] Prepare stock solutions of penicillin V potassium, penicillin G, and amoxicillin at concentrations of 1 mg / mL, and preheat them in a shaker at 50°C for 30 min. Add whole cells to the stock solutions, with a dry weight of 0.45 g / L. Mix thoroughly and continue incubation in a shaker at 120 rpm for 90 min. Samples are taken and analyzed by high-performance liquid chromatography (HPLC), recording the peak area changes for different samples. Repeat the experiment three times in parallel and take the average value.

[0151] The results are shown in Tables 3-5. In this invention, whole-cell catalytic reaction screening of penicillin G acylase and its mutants (F274A, W404G, F274A / W404G, K687G) was conducted. The results showed that compared with penicillin G acylase, F274A, W404G, and F274A / W404G all showed certain enhancements in catalytic activity for penicillin V potassium, penicillin G, and amoxicillin. Among them, the double mutant enzyme F274A / W404G showed the most significant catalytic effect.

[0152] Table 3. Penicillin V potassium degradation data

[0153]

[0154] Table 4. Penicillin G Degradation Data

[0155]

[0156] Table 5 Amoxicillin Degradation Data

[0157]

[0158] 2. Hydrolytic activity of penicillin G acylase and the double mutant enzyme F274A / W404G prepared in Example 1 was investigated:

[0159] Prepare 1 mg / mL penicillin V potassium stock solution and amoxicillin stock solution, respectively, and preheat them in a shaker at 50°C for 30 min. Then, add enzyme solution to the drug solution at a volume ratio of 1:50, mix thoroughly, and continue to react in a shaker at 120 rpm. Samples were taken at different time points and analyzed by high-performance liquid chromatography (HPLC) to calculate the degradation conversion rate of different samples. The experiment was repeated in triplicate, and the average value was taken.

[0160] The enzyme concentration in the enzyme solution is 0.0758 mg / ml.

[0161] The results are as follows Figure 9 (Penicillin V potassium) and Figure 10 As shown in (amoxicillin), compared with the wild-type enzyme, the penicillin G acylase mutant provided by this invention significantly improves the efficiency of catalytic hydrolysis of antibiotics and has broad application prospects.

[0162] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A penicillin G acylase mutant, characterized in that: Based on penicillin G acylase, perform one or two of the following mutations: mutate phenylalanine at position 274 to alanine, or mutate tryptophan at position 404 to glycine. The amino acid sequence of the penicillin G acylase is shown in SEQ ID NO.

1.

2. The penicillin G acylase mutant according to claim 1, characterized in that, The penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO.3; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.

4.

3. The penicillin G acylase mutant according to claim 1, characterized in that, The penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO. 5; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.

6.

4. A penicillin G acylase mutant according to claim 1, characterized in that, The penicillin G acylase mutant has the amino acid sequence shown in SEQ ID NO.7; and / or the penicillin G acylase mutant has the nucleic acid sequence shown in SEQ ID NO.

8.

5. An expression vector containing a nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant according to any one of claims 1-4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5.

7. A method for preparing the penicillin G acylase mutant as described in any one of claims 1-4, characterized in that, The method includes the following steps: Design and synthesize the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase; Design the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant as described in any one of claims 1-4; Primer sets were designed and synthesized based on the target nucleic acid sequence corresponding to the amino acid sequence of penicillin G acylase, the expression vector, and the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase mutant. The target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase was amplified using the primer set via overlap extension PCR technology to obtain the amplification product. The expression vector and amplification product were double-digested, and the digested products were ligated to obtain the recombinant plasmid. The recombinant plasmid was introduced into competent cells to obtain recombinant cells; The recombinant cells were cultured to express the penicillin G acylase mutant.

8. The method according to claim 7, characterized in that, The expression vector is pGEX-6p-1; The primer set includes a set of original amplification primers for amplifying the nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase. The original amplification primer set includes an original upstream primer and an original downstream primer. The original upstream primer has a nucleic acid sequence as shown in SEQ ID NO. 9, and the original downstream primer has a nucleic acid sequence as shown in SEQ ID NO.

10.

9. The method according to claim 7, characterized in that, The primer set further includes an upstream primer and a downstream primer for amplifying the target nucleic acid sequence corresponding to the amino acid sequence of the penicillin G acylase at position 274, wherein the upstream primer has the nucleic acid sequence shown in SEQ ID NO. 11 and the downstream primer has the nucleic acid sequence shown in SEQ ID NO. 12; and / or The primer set also includes an upstream primer and a downstream primer for amplifying the target nucleic acid sequence at site 404, the upstream primer having the nucleic acid sequence shown in SEQ ID NO. 13, and the downstream primer having the nucleic acid sequence shown in SEQ ID NO.

14.

10. The use of a penicillin G acylase mutant as described in any one of claims 1-4 in the catalytic hydrolysis of antibiotics.

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