Recombinant penicillin G acylase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological enzymes, in particular to recombinant penicillin G acylase as well as a preparation method and application thereof. The amino acid sequence of the recombinant penicillin G acylase comprises three mutation sites: a 171 site, a 647 site and a 266 site, the mutation amino acid of the 171 site is tyrosine, the mutation amino acid of the 647 site is arginine, and the mutation amino acid of the 266 site comprises one of glutamine, methionine and arginine. According to the recombinant penicillin G acylase, through three key mutation sites (F171Y, Y647R and S266Q / M / R), the catalytic efficiency of the recombinant penicillin G acylase and the binding affinity of the recombinant penicillin G acylase with a target substrate are remarkably improved on the basis of the multidimensional structural characteristics of collaborative optimization of enzyme-substrate interaction.

Description

Technical Field

[0001] This application relates to the technical field of bioenzymes, and particularly to a recombinant penicillin G acylase, a preparation method thereof, and an application thereof. Background Art

[0002] Penicillin G acylase (PGA) is an important industrial enzyme and is widely used in the industrial production of antibiotics. The main functions of PGA are as follows: (1) Catalyzing hydrolysis reaction: PGA can specifically catalyze the hydrolysis of the side-chain acyl group of penicillin G (or other β-lactam antibiotics), converting 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 efficient 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 donors to achieve the synthesis and preparation of new antibiotics with some 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 with traditional PGA, PGA derived from Bacillus megaterium (BmPGA) has unique catalytic characteristics and thermal stability. However, due to the large demand for β-lactam antibiotics, the current BmPGA has poor activity and low catalytic performance, and cannot continuously and efficiently catalyze the semi-synthesis process of β-lactam antibiotics. With the continuous development of structural biology, the three-dimensional structure of PGA at present and the binding center and binding mode of this three-dimensional structure with the substrate have been relatively clear. Based on the analysis of the current three-dimensional structure, in order to improve the activity and catalytic performance of PGA, specific sites of PGA are mutated to improve the affinity between PGA and the substrate, thereby increasing the ratio of synthase activity to hydrolase activity (S / H) of PGA.

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

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

[0006] In a first aspect, an embodiment of the present application provides a recombinant penicillin G acylase, and 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.

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

[0008] Optionally, the recombinant penicillin G acylase has a first amino acid sequence as shown in SEQ ID NO.1; and / or the recombinant penicillin G acylase has a first nucleotide sequence as shown in SEQ ID NO.2.

[0009] Optionally, the recombinant penicillin G acylase has a second amino acid sequence as shown in SEQ ID NO.3; and / or the recombinant penicillin G acylase has a second nucleotide sequence as shown in SEQ ID NO.4.

[0010] Optionally, the recombinant penicillin G acylase has a third amino acid sequence as shown in SEQ ID NO.5; and / or the recombinant penicillin G acylase has a third nucleotide sequence as shown in SEQ ID NO.6.

[0011] In a second aspect, an embodiment of the present application provides a method for preparing the recombinant penicillin G acylase described in the first aspect, and the method includes: Designing a target nucleotide sequence of the recombinant penicillin G acylase described in the first aspect; Designing a primer set according to an expression vector and the target nucleotide sequence; Using the primer set to amplify the target nucleotide sequence to obtain an amplification product; Performing double digestion on the expression vector and the amplification product respectively to obtain a digested vector and a digested amplification product; Connecting the digested vector and the digested amplification product to obtain a recombinant plasmid; Introducing the recombinant plasmid into competent cells to obtain recombinant cells; Culturing the recombinant cells to express the target nucleotide sequence to obtain recombinant penicillin G acylase.

[0012] Optionally, the primer set includes a first upstream primer and a first downstream primer for amplifying the 171st site of the target nucleotide sequence. The first upstream primer has the nucleotide sequence shown in SEQ ID NO.5, and the first downstream primer has the nucleotide sequence shown in SEQ ID NO.6; and / or The primer set further includes a second upstream primer and a second downstream primer for amplifying the 647th site of the target nucleotide sequence. The second upstream primer has the nucleotide sequence shown in SEQ ID NO.7, and the second downstream primer has the nucleotide sequence shown in SEQ ID NO.8.

[0013] Optionally, the primer set further includes a third upstream primer and a third downstream primer for amplifying the 266th site of the target nucleotide sequence. The third downstream primer has the nucleotide sequence shown in SEQ ID NO.11; the third upstream primer satisfies the following conditions: When the mutated amino acid at the 266th site is glutamine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.12; When the mutated amino acid at the 266th site is methionine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.13; When the mutated amino acid at the 266th site is arginine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.14.

[0014] In a third aspect, an embodiment of the present application provides a recombinant vector of recombinant penicillin G acylase. The recombinant vector includes an expression vector and the nucleotide sequence of the recombinant penicillin G acylase described in the first aspect carried on the expression vector.

[0015] In a fourth aspect, an embodiment of the present application provides a recombinant strain of recombinant penicillin G acylase. The recombinant strain includes the recombinant vector described in the third aspect or the nucleotide sequence of the recombinant penicillin G acylase described in the first aspect.

[0016] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art: A recombinant penicillin G acylase provided by an embodiment of the present application has three mutation sites, which are located at the 171st site, the 647th site, and the 266th site of the recombinant penicillin G acylase respectively. By mutating phenylalanine at the 171st site into tyrosine, a hydroxyl group with an aromatic ring can be formed at the 171st site of the recombinant penicillin G acylase. This hydroxyl group structure can bind to the target substrate in the form of a hydrogen bond to enhance the ability of the recombinant penicillin G acylase to recognize and bind to the target substrate; in addition, by mutating tyrosine at the 647th site into arginine, the branched-chain distribution of the amino acid group at the 647th site of the recombinant penicillin G acylase can be improved, so that a hydrogen bond structure with a shorter distance is formed between the branched chain of the amino acid group and the target substrate. This hydrogen bond structure can improve the binding affinity between the recombinant penicillin G acylase and the target substrate; in addition, by mutating serine at the 266th site into glutamine or methionine arginine, multiple cyclic or chain-like groups can be introduced at the 266th site of the recombinant penicillin G acylase. These cyclic or chain-like groups can optimize the spatial configuration of the binding pocket between the recombinant penicillin G acylase and the target substrate, thereby ensuring the environmental stability of the binding between the target substrate and the recombinant penicillin G acylase and improving the binding affinity between the recombinant penicillin G acylase and the target substrate; in addition, by mutating serine at the 266th site into arginine, arginine side chains can be introduced at the 266th site of the recombinant penicillin G acylase. These arginine side chains can further increase the number of hydrogen bonds for binding between the penicillin G acylase and the target substrate to further improve the binding affinity between the recombinant penicillin G acylase and the target substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings here are incorporated into the description and form a part of this description, showing embodiments consistent with the present application and used together with the description to explain the principles of the present application.

[0018] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a schematic diagram of the molecular structure of a recombinant penicillin G acylase provided by an embodiment of the present application; among them, Figure 1 A is an analysis diagram of the molecular structure of the BmPGA enzyme containing three mutation sites of F171R:S266M:Y647R, Figure 1 B is an analysis diagram of the molecular structure of the BmPGA enzyme containing three mutation sites of F171R:S266Q:Y647R,Figure 1 Figure C is an analysis diagram of the molecular structure of BmPGA enzyme containing three mutation sites of F171R:S266R:Y647R. Figure 1 Figure D is an analysis diagram of the molecular structure of BmPGA enzyme containing three mutation sites of F171Y:S266M:Y647R. Figure 1 Figure E is an analysis diagram of the molecular structure of BmPGA enzyme containing three mutation sites of F171Y:S266Q:Y647R. Figure 1 Figure F is an analysis diagram of the molecular structure of BmPGA enzyme containing three mutation sites of F171Y:S266R:Y647R; in the figure, the dashed line a-a represents a hydrogen bond, the dashed line b-b represents a p-π bond, and the dashed line c-c represents an ionic bond. Figure 2 Figure is a schematic flow diagram of a method for preparing recombinant penicillin G acylase provided by an embodiment of the present application. Figure 3 Figure is a statistical chart of the affinity between BmPGA enzymes with single and multiple mutation sites and the target substrate provided by an embodiment of the present application. Figure 4 Figure is an agarose gel electrophoresis result diagram of the recombinant enzyme of the BmPGA enzyme mutant containing multiple mutation sites provided by an embodiment of the present application; among them, 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 cell solution. Figure 5 Figure is a liquid chromatography detection result diagram of the enzymatic hydrolysis product of the BmPGA enzyme containing multiple mutation sites provided by an embodiment of the present application. Figure 6 Figure is a schematic diagram of the molecular structure of the wild-type BmPGA enzyme provided by an embodiment of the present application. Detailed implementation manners

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0021] The various embodiments of the present application may exist 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 construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values within that range; for example, it should be considered that the range description from 1 to 6 has 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., and single numbers within that range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0022] In this document, terms including "comprising" etc. mean "including but not limited to". Relative terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone; where A and B can be singular or plural. "At least one" means one or more, and "a plurality" means two or more; "at least one kind", "at least one of the following items (pieces)" or similar expressions refer to any combination of these items, including any combination of single items (pieces) or plural items (pieces); for example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both mean: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively. "Parts representation methods" such as parts by weight, parts by mass, etc. represent the proportional relationship between each component. In the proportional relationships involved in this document, the parameters that need to be described by proportion should be understood as the antecedents of the proportion formula in the order of description, and the proportional numbers should be understood as the consequents of the proportion formula. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one by one with the proportional numbers in the proportion formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

[0023] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in this document can be obtained through market purchase or can be prepared by existing methods.

[0024] Figure 1 Exemplarily shown is a schematic diagram of the molecular structure of a recombinant penicillin G acylase provided by an embodiment of the present application; Such as Figure 1As shown in the figure, an embodiment of the present application provides a recombinant penicillin G acylase, and 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.

[0025] It should be noted that this recombinant penicillin G acylase is penicillin G acylase (BmPGA) derived from Bacillus megaterium.

[0026] It should be noted that alanine is present at sites 171, 647, and 266 of PGA derived from Bacillus megaterium. Since the side chain of alanine is a non-polar methyl group, replacing the non-polar methyl group has little impact on the overall structure of the recombinant penicillin G acylase protein. Analyzing the site treatment of alanine can be an effective means to predict the functional sites of penicillin G acylase.

[0027] It should be noted that an embodiment of the present application provides a recombinant penicillin G acylase. Through three key mutation sites (F171Y, Y647R, S266Q / M / R), based on the multi-dimensional structural characteristics of synergistically optimizing enzyme-substrate interactions, the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity with the target substrate are significantly improved. The specific mechanism is as follows: ‌1. F171Y mutation (phenylalanine → tyrosine): Aromatic ring hydroxylation and hydrogen bond network reconstruction:‌ ‌Structural basis‌: Both phenylalanine (F) and tyrosine (Y) contain aromatic rings, but Y introduces a hydroxyl group (-OH) at the para position of the benzene ring. This hydroxyl group enhances substrate recognition through two mechanisms: (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.

[0028] (2)‌π-π stacking optimization‌: The remaining aromatic ring maintains hydrophobic interaction with the phenylacetyl group of the substrate. At the same time, the electron cloud rearrangement induced by the hydroxyl group can enhance the overlap of π-π orbitals, improving the binding energy between the recombinant penicillin G acylase and the target substrate.

[0029] (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 conformation.

[0030] 2. Y647R mutation (tyrosine → arginine): guanidine-mediated short-range strong interaction: (1) Charge complementarity and geometric fit: The phenolic hydroxyl group of the original Y647 is generally neutral, while the guanidine group of arginine (R) is positively charged after protonation and can form a salt bridge with the carboxyl group of the target substrate, which can increase the electrostatic potential energy contribution of this site.

[0031] 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 binding state".

[0032] (2) Steric hindrance elimination: The Y→R mutation can remove a large number of benzene rings, reduce the benzene ring volume of recombinant penicillin G acylase, avoid the steric conflict between recombinant penicillin G acylase and the side chains of some target substrates, make it easier for the target substrate to enter the catalytic center, and improve the catalytic efficiency of the target substrate.

[0033] 3. S266Q / M / R mutation (serine → glutamine / methionine / arginine): binding pocket topology remodeling: (1) Common mechanism: 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.

[0034] 2) Conformational locking effect: The large side chains of Q, M or R will limit the local flexibility of the binding pocket, stabilize the active conformation of the catalytic key residues, and improve the binding strength between the target substrate and recombinant penicillin G acylase.

[0035] (2) Specific mechanism: Q266: The amide group of glutamine will form a hydrogen bond with the thioether bond of the target substrate. At the same time, the long side chain of glutamine can induce the β-lactam ring of the substrate to be closer to the catalytic nucleophilic attack site, improving the catalytic efficiency of recombinant penicillin G acylase.

[0036] M266: The thioether group of methionine can stabilize the non-polar region of the target substrate through hydrophobic effect and participate in sulfur-π interaction, improving the binding affinity of recombinant penicillin G acylase.

[0037] R266: The guanidyl 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 add 2 to 3 hydrogen bonds to the binding of recombinant penicillin G acylase to the target substrate. And through the cation-π interaction, the binding strength between recombinant penicillin G acylase and the target substrate is comprehensively enhanced.

[0038] 3. Synergistic effect and global optimization: Based on the synergistic effect of three mutation sites; (1) Binding energy additivity: The three mutation sites can reduce the total binding free energy.

[0039] (2) Catalytic efficiency improvement: Based on the binding affinity between recombinant penicillin G acylase and the target substrate, the target substrate can bind directionally to penicillin G acylase. Through three mutation sites, the catalytic efficiency kcat / Km value and transition state stability of recombinant penicillin G acylase can be increased by an order of magnitude.

[0040] (3) pH stability broadening: The buffering effect of R647 and R266 enables recombinant penicillin G acylase to maintain high activity at pH 7.5 - 9.0 (the pH range is broadened by 1.5 pH units compared with traditional penicillin G acylase).

[0041] In some alternative embodiments, the mutant amino acid at the 266th site is arginine.

[0042] In these embodiments, the mutant amino acid at the 266th site can be arginine. By introducing an arginine side chain at the 266th site of recombinant penicillin G acylase, the number of binding hydrogen bonds between recombinant penicillin G acylase and the target substrate can be increased to further improve the binding affinity between recombinant penicillin G acylase and the target substrate.

[0043] In some alternative embodiments, the recombinant penicillin G acylase has the first amino acid sequence shown in SEQ ID NO.1; and / or The recombinant penicillin G acylase has the first nucleotide sequence shown in SEQ ID NO.2.

[0044] In these embodiments, the recombinant penicillin G acylase may have the first amino acid sequence shown in SEQ ID NO.1 or the recombinant penicillin G acylase has the first nucleotide sequence shown in SEQ ID NO.2. It can be determined that 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 specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase can be determined, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity with the target substrate based on the multi-dimensional structural characteristics of the synergistic optimization of enzyme-substrate interaction.

[0045] In some alternative embodiments, the recombinant penicillin G acylase has the second amino acid sequence shown in SEQ ID NO.3; and / or The recombinant penicillin G acylase has the second nucleotide sequence shown in SEQ ID NO.4.

[0046] In these embodiments, the recombinant penicillin G acylase may have the second amino acid sequence shown in SEQ ID NO.3 or the recombinant penicillin G acylase has the second nucleotide sequence shown in SEQ ID NO.4. It can be determined that 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 specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase can be determined, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity with the target substrate based on the multi-dimensional structural characteristics of the synergistic optimization of enzyme-substrate interaction.

[0047] In some alternative embodiments, the recombinant penicillin G acylase has the third amino acid sequence shown in SEQ ID NO.5; and / or The recombinant penicillin G acylase has the third nucleotide sequence shown in SEQ ID NO.6.

[0048] In these embodiments, the recombinant penicillin G acylase may have the third amino acid sequence shown in SEQ ID NO.5 or the recombinant penicillin G acylase has the third nucleotide sequence shown in SEQ ID NO.6. It can be determined that 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 specific amino acid sequence or nucleotide sequence of the recombinant penicillin G acylase can be determined, so as to significantly improve the catalytic efficiency of the recombinant penicillin G acylase and its binding affinity with the target substrate based on the multi-dimensional structural characteristics of the synergistic optimization of enzyme-substrate interaction.

[0049] Figure 2 Exemplarily shown is a schematic flow chart of a method for preparing recombinant penicillin G acylase provided by an embodiment of the present application; Based on a general inventive concept, as Figure 2 shown, an embodiment of the present application provides a method for preparing the recombinant penicillin G acylase, and the method includes: S1. Design the target nucleotide sequence of the recombinant penicillin G acylase; S2. Design a primer set according to the expression vector and the target nucleotide sequence; S3. Use the primer set to amplify the target nucleotide sequence to obtain an amplification product; S4. Perform double digestion on the expression vector and the amplification product respectively to obtain a digested vector and a digested amplification product; S5. Connect the digested vector and the digested amplification product to obtain a recombinant plasmid; S6. Introduce the recombinant plasmid into competent cells to obtain recombinant cells; S7. Culture the recombinant cells to express the target nucleotide sequence to obtain recombinant penicillin G acylase.

[0050] This method is directed to the above-mentioned method for preparing recombinant penicillin G acylase. For the specific information of the recombinant penicillin G acylase, reference can be made to the above-mentioned embodiments. Since this method adopts some or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, and will not be elaborated herein one by one.

[0051] In some alternative embodiments, the primer set includes a first upstream primer and a first downstream primer for amplifying the 171st site of the target nucleotide sequence. 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 further includes a second upstream primer and a second downstream primer for amplifying the 647th site of the target nucleotide sequence. The second upstream primer has a nucleotide sequence as shown in SEQ ID NO.9, and the second downstream primer has a nucleotide sequence as shown in SEQ ID NO.10.

[0052] In these embodiments, the first upstream primer of the primer set has the nucleotide sequence shown in SEQ ID NO.7, and the first downstream primer of the primer set has the nucleotide sequence shown in SEQ ID NO.8, and the second upstream primer of the primer set has the nucleotide sequence shown in SEQ ID NO.9, and the second downstream primer of the primer set has the nucleotide sequence shown in SEQ ID NO.10. The target nucleotide sequence is amplified sufficiently by the primer set, thus facilitating the subsequent double digestion and ligation processes to obtain a sufficient amount of amplified product.

[0053] In some alternative embodiments, the primer set further includes a third upstream primer and a third downstream primer for amplifying the 266th site of the target nucleotide sequence. The third downstream primer has the nucleotide sequence shown in SEQ ID NO.11; the third upstream primer satisfies: When the mutated amino acid at the 266th site is glutamine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.12; When the mutated amino acid at the 266th site is methionine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.13; When the mutated amino acid at the 266th site is arginine, the third upstream primer has the nucleotide sequence shown in SEQ ID NO.14.

[0054] In these embodiments, the primer set further includes a third upstream primer and a third downstream primer. First, confirm the nucleotide sequence of the third downstream primer as shown in SEQ ID NO.11, and then according to the type of the mutated amino acid at the 266th site, use the nucleotide sequences shown in SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14 respectively. Through the combination of these designed third upstream primer and third downstream primer, the target nucleotide sequence can be amplified sufficiently, thus facilitating the subsequent double digestion and ligation processes to obtain a sufficient amount of amplified product.

[0055] Based on a general inventive concept, the embodiments of the present application provide a recombinant vector of recombinant penicillin G acylase, which includes an expression vector and the nucleotide sequence of the recombinant penicillin G acylase carried on the expression vector.

[0056] This 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 embodiments. Since this recombinant vector adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated here one by one.

[0057] Based on a general inventive concept, embodiments of the present application provide a recombinant strain of recombinant penicillin G acylase, and the recombinant strain includes the recombinant vector or the nucleotide sequence of the recombinant penicillin G acylase.

[0058] This recombinant strain is realized based on the above-mentioned recombinant penicillin G acylase. For the specific information of this recombinant penicillin G acylase, reference can be made to the above embodiments. Since this recombinant strain adopts some or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated one by one here.

[0059] The following will further elaborate on the present application in combination with specific embodiments. For the experimental methods without specific conditions noted in the following embodiments, they are usually measured according to national standards / industry standards; if there are no corresponding national standards / industry standards, they are carried out according to general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0060] Example 1 1. Selection of amino acid sites: (1) Perform alanine scanning (ABS) on the active pocket region of BmPGA, and calculate the change in binding free energy after each residue is mutated to alanine ( ). Taking ≥0 kcal / mol as the screening criterion, the following key residue sites are identified: TYR170, PHE171, SER266, GLN288, TYR336, ASN510, ARG531, ARG646, TYR647, and LEU710. These sites are considered to play an important role in the binding of BmPGA to the substrate.

[0061] (2) According to these key residue sites, use the CalculateMutation Energy (Binding) module in Discovery Studio 2019 to calculate and analyze the binding affinity of single residue mutations and multi-residue combined mutations with the substrate, and confirm the affinity of different mutants with the substrate; the calculation parameters are set as follows: use the CHARMM force field, the dielectric constant is 10, and the solvent dielectric constant is 80; the energy term scaling factors are 0.5 for electrostatic interaction (Electrostatic), 0.5 for van der Waals force (VDW), and 0.8 for entropy effect (Entropy).

[0062] (3) According to the change in binding free energy ≤ -0.10 kcal / mol was used as the screening criterion to screen mutants with relatively high affinity, including F171R, F171Y, S266R, S266Q, S266M, Y647R, and Y170H. The changes in two-dimensional weak interactions of these mutants were further analyzed. Considering the changes in weak interactions and the magnitude of the affinity with the substrate, it was clarified that the 171st, 266th, and 647th sites of BmPGA are key residue sites, and the types of mutant amino acids at these key residue sites were identified as F171Y, Y647R, and S266Q / M / R.

[0063] Example 2

[0064] 2. Preparation method of recombinant penicillin G acylase: (1) According to the key residue sites screened in Example 1 and the types of corresponding mutant amino acids, the specific amino acid sequence of BmPGA is as follows: When the mutant amino acid at the 171st site is tyrosine, the mutant amino acid at the 647th site is arginine, and the mutant amino acid at the 266th site is arginine, the amino acid sequence of BmPGA is: MKMKWLISVIILFVFIFPQNLVFAGEDKNEGVKVVRDNFGVPHLYAKNKKDLYEAYGYVMAKDRLFQLEMFRRGNEGTVSEIFGEDYLSKDEQSRRDGYSNKEIKKMIDGLDRQPKELIAKFAEGISRYVNEALKDPDDKLSKEFHEYQFLPQKWTSTDVVRVYMVSMTY Y MDNHQELKNAEILAKLEHEYGTEVSRKMFDDLVWKNDPSAPTSIVSEGKPKRESSSQSLQKLSSAVIKASEKVGKERENFVQSSEELGLPLKIG RNAAIVGSEKSATGNALLFSGPQVGFVAPGFLYEVGLHAPGFDMEGSGFIGYPFIMFGANNHFALSATAGYGNVTDIFEEKLNTKNSSQYLYKGKWRDMEKRKESFTVKGDNGEKKTVEKIYYRTVHGPVISRDETNKVAYSKSWSFRGTEAQSMSAYMKANWAKNLKEFENAASEYTMSLNWYYADKKGDIAYYHVGRYPVRNNKIDERIPTPGTGEYEWKGFIPFKENPHVINPKNGYVVNWNNKPSKEWVNGEYSYYWGEDNRVQQYINGMEARGKVTLEDINEINYTASFAQLRANLFKPLLIDVLDKNKSTNGNYAYLIEKLEEWNNLKEDENKDGYYDAGIAAFFDEWWNNLHDKLFMDELGDFYGITKEITDHR R GASLAYKILSKESTNYKWVNVDQEKIIMESTNEVLAKLQSEKGLKAEKWRMPIKTMTFGEKSLIGIPHGYGSMTPIIEMNRGSENHYIEMTPKGPSGFNITPPGQIGFVKKDGTISDHYDDQLVMFAEWKFKPYLFNKKDIYKSAKNVSALNMSK (SEQ ID NO.1), wherein the bold and underlined parts are the mutation sites; the nucleotide sequence of BmPGA is: ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC CGACGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTATTGAAATGAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.2), wherein the bold and underlined parts are the mutation sites; When the mutated amino acids at position 171 are tyrosine, the mutated amino acids at position 647 are arginine, and the mutated amino acids at position 266 are 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 mutation sites; the nucleotide sequence of BmPGA is: ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC CAACGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTATTGAAATGAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.4), wherein the bold and underlined parts are the mutation sites; When the mutated amino acids at position 171 are tyrosine, the mutated amino acids at position 647 are arginine, and the mutated amino acids at position 266 are 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: ATGAAGATGAAGTGGCTAATATCAGTCATAATCCTATTTGTTTTCATTTTTCCTCAAAATCTAGTTTTTGCTGGGGAGGATAAGAATGAAGGGGTCAAAGTAGTACGTGATAATTTTGGAGTACCCCATTTATACGCTAAAAATAAAAAAGATTTATATGAAGCGTATGGATATGTTATGGCAAAGGATCGACTATTTCAGTTGGAGATGTTCCGTCGCGGAAATGAGGGGACCGTTTCAGAAATTTTTGGAGAGGATTATCTTTCAAAAGATGAGCAATCCAGAAGAGATGGATATAGTAATAAAGAAATTAAAAAAATGATTGACGGTCTGGATCGTCAGCCAAAAGAATTAATAGCAAAATTTGCTGAAGGTATTTCACGTTATGTAAATGAAGCTTTAAAAGATCCAGATGATAAACTTTCGAAGGAGTTTCATGAATATCAGTTTTTACCGCAAAAATGGACTTCAACAGATGTTGTCCGTGTTTATATGGTATCCATGACGTAT TAT ATGGATAATCACCAGGAGTTAAAAAACGCAGAGATACTTGCAAAGCTAGAACATGAATATGGGACAGAAGTTTCCCGGAAAATGTTTGATGATTTAGTGTGGAAAAATGATCCTAGCGCTCCTACAAGCATTGTAAGCGAGGGGAAACCAAAAAGGGAATCGTCATCTCAATCCCTTCAAAAACTGTCTTCAGCTGTAATCAAAGCTTCTGAAAAAGTTGGAAAGGAAAGGGAGAATTTTGTCCAATCGTCTGAAGAACTTGGATTACCGTTAAAGATAGGC ATGCGA GGGGCTTCATTAGCATATAAAATATTAAGCAAGGAATCTACAAACTATAAATGGGTGAACGTAGACCAGGAAAAAATAATAATGGAAAGCACAAATGAAGTACTTGCTAAATTGCAATCAGAAAAAGGGTTAAAAGCAGAAAAATGGCGTATGCCTATAAAAACGATGACTTTTGGTGAAAAATCATTGATTGGTATTCCCCACGGGTATGGCTCAATGACTCCAATTATTGAAATGAATCGTGGAAGTGAAAATCATTATATTGAAATGACTCCGAAAGGGCCGAGTGGCTTTAACATCACACCACCTGGTCAAATTGGATTTGTAAAAAAAGATGGAACGATAAGTGACCACTATGATGACCAACTAGTTATGTTCGCCGAATGGAAATTCAAGCCATACTTATTTAACAAGAAAGATATTTATAAATCAGCTAAAAATGTAAGCGCATTAAATATGAGTAAGTAG (SEQ ID NO.6), wherein the bold and underlined parts are the mutation sites.

[0065] (2) According to the nucleotide sequence designed in step (1), synthesize the corresponding target gene, then select a suitable expression vector, and determine the restriction enzyme cleavage sites and design an amplification primer set based on the expression vector and the above nucleotide sequence. The specific sequences of the amplification primer set are as follows: For the case where the mutated amino acid at the 171st site is tyrosine, the first upstream primer: 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); For the case where the mutated amino acid at the 647th site is arginine, the second upstream primer: CCGATCATCGT CGA GGGGCTTC (SEQ ID NO.9), wherein the bold and underlined parts are the mutation sites; the second downstream primer: CTACTTACTCATATTTAATGCGC (SEQ ID NO.10); For the case where the mutated amino acid at the 266th site is glutamine, the third upstream primer: TTAAAGATAGGC CAAAATGCCG (SEQ ID NO.12); The third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO.11); For the case where the mutated amino acid at the 266th site is methionine, the third upstream primer: TAAAGATAGGC ATG AATGCCGCC (SEQ ID NO.13); The third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO.11); For the case where the mutated amino acid at the 266th site is arginine, the third upstream primer: AGATAGGC CGA AATGCCGCCAT (SEQ ID NO.14); The third downstream primer: GAAGCCCCTCGACGATGATCGG (SEQ ID NO.11).

[0066] In addition, a set of original amplification primers was designed for the non-mutated original nucleotide sequence of BmPGA. The specific sequences of the original amplification primer set are as follows: The upstream primer of the original amplification primer set: ATGAAGATGAAGTGGCTAATATC (SEQ ID NO.15); The downstream primer of the original amplification primer set: CTACTTACTCATATTTAATGCGC (SEQ ID NO.10).

[0067] (3) Amplify the target gene using the designed amplification primer sets to obtain amplification products: Using the different primers designed above, for different combinations of mutation sites, use the corresponding upstream and downstream primers, and perform PCR amplification according to the amplification system shown in Table 1 and the amplification program shown in Table 2, and obtain amplification products of different combinations of mutation sites.

[0068] Table 1 PCR amplification reaction system

[0069] Table 2 PCR amplification reaction program

[0070] (4) Select the corresponding endonuclease group according to the restriction sites, and use this endonuclease group to digest the expression vector and the amplification products respectively to obtain digested vectors and digested products: According to the designed BmPGA nucleotide sequence and the original BmPGA nucleotide sequence, different double digestion systems were selected. In this application, the Ndel and Xhol double digestion system was used, and then the pEASY-T3 vector was used as the expression vector. 0.5 μL of Ndel restriction endonuclease and 0.5 μL of Xhol restriction endonuclease were used to digest 4.0 μL of the amplification product and 1.5 μL of the expression vector respectively under water bath conditions (the digestion temperature was 37 °C and the digestion time was 1 h) to obtain the digested vector and the digested product.

[0071] (5)The digested vector and the digested product were ligated under the action of DNA ligase to obtain the recombinant plasmid: Take 4 μL of the digested product and 1 μL of the digested vector and mix them, then add 1.5 μL of DNA ligase for ligation to obtain the recombinant plasmid vector; (6)The recombinant plasmid was introduced into competent cells to obtain recombinant cells: Select 50 μL of competent cells of Bacillus megaterium (BM) and add 5 μL of the recombinant plasmid vector for mixing to obtain a mixed product; the mixed product was treated under ice bath conditions for 40 min, and then transferred to a water bath environment at 42 °C (temperature error within ±0.3 °C) for heat shock treatment for 30 s to allow the recombinant vector to enter the competent cells to obtain heat shock cells; then the heat shock cells were immediately transferred to an ice bath environment and allowed to stand for 2 min to obtain recombinant cells.

[0072] (7)The recombinant cells were cultured in a medium to express the target gene to obtain recombinant penicillin G acylase: Add the recombinant cells to 500 μL of LB recovery medium (without antibiotics) and shake 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 the recombinant bacterial solution; Then the recombinant bacterial solution was inoculated onto an LB resistant plate (containing 100 μg / mL of kanamycin) at an inoculation amount of 2%, and cultured for another 14 d. Then, a flame-sterilized toothpick was used to spot the surface of the typical single colonies (diameter 1 mm - 2 mm) on the LB resistant plate, avoiding contact with adjacent colonies or scratched areas of the agar to obtain recombinant bacterial cells; The selected recombinant bacterial cells were inoculated into a liquid medium and cultured for another 7 d to obtain the seed culture solution; The seed culture solution was inoculated into 200 mL of fermentation medium and shake cultured at 37 °C and 180 rpm for 4 h, and then induced overnight with IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.3 mM for 12 h to obtain the fermentation broth; Purify and dry the fermentation broth to obtain recombinant penicillin G acylase.

[0073] Related experiments and effect data: 1. For the experiment in Example 1, the specific data are as follows: (1) Affinity of different mutants: The binding affinity of different mutants with the substrate is as Figure 3 shown. The results show that mutants with multiple sites have an obvious synergistic effect, and in addition, the stability of the expressed BmPGA is better than that of single-site mutants. However, the multiple-site mutants will have a certain impact on the overall structure and biological activity of BmPGA. Based on this, further experimental analysis is carried out.

[0074] 2. For the selected mutants, according to different combinations of mutation sites, the mutant containing the F171Y:S266R:Y647R mutation sites is denoted as mutant 1, the mutant containing the F171Y:S266Q:Y647R mutation sites is denoted as mutant 2, and the mutant containing the F171Y:S266M:Y647R mutation sites is denoted as mutant 3. The results of agarose gel electrophoresis of their recombinant cells are as Figure 4 shown.

[0075] In addition, the catalytic ability of these BmPGA enzymes as catalysts is investigated. The specific process is as follows: Weigh 3 mg of the substrate, add 4 mg of sodium carbonate to the substrate, then add 1 mL of water to dissolve these mixtures, and then add the organic reagent required for EA extraction of the substrate in three portions to obtain the substrate to be enzymatically hydrolyzed; add the above BmPGA enzyme and the Figure 6 unmutated BmPGA enzyme as shown to the substrate to be enzymatically hydrolyzed and carry out enzymatic hydrolysis at 30 °C for 1 h to obtain the enzymatic hydrolysis product; detect the content of these enzymatic hydrolysis products by liquid chromatography to clarify their catalytic performance. The results are as Figure 5 shown, which shows that the BmPGA enzyme mutant containing the three mutation sites of F171Y:S266R:Y647R has good catalytic ability, and its catalytic ability is increased by 5 times or more compared with the unmutated BmPGA enzyme. At the same time, the substrate conversion rate of this BmPGA enzyme mutant is above 98%.

[0076] 3. For the selected mutants, the folding free energy and two-dimensional weak interaction changes of these mutants are investigated. The results are as Figure 6 shown, indicating that the combination of the three mutation sites of F171Y:S266R:Y647R can meet the stable expression, stable biological activity and stable overall structure of BmPGA.

[0077] In summary, a recombinant penicillin G acylase provided by an embodiment of the present application significantly improves the functional stability of the recombinant penicillin G acylase and its binding affinity with the target substrate based on the multi-dimensional structural characteristics of synergistically optimizing enzyme-substrate interactions through three key mutation sites (F171Y, Y647R, S266Q / M / R).

[0078] In addition, a recombinant penicillin G acylase provided by an embodiment of the present application can be used as an efficient catalytic enzyme in related industries of penicillin G acylase, such as in the catalytic industry of penicillin.

[0079] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features claimed in the present application.

Claims

1. A recombinant penicillin G acylase, wherein the amino acid sequence of the recombinant penicillin G acylase comprises three mutation sites: site 171, site 647 and site 266, wherein the mutant amino acid at site 171 is tyrosine, the mutant amino acid at site 647 is arginine, and the mutant amino acid at site 266 comprises one of the following: glutamine, methionine and arginine.

2. The recombinant penicillin G acylase according to claim 1, characterized in that The mutant amino acid at position 266 is arginine.

3. The recombinant penicillin G acylase according to claim 2, characterized in that The recombinant penicillin G acylase has a first amino acid sequence as shown in SEQ ID NO.1; and / or The recombinant penicillin G acylase has a first nucleotide sequence as shown in SEQ ID NO.

2.

4. The recombinant penicillin G acylase according to claim 1, characterized in that The recombinant penicillin G acylase has the second amino acid sequence shown in SEQ ID NO.3; and / or The recombinant penicillin G acylase has a second nucleotide sequence as shown in SEQ ID NO.

4.

5. The recombinant penicillin G acylase according to claim 1, characterized in that The recombinant penicillin G acylase has the third amino acid sequence shown in SEQ ID NO.5; and / or The recombinant penicillin G acylase has a third nucleotide sequence as shown in SEQ ID NO.

6.

6. A method for preparing the recombinant penicillin G acylase according to any one of claims 1 to 5, the method comprising: Designing a target nucleotide sequence of the recombinant penicillin G acylase according to any one of claims 1 to 5; Designing a primer set according to the expression vector and the target nucleotide sequence; Using the primer set to PCR amplify the target nucleotide sequence to obtain an amplified product; The expression vector and the amplified product are double-digested to obtain a digested vector and a digested amplified product; Connecting the restriction digested vector and the restriction 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, thereby obtaining recombinant penicillin G acylase.

7. The method according to claim 6, characterized in that The primer set comprises a first upstream primer and a first downstream primer for amplifying site 171 of the target nucleotide sequence, 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.

8. The method according to claim 6, characterized in that The primer set further includes a third upstream primer and a third downstream primer for amplifying the 266th site 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: 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; When the mutant amino acid at position 266 is methionine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.13; When the mutant amino acid at position 266 is arginine, the third upstream primer has a nucleotide sequence as shown in SEQ ID NO.

14. 9 . A recombinant vector of recombinant penicillin G acylase, comprising an expression vector and the nucleotide sequence of the recombinant penicillin G acylase according to claim 1 , which is carried on the expression vector. 10 . A recombinant strain of recombinant penicillin G acylase, comprising the recombinant vector according to claim 9 or the nucleotide sequence of the recombinant penicillin G acylase according to any one of claims 1 to 5 .

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