A recombinant penicillin G acylase and its preparation method and application

Abstract

The present application relates to the field of bioenzyme technology, and in particular to a recombinant penicillin G acylase, a preparation method thereof, and an application thereof. The amino acid sequence of the recombinant penicillin G acylase includes three mutation sites: site 171, site 647, and site 266. The mutated amino acid at site 171 is tyrosine, the mutated amino acid at site 647 is arginine, and the mutated amino acid at site 266 includes one of the following: glutamine, methionine, and arginine. The recombinant penicillin G acylase significantly improves the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity with the target substrate through three key mutation sites (F171Y, Y647R, S266Q / M / R) based on the multidimensional structural characteristics of the synergistic optimization of the enzyme-substrate interaction.

Description

Technical Field

[0001] The present application relates to the field of bioenzyme technology, and in particular to a recombinant penicillin G acylase and a preparation method and application thereof. Background Art

[0002] Penicillin G acylase (PGA) is an important industrial enzyme widely used in the industrial production of antibiotics. PGA has the following main functions: (1) Catalytic hydrolysis reaction: PGA can specifically catalyze the hydrolysis of the side chain acyl group of penicillin G (or other β-lactam antibiotics) and convert it into 6-aminopenicillanic acid (6-APA). These 6-APAs are essential key intermediates in the synthesis of various semi-synthetic penicillins such as ampicillin and amoxicillin, thus laying the foundation for the production of a variety of new and highly effective semi-synthetic antibiotics; (2) Synthesis reaction: In addition to the above-mentioned catalytic hydrolysis reaction, under specific conditions, PGA can also be used to catalyze the reverse reaction, that is, the synthesis of new β-lactam antibiotics from 6-APA and the corresponding acyl donor, so as to achieve the synthesis and preparation of new antibiotics with specific antibacterial activity requirements. Therefore, penicillin G acylase has become a key biocatalyst for the industrial production of semi-synthetic β-lactam antibiotics.

[0003] Compared to traditional PGAs, PGA derived from Bacillus megaterium (BmPGA) possesses unique catalytic properties and thermal stability. However, due to the high demand for β-lactam antibiotics, current BmPGA exhibits poor activity and catalytic performance, making it incapable of continuously and efficiently catalyzing the semi-synthesis of β-lactam antibiotics. With the continuous advancement of structural biology, the three-dimensional structure of PGA, as well as its binding sites and modes with substrates, have become more clearly understood. Based on this current three-dimensional structural analysis, to enhance the activity and catalytic performance of PGA, specific sites in PGA are mutated to increase its affinity for substrates, thereby increasing the ratio of PGA synthase activity to hydrolase activity (S / H).

[0004] However, most existing technologies mutate large side chain amino acids near the substrate binding pocket into small side chain amino acids. In addition, for the functional stability of PGA, the number of mutation sites is mostly maintained at 1 or 2. It is difficult to effectively improve the affinity of PGA with the substrate through these mutation sites, which ultimately leads to poor performance of PGA's synthase activity and hydrolase activity. Summary of the Invention

[0005] The present application provides a recombinant penicillin G acylase and a preparation method and application thereof, in order to solve the following technical problem: how to improve the affinity between penicillin G acylase and a substrate.

[0006] In a first aspect, an embodiment of the present application provides a recombinant penicillin G acylase, the amino acid sequence of the recombinant penicillin G acylase comprising three mutation sites: site 171, site 647, and site 266, the mutated amino acid at site 171 being tyrosine, the mutated amino acid at site 647 being arginine, and the mutated amino acid at site 266 comprising one of the following: glutamine, methionine, and arginine.

[0007] Optionally, the mutated amino acid at position 266 is arginine.

[0008] Optionally, the recombinant penicillin G acylase has the first amino acid sequence shown in SEQ ID NO.1; and / or

[0009] The recombinant penicillin G acylase has a first nucleotide sequence as shown in SEQ ID NO.2.

[0010] Optionally, the recombinant penicillin G acylase has the second amino acid sequence shown in SEQ ID NO.3; and / or

[0011] The recombinant penicillin G acylase has a second nucleotide sequence as shown in SEQ ID NO.4.

[0012] Optionally, the recombinant penicillin G acylase has the third amino acid sequence shown in SEQ ID NO.5; and / or

[0013] The recombinant penicillin G acylase has the third nucleotide sequence shown as SEQ ID NO.6.

[0014] In a second aspect, the present invention provides a method for preparing the recombinant penicillin G acylase according to the first aspect, the method comprising:

[0015] Designing the target nucleotide sequence of the recombinant penicillin G acylase according to the first aspect;

[0016] Designing a primer set based on the expression vector and the target nucleotide sequence;

[0017] Using the primer set to amplify the target nucleotide sequence to obtain an amplified product;

[0018] Performing double enzyme digestion on the expression vector and the amplified product to obtain an enzyme-digested vector and an enzyme-digested amplified product;

[0019] Connecting the enzyme-digested vector and the enzyme-digested amplified product to obtain a recombinant plasmid;

[0020] introducing the recombinant plasmid into competent cells to obtain recombinant cells;

[0021] The recombinant cell is cultured to express the target nucleotide sequence, thereby obtaining recombinant penicillin G acylase.

[0022] Optionally, the primer set includes a first upstream primer and a first downstream primer for amplifying site 171 of the target nucleotide sequence, the first upstream primer having a nucleotide sequence as shown in SEQ ID NO.5, and the first downstream primer having a nucleotide sequence as shown in SEQ ID NO.6; and / or

[0023] The primer set also includes a second upstream primer and a second downstream primer for amplifying site 647 of the target nucleotide sequence, the second upstream primer has a nucleotide sequence as shown in SEQ ID NO.7, and the second downstream primer has a nucleotide sequence as shown in SEQ ID NO.8.

[0024] Optionally, the primer set further includes a third upstream primer and a third downstream primer for amplifying site 266 of the target nucleotide sequence, wherein the third downstream primer has a nucleotide sequence as shown in SEQ ID NO.11; the third upstream primer satisfies:

[0025] When the mutant amino acid at position 266 is glutamine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.12;

[0026] When the mutated amino acid at position 266 is methionine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.13;

[0027] When the mutated amino acid at position 266 is arginine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.14.

[0028] In a third aspect, an embodiment of the present application provides a recombinant vector of recombinant penicillin G acylase, wherein the recombinant vector comprises an expression vector and the nucleotide sequence of the recombinant penicillin G acylase according to the first aspect carried on the expression vector.

[0029] In a fourth aspect, an embodiment of the present application provides a recombinant strain of recombinant penicillin G acylase, wherein the recombinant strain comprises the recombinant vector described in the third aspect or the nucleotide sequence of the recombinant penicillin G acylase described in the first aspect.

[0030] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0031] The present application provides a recombinant penicillin G acylase having three mutation sites, which are located at sites 171, 647, and 266 of the recombinant penicillin G acylase, respectively. By mutating the phenylalanine at site 171 to tyrosine, a hydroxyl group with an aromatic ring can be formed at site 171 of the recombinant penicillin G acylase. This hydroxyl group can bind to the target substrate through hydrogen bonds, thereby enhancing the ability of the recombinant penicillin G acylase to recognize and bind to the target substrate. In addition, by mutating the tyrosine at site 647 to arginine, the branched chain distribution of the amino acid group at site 647 of the recombinant penicillin G acylase can be improved, so that a shorter hydrogen bond structure is formed between the branched chain of the amino acid group and the target substrate. This hydrogen bond structure can improve the ability of the recombinant penicillin G acylase to recognize and bind to the target substrate. The invention relates to a novel method for improving the binding affinity between recombinant penicillin G acylase and the target substrate; in addition, the serine at position 266 is mutated to glutamine or methionine arginine, and multiple cyclic or chain groups can be introduced into position 266 of the recombinant penicillin G acylase, and these cyclic or chain groups can optimize the spatial configuration of the binding pocket between the recombinant penicillin G acylase and the target substrate, thereby ensuring the stability of the environment for the binding of the target substrate to the recombinant penicillin G acylase, and improving the binding affinity between the recombinant penicillin G acylase and the target substrate; in addition, the serine at position 266 is mutated to arginine, and arginine side chains can be introduced into position 266 of the recombinant penicillin G acylase, and these arginine side chains can further increase the number of hydrogen bonds between the penicillin G acylase and the target substrate, so as to further improve the binding affinity between the recombinant penicillin G acylase and the target substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0033] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0034] Figure 1 A schematic diagram of the molecular structure of a recombinant penicillin G acylase provided in an embodiment of the present application; wherein, Figure 1 A is the molecular structure analysis diagram of BmPGA enzyme containing three mutation sites: F171R: S266M: Y647R. Figure 1 B is the molecular structure analysis diagram of BmPGA enzyme containing three mutation sites: F171R: S266Q: Y647R. Figure 1 C is the molecular structure analysis diagram of BmPGA enzyme containing three mutation sites: F171R: S266R: Y647R. Figure 1 D is the molecular structure analysis diagram of BmPGA enzyme containing three mutation sites: F171Y: S266M: Y647R. Figure 1 E is the molecular structure analysis diagram of BmPGA enzyme containing three mutation sites: F171Y: S266Q: Y647R. Figure 1 F is a molecular structure analysis diagram of the BmPGA enzyme containing three mutation sites: F171Y:S266R:Y647R. In the figure, the aa dotted line represents a hydrogen bond, the bb dotted line represents a p-π bond, and the cc dotted line represents an ionic bond.

[0035] Figure 2 A schematic flow chart of a method for preparing recombinant penicillin G acylase provided in an embodiment of the present application;

[0036] Figure 3 Statistical graphs of the affinity of BmPGA enzymes with single and multiple mutation sites and target substrates provided in the examples of this application;

[0037] Figure 4 Agarose gel electrophoresis results of the recombinant enzyme of the BmPGA enzyme mutant containing multiple mutation sites provided in the examples of the present application; wherein, lane 1 is the uninduced bacterial solution, lane 2 is the whole bacterial solution, lane 3 is the unpurified supernatant solution, lane 4 is the purified supernatant solution, and lane 5 is the bacterial solution;

[0038] Figure 5 This is a diagram showing the results of liquid chromatography detection of the enzymatic hydrolysate of the BmPGA enzyme containing multiple mutation sites provided in the examples of the present application;

[0039] Figure 6 Schematic diagram of the molecular structure of the wild-type BmPGA enzyme provided in the examples of this application. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0041] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range; for example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0042] As used herein, the terms "including," "comprising," and "including" mean "including but not limited to." Relational terms such as "first" and "second" are used solely to distinguish one entity or operation from another and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or" describes an association between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, and B exists alone; A and B can be singular or plural. "At least one" means one or more, "plurality" means two or more, "at least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can each be single or plural. "Parts" notation, such as parts by weight or parts by mass, indicates the proportional relationship between components. For proportional relationships described herein, the parameters that require ratio description should be understood as the first term of the proportional formula, in the order in which they are described, and the proportional figures should be understood as the second term. For example, if the mass ratio of substances A, B, and C is 1:2:3, then substances A, B, and C should correspond to the proportional figures in the proportional formula, in the order in which they are described. That is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0043] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this article can be purchased from the market or prepared by existing methods.

[0044] Figure 1 The molecular structure diagram of a recombinant penicillin G acylase provided in the examples of the present application is exemplarily shown;

[0045] like Figure 1 As shown, an embodiment of the present application provides a recombinant penicillin G acylase, the amino acid sequence of the recombinant penicillin G acylase includes three mutation sites: site 171, site 647 and site 266, the mutated amino acid at site 171 is tyrosine, the mutated amino acid at site 647 is arginine, and the mutated amino acid at site 266 includes one of the following: glutamine, methionine and arginine.

[0046] It should be noted that the recombinant penicillin G acylase is penicillin G acylase (BmPGA) derived from Bacillus megaterium.

[0047] It should be noted that PGA from Bacillus megaterium contains alanine at positions 171, 647, and 266. Since the side chain of alanine is a non-polar methyl group, replacing the non-polar methyl group has little effect on the overall structure of the recombinant penicillin G acylase protein. Site processing analysis of alanine can be used as an effective means to predict the functional sites of penicillin G acylase.

[0048] It should be noted that the recombinant penicillin G acylase provided in the examples of this application significantly improves the catalytic efficiency and binding affinity of the recombinant penicillin G acylase with the target substrate by synergistically optimizing the multidimensional structural characteristics of the enzyme-substrate interaction through three key mutation sites (F171Y, Y647R, and S266Q / M / R). The specific mechanism is as follows:

[0049] ‌1. F171Y mutation (phenylalanine → tyrosine): aromatic ring hydroxylation and hydrogen bond network reconstruction:‌

[0050] Structural basis: Both phenylalanine (F) and tyrosine (Y) contain aromatic rings, but Y has a hydroxyl group (-OH) introduced at the para position of the phenyl ring. This hydroxyl group enhances substrate recognition through two mechanisms:

[0051] (1) Directed hydrogen bond formation: The hydroxyl group of tyrosine forms a strong hydrogen bond with the carbonyl oxygen of the phenylacetyl group of penicillin G (or the amino group of the β-lactam ring), precisely anchoring the polar region of the substrate.

[0052] (2) π-π stacking optimization: The retained aromatic ring maintains hydrophobic interaction with the substrate phenylacetyl group, while the hydroxyl-induced electron cloud rearrangement can enhance the overlap of π-π orbitals and improve the binding energy between recombinant penicillin G acylase and the target substrate.

[0053] (3) Dynamic effect: The hydroxyl group of Y171 can stabilize the local conformation of the substrate binding pocket through a water-mediated hydrogen bond network, reducing the entropy loss of the bound state conformation.

[0054] ‌2. Y647R mutation (tyrosine → arginine): strong short-range interaction mediated by guanidine group:‌

[0055] (1) Charge complementarity and geometric adaptation:

[0056] The phenolic hydroxyl group of the original Y647 is generally neutral, while the guanidine group of arginine (R) is positively charged after protonation, which can form a salt bridge with the carboxylic acid group of the target substrate, thereby enhancing the electrostatic potential energy contribution of this site.

[0057] In addition, the flexible side chain of R647 can shorten the hydrogen bond distance between recombinant penicillin G acylase and the target substrate through conformational adjustment, forming a more stable "low potential energy binding state".

[0058] (2) Elimination of steric hindrance: The Y→R mutation can remove a large number of benzene rings, reduce the volume of the benzene ring of recombinant penicillin G acylase, avoid steric conflicts between recombinant penicillin G acylase and some side chains of target substrates, make it easier for target substrates to enter the catalytic center, and improve the catalytic efficiency of target substrates.

[0059] ‌3. S266Q / M / R mutation (serine → glutamine / methionine / arginine): remodeling of binding pocket topology:‌

[0060] (1) Common mechanisms:

[0061] 1) Volume-filling effect: Replacing the small side chain of S266 with Q, M, or R can fill the hydrophobic cavity of the binding pocket of recombinant penicillin G acylase, induce the target substrate to adopt a more optimal orientation rule, and improve the binding affinity between the target substrate and recombinant penicillin G acylase.

[0062] 2) Conformational locking effect: The large side chains of Q, M, or R restrict the local flexibility of the binding pocket, stabilize the active conformation of the key catalytic residues, and enhance the binding strength between the target substrate and recombinant penicillin G acylase.

[0063] (2) Specific mechanism:

[0064] ‌Q266‌: The amide group of glutamine forms a hydrogen bond with the thioether bond of the target substrate. At the same time, the long side chain of glutamine can induce the substrate β-lactam ring to be closer to the catalytic nucleophilic attack site, thereby improving the catalytic efficiency of recombinant penicillin G acylase.

[0065] ‌M266‌: The thioether group of methionine can stabilize the non-polar region of the target substrate through a hydrophobic effect and participate in sulfur-π interactions, thereby improving the binding affinity of recombinant penicillin G acylase.

[0066] ‌R266‌: The guanidine group of arginine can form a three-center hydrogen bond network with the hydroxyl group of the C6 chain of some target substrates and water molecules, which can increase 2 to 3 hydrogen bonds for the binding of recombinant penicillin G acylase to the target substrate, and comprehensively enhance the binding strength of recombinant penicillin G acylase to the target substrate through cation-π interaction.

[0067] 3. Synergistic effect and global optimization: based on the synergistic effect of three mutation sites;

[0068] (1) Binding energy additivity: The three mutation sites can reduce the total binding free energy.

[0069] (2) Improved catalytic efficiency: Based on the binding affinity between recombinant penicillin G acylase and the target substrate, the target substrate can bind to penicillin G acylase in a directional manner. The catalytic efficiency kcat / Km value and transition state stability of recombinant penicillin G acylase can be increased by orders of magnitude through three mutation sites.

[0070] (3) Broadened pH stability: The buffering effect of R647 and R266 enables the recombinant penicillin G acylase to maintain high activity at pH 7.5-9.0 (a pH range that is 1.5 pH units wider than that of traditional penicillin G acylase).

[0071] In some optional embodiments, the mutated amino acid at position 266 is arginine.

[0072] In these embodiments, the mutant amino acid at position 266 can be arginine. By introducing an arginine side chain at position 266 of the recombinant penicillin G acylase, the number of hydrogen bonds between the recombinant penicillin G acylase and the target substrate can be increased, thereby further improving the binding affinity between the recombinant penicillin G acylase and the target substrate.

[0073] In some optional embodiments, the recombinant penicillin G acylase has the first amino acid sequence shown in SEQ ID NO.1; and / or

[0074] The recombinant penicillin G acylase has a first nucleotide sequence as shown in SEQ ID NO.2.

[0075] In these embodiments, the recombinant penicillin G acylase may have a first amino acid sequence as shown in SEQ ID NO.1 or the recombinant penicillin G acylase may have a first nucleotide sequence as shown in SEQ ID NO.2. When the mutated amino acid at position 171 is tyrosine, the mutated amino acid at position 647 is arginine, and the mutated amino acid at position 266 is arginine, the specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase may be determined, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity to the target substrate based on the synergistic optimization of the multidimensional structural characteristics of the enzyme-substrate interaction.

[0076] In some optional embodiments, the recombinant penicillin G acylase has the second amino acid sequence shown in SEQ ID NO. 3; and / or

[0077] The recombinant penicillin G acylase has a second nucleotide sequence as shown in SEQ ID NO.4.

[0078] In these embodiments, the recombinant penicillin G acylase may have a second amino acid sequence as shown in SEQ ID NO.3 or the recombinant penicillin G acylase may have a second nucleotide sequence as shown in SEQ ID NO.4. When the mutated amino acid at position 171 is tyrosine, the mutated amino acid at position 647 is arginine, and the mutated amino acid at position 266 is glutamine, the specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase may be clarified, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity to the target substrate based on the synergistic optimization of the multidimensional structural characteristics of the enzyme-substrate interaction.

[0079] In some optional embodiments, the recombinant penicillin G acylase has the third amino acid sequence shown in SEQ ID NO.5; and / or

[0080] The recombinant penicillin G acylase has the third nucleotide sequence shown as SEQ ID NO.6.

[0081] In these embodiments, the recombinant penicillin G acylase may have a third amino acid sequence as shown in SEQ ID NO.5 or the recombinant penicillin G acylase may have a third nucleotide sequence as shown in SEQ ID NO.6. When the mutated amino acid at position 171 is tyrosine, the mutated amino acid at position 647 is arginine, and the mutated amino acid at position 266 is methionine, the specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase may be clarified, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity to the target substrate based on the synergistic optimization of the multidimensional structural characteristics of the enzyme-substrate interaction.

[0082] Figure 2 The following is a schematic flow chart of a method for preparing recombinant penicillin G acylase provided in an embodiment of the present application;

[0083] Based on a general inventive concept, such as Figure 2 As shown, the present embodiment provides a method for preparing the recombinant penicillin G acylase, the method comprising:

[0084] S1. Designing the target nucleotide sequence of the recombinant penicillin G acylase;

[0085] S2. Designing a primer set based on the expression vector and the target nucleotide sequence;

[0086] S3. using the primer set to amplify the target nucleotide sequence to obtain an amplified product;

[0087] S4. The expression vector and the amplified product are double-digested to obtain a digested vector and a digested amplified product;

[0088] S5. Connecting the digested vector and the digested amplified product to obtain a recombinant plasmid;

[0089] S6. introducing the recombinant plasmid into competent cells to obtain recombinant cells;

[0090] S7. Cultivating the recombinant cell to express the target nucleotide sequence and obtain recombinant penicillin G acylase.

[0091] This method is a method for preparing the above-mentioned recombinant penicillin G acylase. The specific information of the recombinant penicillin G acylase can be referred to the above-mentioned embodiments. Since this method adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described in detail here.

[0092] In some optional embodiments, the primer set includes a first upstream primer and a first downstream primer for amplifying site 171 of the target nucleotide sequence, the first upstream primer having a nucleotide sequence as shown in SEQ ID NO.7, and the first downstream primer having a nucleotide sequence as shown in SEQ ID NO.8; and / or

[0093] The primer set also includes a second upstream primer and a second downstream primer for amplifying site 647 of the target nucleotide sequence, the second upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, and the second downstream primer having a nucleotide sequence as shown in SEQ ID NO.10.

[0094] In these embodiments, the first upstream primer of the primer set has a nucleotide sequence as shown in SEQ ID NO.7, and the first downstream primer of the primer set has a nucleotide sequence as shown in SEQ ID NO.8, and the second upstream primer of the primer set has a nucleotide sequence as shown in SEQ ID NO.9, and the second downstream primer of the primer set has a nucleotide sequence as shown in SEQ ID NO.10. The target nucleotide sequence is fully amplified by the primer set, thereby facilitating the subsequent double enzyme digestion and ligation process to obtain a sufficient amount of amplification product.

[0095] In some optional embodiments, the primer set further includes a third upstream primer and a third downstream primer for amplifying position 266 of the target nucleotide sequence, wherein the third downstream primer has a nucleotide sequence as shown in SEQ ID NO.11; the third upstream primer satisfies:

[0096] When the mutant amino acid at position 266 is glutamine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.12;

[0097] When the mutated amino acid at position 266 is methionine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.13;

[0098] When the mutated amino acid at position 266 is arginine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.14.

[0099] In these embodiments, the primer set also includes a third upstream primer and a third downstream primer. The nucleotide sequence of the third downstream primer is first confirmed as shown in SEQ ID NO.11, and then, according to the type of the mutated amino acid at position 266, the nucleotide sequences shown in SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 are used respectively. By combining these designed third upstream primers and third downstream primers, the target nucleotide sequence can be fully amplified, thereby facilitating the subsequent double enzyme digestion and ligation process to obtain a sufficient amount of amplified product.

[0100] Based on a general inventive concept, an embodiment of the present application provides a recombinant vector of recombinant penicillin G acylase, wherein the recombinant vector comprises an expression vector and a nucleotide sequence of the recombinant penicillin G acylase carried on the expression vector.

[0101] The recombinant vector is realized based on the above-mentioned recombinant penicillin G acylase. The specific information of the recombinant penicillin G acylase can be referred to the above-mentioned embodiments. Since the recombinant vector adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described in detail here.

[0102] Based on a general inventive concept, an embodiment of the present application provides a recombinant strain of recombinant penicillin G acylase, wherein the recombinant strain comprises the recombinant vector or the nucleotide sequence of the recombinant penicillin G acylase.

[0103] The recombinant strain is realized based on the above-mentioned recombinant penicillin G acylase. The specific information of the recombinant penicillin G acylase can be referred to the above-mentioned embodiments. Since the recombinant strain adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be described in detail here.

[0104] The present application is further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national / industry standards. If there are no corresponding national / industry standards, the methods are carried out in accordance with commonly used international standards, conventional conditions, or conditions recommended by the manufacturer.

[0105] Example 1

[0106] 1. Selection of amino acid sites:

[0107] (1) Alanine scanning (ABS) was performed on the active pocket region of BmPGA to calculate the binding free energy change after each residue was mutated to alanine ( ).by The screening criteria of ≥0 kcal / mol identified the following key residue sites: TYR170, PHE171, SER266, GLN288, TYR336, ASN510, ARG531, ARG646, TYR647 and LEU710, which are believed to play an important role in the binding of BmPGA to the substrate.

[0108] (2) Based on these key residue sites, the CalculateMutation Energy (Binding) module in Discovery Studio 2019 was used to calculate and analyze the binding affinity of single residue mutations and multi-residue combined mutations to the substrate to confirm the affinity of different mutants to the substrate; the calculation parameters were set as follows: using the CHARMM force field, a dielectric constant of 10, and a solvent dielectric constant of 80; the energy term scaling factors were 0.5 for electrostatic interaction (Electrostatic), 0.5 for van der Waals force (VDW), and 0.8 for entropy effect (Entropy).

[0109] (3) According to the change of binding free energy ≤-0.10 kcal / mol was used as the screening criterion to screen mutants with higher affinity, including F171R, F171Y, S266R, S266Q, S266M, Y647R and Y170H. The two-dimensional weak interaction changes of these mutants were further analyzed. Taking into account the changes in weak interactions and the size of substrate affinity, it was determined that positions 171, 266 and 647 of BmPGA were key residue positions, and the mutant amino acids at these key residue positions were identified as F171Y, Y647R and S266Q / M / R.

[0110] Example 2

[0111] 2. Preparation of recombinant penicillin G acylase:

[0112] (1) Based on the key residue sites and the types of corresponding mutant amino acids screened in Example 1, the specific amino acid sequence of BmPGA is as follows:

[0113] When the mutant amino acid at position 171 is tyrosine, the mutant amino acid at position 647 is arginine, and the mutant amino acid at position 266 is arginine, the amino acid sequence of BmPGA is: MKMKWLISVIILFVFIFPQNLVFAGEDKNEGVKVVRDNFGVPHLYAKNKKDLYEAYGYVMAKDRLFQLEMFRRGNEGTVSEIFGEDYLSKDEQSRRDGYSNKEIKKMIDGLDRQPKELIAKFAEGISRYVNEALKDPDDKLSKEFHEYQFLPQKWTSTDVVRVYMVSMTY Y MDNHQELKNAEILAKLEHEYGTEVSRKMFDDLVWKNDPSAPTSIVSEGKPKRESSSQSLQKLSSAVIKASEKVGKERENFVQSSEELGLPLKIG R NAAIVGSEKSATGNALLFSGPQVGFVAPGFLYEVGLHAPGFDMEGSGFIGYPFIMFGANNHFALSATAGYGNVTDIFEEKLNTKNSSQYLYKGKWRDMEKRKESFTVKGDNGEKKTVEKIYYRTVHGPVISRDETNKVAYSKSWSFRGTEAQSMSAYMKANWAKNLKEFENAASEYTMSLNWYYADKKGD IAYYHVGRYPVRNNKIDERIPTPGTGEEYEWKGFIPFKENPHVINPKNGYVVNWNNKPSKEWVNGEYSYYWGEDNRVQQYINGMEARGKVTLEDINEINYTASFAQLRANLFKPLLIDVLDKNKSTNGNYAYLIEKLEEWNNLKEDENKDGYYDAGIAAFFDEWWNNLHDKLFMDELGDFYGITKEITDHR R GASLAYKILSKESTNYKWVNVDQEKIIMESTNEVLAKLQSEKGLKAEKWRMPIKTMTFGEKSLIGIPHGYGSMTPIIEMNRGSENHYIEMTPKGPSGFNITPPGQIGFVKKDGTISDHYDDQLVMFAEWKFKPYLFNKKDIYKSAKNVSALNMSK (SEQ ID NO. 1), wherein the bold and underlined parts are mutation sites; the nucleotide sequence of BmPGA is:

[0114] ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC CGA CGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTGAAAT GAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.2), where the bold and underlined parts are the mutation points;

[0115] When the mutant amino acid at position 171 is tyrosine, the mutant amino acid at position 647 is arginine, and the mutant amino acid at position 266 is glutamine, the amino acid sequence of BmPGA is: MKMKWLISVIILFVFIFPQNLVFAGEDKNEGVKVVRDNFGVPHLYAKNKKDLYEAYGYVMAKDRLFQLEMFRRGNEGTVSEIFGEDYLSKDEQSRRDGYSNKEIKKMIDGLDRQPKELIAKFAEGISRYVNEALKDPDDKLSKEFHEYQFLPQKWTSTDVVRVYMVSMTY Y MDNHQELKNAEILAKLEHEYGTEVSRKMFDDLVWKNDPSAPTSIVSEGKPKRESSSQSLQKLSSAVIKASEKVGKERENFVQSSEELGLPLKIG QNAAIVGSEKSATGNALLFSGPQVGFVAPGFLYEVGLHAPGFDMEGSGFIGYPFIMFGANNHFALSATAGYGNVTDIFEEKLNTKNSSQYLYKGKWRDMEKRKESFTVKGDNGEKKTVEKIYYRTVHGPVISRDETNKVAYSKSWSFRGTEAQSMSAYMKANWAKNLKEFENAASEYTMSLNWYYADKKGDIAYYHVGRYPVRNNKIDERIPTPGTGEYEWKGFIPFKENPHVINPKNGYVVNWNNKPSKEWVNGEYSYYWGEDNRVQQYINGMEARGKVTLEDINEINYTASFAQLRANLFKPLLIDVLDKNKSTNGNYAYLIEKLEEWNNLKEDENKDGYYDAGIAAFFDEWWNNLHDKLFMDELGDFYGITKEITDHR R GASLAYKILSKESTNYKWVNVDQEKIIMESTNEVLAKLQSEKGLKAEKWRMPIKTMTFGEKSLIGIPHGYGSMTPIIEMNRGSENHYIEMTPKGPSGFNITPPGQIGFVKKDGTISDHYDDQLVMFAEWKFKPYLFNKKDIYKSAKNVSALNMSK (SEQ ID NO.3), wherein the bold and underlined parts are the mutation sites; the nucleotide sequence of BmPGA is:

[0116] ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC CAA CGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTGAAAT GAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.4), where the bold and underlined parts are the mutation points;

[0117] When the mutant amino acid at position 171 is tyrosine, the mutant amino acid at position 647 is arginine, and the mutant amino acid at position 266 is methionine, the amino acid sequence of BmPGA is: MKMKWLISVIILFVFIFPQNLVFAGEDKNEGVKVVRDNFGVPHLYAKNKKDLYEAYGYVMAKDRLFQLEMFRRGNEGTVSEIFGEDYLSKDEQSRRDGYSNKEIKKMIDGLDRQPKELIAKFAEGISRYVNEALKDPDDKLSKEFHEYQFLPQKWTSTDVVRVYMVSMTY Y MDNHQELKNAEILAKLEHEYGTEVSRKMFDDLVWKNDPSAPTSIVSEGKPKRESSSQSLQKLSSAVIKASEKVGKERENFVQSSEELGLPLKIG MNAAIVGSEKSATGNALLFSGPQVGFVAPGFLYEVGLHAPGFDMEGSGFIGYPFIMFGANNHFALSATAGYGNVTDIFEEKLNTKNSSQYLYKGKWRDMEKRKESFTVKGDNGEKKTVEKIYYRTVHGPVISRDETNKVAYSKSWSFRGTEAQSMSAYMKANWAKNLKEFENAASEYTMSLNWYYADKKGDIAYYHVGRYPVRNNKIDERIPTPGTGEYEWKGFIPFKENPHVINPKNGYVVNWNNKPSKEWVNGEYSYYWGEDNRVQQYINGMEARGKVTLEDINEINYTASFAQLRANLFKPLLIDVLDKNKSTNGNYAYLIEKLEEWNNLKEDENKDGYYDAGIAAFFDEWWNNLHDKLFMDELGDFYGITKEITDHR R GASLAYKILSKESTNYKWVNVDQEKIIMESTNEVLAKLQSEKGLKAEKWRMPIKTMTFGEKSLIGIPHGYGSMTPIIEMNRGSENHYIEMTPKGPSGFNITPPGQIGFVKKDGTISDHYDDQLVMFAEWKFKPYLFNKKDIYKSAKNVSALNMSK (SEQ ID NO.5), wherein the bold and underlined parts are the mutation sites; the nucleotide sequence of BmPGA is:

[0118] ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC ATG CGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTGAAAT GAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.6), where the bold and underlined parts are the mutation points.

[0119] (2) Based on the nucleotide sequence designed in step (1), synthesize the corresponding target gene, then select a suitable expression vector, and determine the restriction site and design the amplification primer set based on the expression vector and the above nucleotide sequence. The specific sequence of the amplification primer set is as follows:

[0120] For the case where the mutant amino acid at position 171 is tyrosine, the first upstream primer is ATGACGTAT TAT ATGGATAATC (SEQ ID NO. 7), wherein the bold and underlined parts are the mutation sites; the first downstream primer: GCCTATCTTTAACGGTAATCCA (SEQ ID NO. 8);

[0121] For the case where the mutant amino acid at position 647 is arginine, the second upstream primer is: CCGATCATCGT CGA GGGGCTTC (SEQ ID NO. 9), where the bold and underlined parts are the mutation sites; the second downstream primer: CTACTTACTCATATTTAATGCGC (SEQ ID NO. 10);

[0122] For the case where the mutant amino acid at position 266 is glutamine, the third upstream primer is: TTAAAGATAGGC CAA AATGCCG (SEQ ID NO. 12); third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO. 11);

[0123] For the case where the mutant amino acid at position 266 is methionine, the third upstream primer is: TAAAGATAGGC ATG AATGCCGCC (SEQ ID NO. 13); third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO. 11);

[0124] For the case where the mutant amino acid at position 266 is arginine, the third upstream primer is: AGATAGGC CGA AATGCCGCCAT (SEQ ID NO. 14); the third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO. 11).

[0125] In addition, an original amplification primer set was designed based on the original nucleotide sequence of BmPGA without mutation. The specific sequence of the original amplification primer set is as follows:

[0126] The upstream primer of the original amplification primer set is ATGAAGATGAAGTGGCTAATATC (SEQ ID NO. 15); the downstream primer of the original amplification primer set is CTACTTACTCATATTTAATGCGC (SEQ ID NO. 10).

[0127] (3) Use the designed amplification primer set to amplify the target gene to obtain the amplification product:

[0128] Using the different primers designed above, according to the combination of different mutation sites, corresponding upstream and downstream primers were used to perform PCR amplification according to the amplification system shown in Table 1 and the amplification program shown in Table 2, and amplification products of different mutation site combinations were obtained.

[0129] Table 1 PCR amplification reaction system

[0130]

[0131] Table 2 PCR amplification reaction procedure

[0132]

[0133] (4) Select the corresponding endonuclease group according to the restriction site, and use the endonuclease group to digest the expression vector and the amplified product respectively to obtain the digested vector and digested product:

[0134] Different double enzyme digestion systems were selected according to the designed BmPGA nucleotide sequence and the original BmPGA nucleotide sequence. This application used the Ndel and Xhol double enzyme digestion system, and then used the pEASY-T3 vector as the expression vector; 4.0 μL of amplified product and 1.5 μL of expression vector were digested with 0.5 μL of Ndel restriction endonuclease and 0.5 μL of Xhol restriction endonuclease respectively under water bath conditions (digestion temperature was 37 ° C, digestion time was 1 h) to obtain digestion vectors and digestion products.

[0135] (5) Connect the digested vector and digested product with DNA ligase to obtain a recombinant plasmid:

[0136] Take 4 μL of the enzyme-digested product and 1 μL of the enzyme-digested vector and mix them, then add 1.5 μL of DNA ligase for ligation to obtain the recombinant plasmid vector;

[0137] (6) Introduce the recombinant plasmid into competent cells to obtain recombinant cells:

[0138] 50 μL of competent cells of Bacillus megaterium (BM) were selected, and 5 μL of recombinant plasmid vector was added and mixed to obtain a mixed product; the mixed product was treated in an ice bath for 40 minutes, and then transferred to a 42°C water bath environment (temperature error within ±0.3°C) for heat shock treatment for 30 seconds to allow the recombinant vector to enter the competent cells, thereby obtaining heat-shocked cells; the heat-shocked cells were then immediately transferred to an ice bath environment and allowed to stand for 2 minutes to obtain recombinant cells.

[0139] (7) Cultivating the recombinant cells in a culture medium to express the target gene and obtain recombinant penicillin G acylase:

[0140] Add the recombinant cells to 500 μL of LB recovery medium (without antibiotics) and culture at 37°C and 180 rpm for 2 h to activate the expression of the kanamycin resistance gene (the pET vector itself carries the KanR gene) to obtain a recombinant bacterial solution.

[0141] The recombinant bacterial suspension was then inoculated onto LB-resistant plates (containing 100 μg / mL karatomycin) at a 2% inoculum volume and cultured for 14 days. A flame-sterilized toothpick was then used to pick a typical single colony (1–2 mm in diameter) from the surface of the LB-resistant plate, avoiding contact with adjacent colonies or scratched areas on the agar, to obtain the recombinant bacteria.

[0142] The selected recombinant bacteria were inoculated into liquid culture medium and cultured for 7 days to obtain seed culture solution;

[0143] The seed culture was inoculated into 200 mL of fermentation medium, cultured at 37°C and 180 rpm with shaking for 4 h, and then induced overnight with IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.3 mM for 12 h to obtain fermentation broth.

[0144] The fermentation broth is purified and dried to obtain recombinant penicillin G acylase.

[0145] Related experiments and effect data:

[0146] 1. For the experiment in Example 1, the specific data are as follows:

[0147] (1) Affinity of different mutants:

[0148] The binding affinity of different mutants to substrates is as follows Figure 3 The results show that multi-site mutants have a significant synergistic effect, and the stability of the expressed BmPGA is superior to that of single-site mutants. However, multi-site mutants may have some impact on the overall structure and biological activity of BmPGA. Based on this, the next step of experimental analysis was carried out.

[0149] 2. For the selected mutants, according to the different combinations of mutation sites, the mutant containing F171Y:S266R:Y647R mutation site was recorded as mutant 1, the mutant containing F171Y:S266Q:Y647R mutation site was recorded as mutant 2, and the mutant containing F171Y:S266M:Y647R mutation site was recorded as mutant 3. The results of agarose gel electrophoresis of the recombinant cells are shown in the figure. Figure 4 shown.

[0150] In addition, the catalytic ability of these BmPGA enzymes as catalysts was investigated. The specific process is as follows:

[0151] Weigh 3 mg of substrate and add 4 mg of sodium carbonate to the substrate, then add 1 mL of water to dissolve the mixture, and then add the organic reagent needed to extract the substrate with EA three times to obtain the substrate to be enzymatically hydrolyzed; Figure 6 The unmutated BmPGA enzyme shown was added to the substrate to be hydrolyzed and hydrolyzed at 30°C for 1 hour to obtain hydrolysis products; the content of these hydrolysis products was detected by liquid chromatography to determine their catalytic performance. The results are as follows: Figure 5As shown, this indicates that the BmPGA enzyme mutant containing the three mutation sites F171Y:S266R:Y647R has good catalytic ability, and its catalytic ability is improved by 5 times or more compared with the unmutated BmPGA enzyme. At the same time, the substrate conversion rate of the BmPGA enzyme mutant is above 98%.

[0152] 3. For the selected mutants, the changes in folding free energy and two-dimensional weak interactions of these mutants were investigated. The results are as follows: Figure 6 As shown, the combination of the three mutation sites F171Y:S266R:Y647R can meet the requirements of stable expression, stable biological activity and stable overall structure of BmPGA.

[0153] In summary, the embodiments of the present application provide a recombinant penicillin G acylase, which significantly improves the functional stability of the recombinant penicillin G acylase and its binding affinity to the target substrate by synergistically optimizing the multidimensional structural characteristics of the enzyme-substrate interaction through three key mutation sites (F171Y, Y647R, and S266Q / M / R).

[0154] In addition, the recombinant penicillin G acylase provided in the embodiments of the present application can be used as an efficient catalytic enzyme in penicillin G acylase-related industries, such as the penicillin catalysis industry.

[0155] The foregoing is merely a detailed description of the present invention, intended to enable those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein, but rather is intended to conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A recombinant penicillin G acylase, wherein the recombinant penicillin G acylase has the first amino acid sequence shown in SEQ ID NO. 1; or The recombinant penicillin G acylase has the second amino acid sequence shown in SEQ ID NO.3; or The recombinant penicillin G acylase has the third amino acid sequence shown in SEQ ID NO.

5.

2. A method for preparing the recombinant penicillin G acylase according to claim 1, comprising: Designing a target nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase according to claim 1; Designing a primer set based on the target nucleotide sequence corresponding to the expression vector and the amino acid sequence of the recombinant penicillin G acylase; using the primer set to amplify the target nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase to obtain an amplified product; Performing double enzyme digestion on the expression vector and the amplified product to obtain an enzyme-digested vector and an enzyme-digested amplified product; Connecting the enzyme-digested vector and the enzyme-digested amplified product to obtain a recombinant plasmid; introducing the recombinant plasmid into competent cells to obtain recombinant cells; The recombinant cell is cultured to express the target nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase, thereby obtaining the recombinant penicillin G acylase.

3. The method according to claim 2, characterized in that The primer set comprises a first upstream primer and a first downstream primer for amplifying position 171 of the target nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase, wherein the first upstream primer has a nucleotide sequence as shown in SEQ ID NO. 7, and the first downstream primer has a nucleotide sequence as shown in SEQ ID NO. 8; and / or The primer set also includes a second upstream primer and a second downstream primer for amplifying site 647 of the target nucleotide sequence, the second upstream primer having a nucleotide sequence as shown in SEQ ID NO.9, and the second downstream primer having a nucleotide sequence as shown in SEQ ID NO.

10.

4. The method according to claim 2, characterized in that The primer set further includes a third upstream primer and a third downstream primer for amplifying position 266 of the target nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase, wherein the third downstream primer has a nucleotide sequence as shown in SEQ ID NO. 11; the third upstream primer satisfies: When the mutated amino acid at position 266 is glutamine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.12; When the mutated amino acid at position 266 is methionine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO. 13; When the mutated amino acid at position 266 is arginine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.

14.

5. A recombinant vector of recombinant penicillin G acylase, comprising an expression vector and a nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase according to claim 1 carried on the expression vector.

6. A recombinant strain of recombinant penicillin G acylase, comprising the recombinant vector according to claim 5 or a nucleotide sequence corresponding to the amino acid sequence of the recombinant penicillin G acylase according to claim 1.

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