WO2021096298A1 - Penicillin g acylase mutant having increased cefazoline productivity and use thereof - Google Patents

Abstract

The present invention relates to a penicillin G acylase mutant having increased cefazoline productivity and a use thereof, wherein Achromobacter sp. CCM 4824-derived penicillin G acylase mutants having unique mutation forms disclosed in the present invention have a very high synthesis activity specifically for cefazoline, compared to wild-type enzyme thereof and other known mutant forms, and have a remarkably high synthesis ratio (S/H ratio) compared to the degradation activity of a substrate used for synthesis of cefazoline, and thus the productivity of cefazoline is remarkably increased to a significant level in industrial applications.

Description

Penicillin G acylase variants with increased cefazoline productivity and their use

This application claims priority to Korean Patent Application No. 10-2019-0147086 filed on November 15, 2019, and the entire specification is a reference to this application.

The present invention relates to a penicillin G acylase variant with increased cephazoline productivity and its use, and more particularly, to an alpha subunit represented by SEQ ID NO: 1; And in the wild-type penicillin G acylase protein (from Achromobacter sp. CCM 4824) sequence comprising the beta subunit represented by SEQ ID NO: 2, A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA, T388βA, R478βH, It relates to a mutant penicillin G acylase, characterized in that it comprises one or more mutations selected from the group consisting of E486βA, D541βE, and Y555βC, and a method for synthesizing (manufacturing) cefazoline using the same.

In the process of synthesizing a semisynthetic β-lactam compound, a compound having a nuclear structure of β-lactam and a compound bonded thereto to become a side chain (acyl donor) are used as substrates. When penicillin G acylase is added to the substrate mixture, an enzymatic coupling reaction between the activated side chain and the β-lactam nucleus occurs. Penicillin G acylase enzyme is β- from the activated side chain (i.e., the acyl donor). It promotes the transfer of acyl groups to the lactam nucleus (acylation). On the other hand, the enzyme also possesses the activity of hydrolyzing the synthesized β-lactam compound with the activated side chain used as an acyl donor, which causes a problem in the productivity of the semisynthetic β-lactam compound. Particularly, considering that it is advantageous in the production process and production efficiency to perform the synthesis reaction in a ratio of 1:1 in terms of industrial and economic terms, in particular, the substrate decomposition reaction is lowered so that more substrates can be used in the synthesis reaction. Is an important challenge.

In particular, in the conventional enzymatic synthesis method of cefazoline, an ester compound in which a group such as methyl, ethyl, or propyl is bonded to TzAA as a reaction substance has been used. In the case of synthesizing cefazoline from the ester compound of TzAA and MMTD-7-ACA according to conventional methods and means, the synthesis proceeds by the enzymatic activity of penicillin zamidase (penicillin G acylase) and at the same time TzAA The ester compound of is naturally hydrolyzed, that is, the reaction of penicillin g amidase (penicillin G acylase) used as a biocatalyst to hydrolyze on the one hand to decompose it into tetrazol-1-acetic acid and the corresponding alcohol. It happens competitively. A specific example of this reaction is shown in FIG. 2. As shown in FIG. 2, as a substrate for synthesizing (S) cefazoline through an enzymatic reaction, for example, tetrazolyl acetic acid methyl ester (abbreviated herein as "TzAAMe") and 7 -Amino-3-(2-methyl-1,3,4-thiadiazol-5-yl)-thiomethyl-3-cefem-4-carboxyl acid (7-amino-3-(2-methyl-1 ,3,4-thiadiazol-5-yl)-thiomethyl-3-cephem-4-carboxylic acid, abbreviated herein as "TDA") may be used. Substrate TzAAMe is also hydrolyzed by enzymes and converted into tetrazolyl acetic acid (tetrazolyl acetic acid, abbreviated herein as "TzAA") (H1), and the synthesized cefazoline is also hydrolyzed by enzymes (H2 ), so that TzAA is created. Therefore, in order to increase the productivity of cefazoline, it is important to increase the synthesis reaction (S) and lower the decomposition reactions (H1, H2).

Therefore, it is possible to reduce the amount of TzAA ester compound used by reducing the rate of hydrolysis as much as possible.In order to reduce the rate of hydrolysis, it is known that it is desirable to proceed the enzymatic reaction at as low pH as possible, but this method is used to synthesize cefazoline. Since the reaction is limited due to the optimal conditions of the reaction and the solubility of MMTD-7-ACA, so far, when synthesizing cefazoline, TzAAMe was added at an equivalent ratio of MMTD-7-ACA more than twice to proceed with the reaction. .

In addition, even if the same enzyme is used, a significant difference may occur in the synthetic yield depending on the specific type of antibiotic. Antibiotics having a benzyl ring such as cephalexin, cefaclor and cephradine and antibiotics having a phenolic ring such as amoxicillin, cefadroxil and cefprozil have high structural similarities, but cefazoline has a tetrazole ring. Since there is a significant structural difference from the above-described antibiotics, a situation arises in which the efficiency of obtaining cefazoline is relatively remarkably low even when the same enzyme is used. For example, in the case of the enzyme disclosed in KR101985911 (Patent Document 1), high-efficiency multi-synthesis capability for semi-synthetic β-lactam antibiotics of ceparexin, cefprozil, cepachlor, cepradin, cepadroxil, and amoxicillin It has been known to have excellent productivity, but cefazoline did not significantly increase productivity efficiency to the extent that it has utility at an industrial level. So far, no enzyme with high cefazoline productivity has been known enough for application at an industrial level.

[Prior technical literature]

[Patent Literature]

(Patent Document 1) KR101985911B

Accordingly, the inventors of the present invention were researching a method to increase cefazoline productivity, while the penicillin G acylase mutant having the unique mutation disclosed in the present invention was specifically compared to the wild type (from Achromobacter sp. CCM 4824 strain) compared to the wild type (from Achromobacter sp. CCM 4824 strain). The present invention was completed by confirming that the synthesizing ability for sleepy was significantly increased and the S/H ratio was improved to a remarkably high level, thereby having high-efficiency productivity for cefazoline.

Accordingly, an object of the present invention is an alpha subunit represented by SEQ ID NO: 1; And among the wild-type penicillin G acylase protein sequence containing the beta subunit represented by SEQ ID NO: 2, the group consisting of A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA, T388βA, R478βH, E486βA, D541βE and Y555βC It is to provide a mutant penicillin G acylase, characterized in that it comprises one or more mutations selected from.

Another object of the present invention is to provide a polynucleotide encoding the mutant penicillin G acylase.

Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide.

Another object of the present invention is to provide a host cell transformed with the expression vector.

Another object of the present invention is to provide a composition for preparing cefazolin comprising the mutant penicillin G acylase of the present invention.

In addition, to provide a composition for producing cefazolin (cefazolin) consisting of the mutant penicillin G acylase of the present invention.

In addition, to provide a composition for producing cefazolin (cefazolin) consisting essentially of the mutant penicillin G acylase of the present invention.

Another object of the present invention is to enzymatic cefazolin from tetrazolyl acetic acid ester and 3-[5-methyl-1,3,4-thiodiazol-2-yl]-7-aminocephalosporic acid. In the method for synthesizing by way, it is to provide an enzymatic synthesis (manufacturing) method of cefazoline, characterized in that the mutant penicillin G acylase of the present invention is used.

Another object of the present invention is to provide the use of the mutant penicillin G acylase of the present invention for producing cefazolin.

In order to achieve the above object, the present invention

An alpha subunit represented by SEQ ID NO: 1; And among the wild-type penicillin G acylase protein sequence containing the beta subunit represented by SEQ ID NO: 2, the group consisting of A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA, T388βA, R478βH, E486βA, D541βE and Y555βC It provides a mutant penicillin G acylase, characterized in that it comprises one or more mutations selected from.

In order to achieve another object of the present invention, the present invention provides a polynucleotide that darkens the mutant penicillin G acylase.

In order to achieve another object of the present invention, the present invention provides a recombinant expression vector comprising the polynucleotide.

In order to achieve another object of the present invention, the present invention provides a host cell transformed with the expression vector.

In order to achieve another object of the present invention, the present invention provides a composition for preparing cefazolin comprising the mutant penicillin G acylase of the present invention.

In addition, the present invention provides a composition for producing cefazolin consisting of the mutant penicillin G acylase of the present invention.

In addition, the present invention provides a composition for producing cefazolin consisting essentially of the mutant penicillin G acylase of the present invention.

In order to achieve another object of the present invention, the present invention is separated from tetrazolyl acetic acid ester and 3-[5-methyl-1,3,4-thiodiazol-2-yl]-7-aminocephalosporic acid. In the method of enzymatically synthesizing cefazolin, there is provided a method for enzymatic synthesis (manufacturing) of cefazoline, characterized in that the mutant penicillin G acylase of the present invention is used.

In order to achieve another object of the present invention, the present invention provides the use of the mutant penicillin G acylase of the present invention for producing cefazolin.

Hereinafter, the present invention will be described in detail.

As used herein, the term "protein" is used interchangeably with "polypeptide" or "peptide", and refers to a polymer of amino acid residues as commonly found in proteins in nature.

In the present invention, "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides in the form of single- or double-stranded. Unless otherwise limited, known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides are also included.

In the present specification, the term "expression" refers to the production of a protein or nucleic acid in a cell.

One letter (three letters) of amino acids as used herein means the following amino acids according to the standard abbreviation regulations in the field of biochemistry: A(Ala): alanine; C(Cys): cysteine; D(Asp): aspartic acid; E(Glu): glutamic acid; F(Phe): phenylalanine; G(Gly): glycine; H(His): histidine; I(IIe): isoleucine; K(Lys): lysine; L(Leu): leucine; M(Met): methionine; N(Asn): asparagine; P(Pro): proline; Q(Gin): glutamine; R(Arg): arginine; S(Ser): serine; T(Thr): threonine; V(Val): valine; W(Trp): tryptophan; Y(Tyr): Tyrosine.

As used herein, "(one amino acid position) (one amino acid position) (one amino acid position)" means that the amino acid previously marked at the corresponding amino acid position of the natural (wild type) polypeptide is substituted with the amino acid marked later. For example, I156αN indicates that isoleucine corresponding to No. 156 of the natural polypeptide α-subunit (in the present invention, wild-type APA α-subunit of SEQ ID NO: 1) is substituted with asparagine, and M3βV is a natural polypoly. It means that the third methionine of the peptide α-subunit (in the present invention, wild-type APA β-subunit of SEQ ID NO: 2) is substituted with valine.

In the present specification, the term MMTD-7-ACA refers to a reaction product of MMTD (Mercepto-5-methyl-1,3,4- thiadiazole) and 7-ACA (7-amino cephalosporanic acid), and "3-[ It is also expressed as "5-methyl-1,3,4-thiodiazol-2-yl]-7-aminocephalosporic acid" and the like. It is used as a substrate in the synthesis of cefazoline antibiotics and is a compound having a β-lactam nuclear structure. The MMTD and 7-ACA reaction product are known in various expressions in the art, but in the present invention, it is meant to include all substances considered to be substantially equivalent in the art. Although not limited thereto, as an example in the present invention, TDA (7-amino-3-(2-methyl-1,3,4-thiadiazol-5-yl)-thiomethyl-3-cephem-4-carboxylic acid) is preferably Can be used.

In the present specification, the term "TzAA ester" compound refers to "tetrazolyl acetic acid ester", and refers to an ester compound in which a group such as methyl, ethyl, propyl, etc. is bonded to TzAA, and in the present specification, "tetrazol-1-acetic acid It may also be expressed as "ester" or the like. It is used as a substrate in the synthesis of cefazoline and acts as an acyl donor. The "TzAA ester" compound is not limited thereto, but TzAAMe (tetrazole-1-acetic acid methyl ester) may be preferably used as an example in the present invention.

In the present invention, the productivity for cefazoline is Increased Achromobacter sp . CCM Penicillin G from 4824 Acylase Variant to provide.

In the present specification, Achromobacter sp. The penicillin G acylase protein derived from CCM 4824 was referred to as "APA", and the polynucleotide or gene sequence encoding it was referred to as "apa".

Achromobacter sp. The penicillin G acylase (APA) wild type derived from CCM 4824 includes an α-subunit represented by SEQ ID NO: 1 and a β-subunit represented by SEQ ID NO: 2. Specifically, wild-type APA is a precursor type, consisting of an alpha subunit represented by SEQ ID NO: 1 and a beta subunit represented by SEQ ID NO: 2 connected by a spacer peptide (e.g., SEQ ID NO: 3). It is expressed as a single-chain polypeptide (e.g., SEQ ID NO: 4), and then the spacer peptide is removed through autocatalytic processing in the cell to form a mature form consisting of the alpha subunit and the beta subunit. ) To have an active dimer form. For example, the precursor-type wild-type APA represented by SEQ ID NO: 4 may be encoded by, for example, a polynucleotide of a base sequence known as NCBI genbank AY919310.1, but is not limited thereto.

Achromobacter sp. It has been reported that penicillin G acylase (APA wild type) derived from CCM 4824 strain has superior advantages compared to the known homologous enzymes derived from other organisms. For example, it has been reported that it has excellent thermal stability compared to enzymes derived from Achromobacter xylosoxidans, and has better catalytic activity for the synthesis of β-lactam antibiotics compared to enzymes derived from E. coli at low substrate concentrations (Stanislav Becka et al. , Penicillin G acylase from Achromobacter sp.CCM 4824, Appl Microbiol Biotechnol DOI 10.1007/s00253-013-4945-3). However, Achromobacter sp. The penicillin G acylase (APA) wild-type protein derived from the CCM 4824 strain also has a problem in that, as described in the examples herein, substrate and product degradation activity occurs at a considerable level compared to the synthetic activity. On the other hand, there have been attempts to manufacture and use variants for penicillin G acylase derived from various microorganisms in the past, but these are only reported conversion rates without taking into account decomposition reactions and S/H, or focusing only on synthetic reactions. There were limitations in that it was developed by focusing only on each specific reaction, such as being tailored and developed. Therefore, in fact, they still had insufficient effect in application to the industrial level, for example, some amino acid variants disclosed in the CN105483105A patent disclosed that the substrate conversion rate for a small number of antibiotics such as amoxicillin was 99% after 90 to 120 minutes. However, in reality, the mutant was only about 2 times improved in the synthesis ability compared to the wild type, and the S/H value for this synthetic ability was only about 3.9 times (based on amoxicillin). The degree of such improvement is limited in terms of process efficiency when applied to an actual industrial unit. In other words, in order to determine the comparative advantage of the enzyme improvement, it is necessary to evaluate not only the increase in synthetic activity, but also the decrease in the decomposition activity and the increase in the S/H value.

On the other hand, as a representative example of the mutants of the present invention, the ZSH1-13 mutant not only significantly increased the sefazoline synthesis activity by 12.5 times compared to the wild type, but also showed a 6.1 times S/H ratio compared to the wild type. In addition, as another example, the ZSH2-5 mutant (developed based on the ZSH1-13 mutant) significantly increased the synthesis activity of cefazoline 43 times compared to the wild type, and in addition to this synthetic activity, the S/H ratio was wild type. Compared to 19.8 times. In particular, in the case of the finally selected ZSH3-1 variant (developed based on the ZSH2-5 variant) among the various variants generated through countless steps, the cefazoline synthesis activity significantly increased 65 times compared to the wild type. In addition to the synthetic activity, the S/H ratio was 36.7 times that of the wild type. Accordingly, according to the mutant APA provided by the present invention, cefazoline can be produced with high efficiency. This is a remarkable improvement that can be used in the production process, and represents a significant level of technological advancement compared to the existing technology.

As described above, Achromobacter sp. The penicillin G acylase variants derived from CCM 4824 are characterized by having a high-efficiency synthesis ability against cefazoline. In the present invention, the term "high efficiency" means that the production amount and speed of the synthetic product is increased, as well as the S/H ratio is high.

Accordingly, the APA mutation form first disclosed in the present invention is as follows. The mutant penicillin G acylase of the present invention (hereinafter, referred to as mutant APA in the present specification) is a wild-type penicillin G acyl comprising an alpha subunit represented by SEQ ID NO: 1 and a beta subunit represented by SEQ ID NO: 2 In the Rase protein sequence, A116α, I156α, N169α, M3β, S54β, T64β, H173β, T251β, T388β, R478β, E486β, D541β, and Y555β. It is characterized by that.

Specifically, the mutation is in the wild-type penicillin G acylase protein sequence comprising the alpha subunit represented by SEQ ID NO: 1 and the beta subunit represented by SEQ ID NO: 2, A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN , T251βA, T388βA, R478βH, E486βA, D541βE, and Y555βC.

For example, in the wild-type penicillin G acylase protein sequence comprising the alpha subunit represented by SEQ ID NO: 1 and the beta subunit represented by SEQ ID NO: 2, a polypeptide comprising an A116αT substitution, a poly comprising an I156αN substitution One mutation site such as a peptide, a polypeptide including an N169αT substitution, a polypeptide including an H173βN substitution, a polypeptide including a T251βA substitution, a polypeptide including an E486βA substitution, or a polypeptide including a Y555βC substitution, etc. ), or

For example, in the wild-type penicillin G acylase protein sequence comprising the alpha subunit represented by SEQ ID NO: 1 and the beta subunit represented by SEQ ID NO: 2, A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA , T388βA, R478βH, E486βA, D541βE and Y555βC 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 randomly selected from the group consisting of It may have multiple mutation sites, such as a polypeptide including dog mutation sites, but is not limited thereto.

Preferably, the mutant penicillin G acylase (mutant APA) of the present invention may include six mutant sites of I156αN, N169αT, H173βN, T251βA, E486βA and Y555βC in the wild-type penicillin G acylase protein sequence (ZSH1- 13). More preferably, the mutant penicillin G acylase of the present invention may include an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 6. As a preferred form thereof, i) a single-chain polypeptide (e.g., an alpha subunit represented by SEQ ID NO: 5) and a beta subunit represented by SEQ ID NO: 6 are linked by a spacer peptide (e.g., SEQ ID NO: 3). For example, it may be provided as a precursor protein in the form of SEQ ID NO: 7), or ii) a mature form consisting of an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 6 It may be provided as a protein, but is not limited thereto.

In addition, the present invention may include modification of additional amino acid residues in the mutant APA. The term “modification” refers to deletion, substitution or addition of amino acid residues. The modification of the additional amino acid residue is carried out for the purpose of increasing the sefazoline synthesis activity of the mutant APA of the present invention and reducing the degrading activity of the substrate used for the synthesis, and a modified form of the amino acid residue that can achieve the above object (Type) and position are not particularly limited.

Preferably, one or more mutations selected from the group consisting of G49αF, T201αS, M3βV, T64βA, T225βN and T388βA among the mutant APA sequences comprising six mutation sites of I156αN, N169αT, H173βN, T251βA, E486βA and Y555βC compared to the wild type It may be to further include. More preferably, one or more selected from the group consisting of G49αF, T201αS, M3βV, T64βA, T225βN and T388βA among the mutant APA sequences including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 6 It may be to further include a mutation.

For example, in the mutant APA sequence including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 6, a polypeptide including a G49αF substitution, a polypeptide including a T201αS substitution, and an M3βV substitution A polypeptide including, a polypeptide including a T64βA substitution, a polypeptide including a T225βN substitution, or a polypeptide including a T388βA substitution, may additionally include one mutation site, or

For example, among the mutant APA sequences including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 6, 2 randomly selected from the group consisting of G49αF, T201αS, M3βV, T64βA, T225βN and T388βA It may be one that additionally includes multiple mutation sites, such as a polypeptide including dog mutations, three mutations, four mutations, five mutations, or six mutations, but is not limited thereto.

Most preferably, the mutant APA of the present invention is in the mutant APA including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 6, three mutation (substitution) sites of M3βV, T64βA and T388βA. It may further include (including a total of 9 substitution sites compared to the wild type, see ZSH2-5). More preferably, the variant APA of the present invention may include an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 8. As a preferred form of provision thereof, i) a single-chain polypeptide (e.g., an alpha subunit represented by SEQ ID NO: 5) and a beta subunit represented by SEQ ID NO: 8 are linked by a spacer peptide (e.g., SEQ ID NO: 3). For example, it may be provided as a precursor protein in the form of SEQ ID NO: 9), or ii) may be provided as a mature protein consisting of an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 8, , Is not limited thereto.

In addition, the present invention provides a polypeptide comprising a further mutation with respect to the variant APA. Preferably, in the mutant APA sequence including nine mutation sites of I156αN, N169αT, M3βV, T64βA, H173βN, T251βA, T388βA, E486βA and Y555βC compared to the wild type, S54βG, S150βT, N226βT, A310βD, R478βH and D541βD It may be to further include one or more mutations selected from. More preferably, one or more selected from the group consisting of S54βG, S150βT, N226βT, A310βD, R478βH and D541βE among the mutant APA sequences including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 8 It may be to further include a mutation.

For example, among the mutant APA sequences including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 8, a polypeptide including S54βG substitution, a polypeptide including S150βT substitution, and N226βT substitution It may be one that additionally includes one mutation site, such as a polypeptide including a polypeptide, a polypeptide including an A310βD substitution, a polypeptide including a R478βH substitution, or a polypeptide including a D541βE substitution, or

For example, among the mutant APA sequences including the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 8, 2 randomly selected from the group consisting of S54βG, S150βT, N226βT, A310βD, R478βH and D541βE It may have multiple mutation sites, such as a polypeptide including dog mutations, three mutations, four mutations, five mutations, or six mutation sites, but is not limited thereto.

Most preferably, the mutant APA of the present invention is in the mutant APA comprising the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 8, three mutation (substitution) sites of S54βG, R478βH and D541βE. It may be to further include (including a total of 12 substitution sites compared to the wild type, see ZSH3-1). More preferably, the variant APA of the present invention may include an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 10. As a preferred form of provision thereof, i) a single-chain polypeptide consisting of an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 10 are linked by a spacer peptide (eg, SEQ ID NO: 3). For example, it may be provided as a precursor protein in the form of SEQ ID NO: 11), or ii) may be provided as a mature protein consisting of an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 10. , Is not limited thereto.

On the other hand, the present invention is the mutant penicillin G Acylase (mutant APA) Encoding polynucleotides are provided.

The combination of bases constituting the polynucleotide is not particularly limited as long as the polynucleotide can encode the mutant APA polypeptide of the present invention. When an amino acid sequence is identified, techniques for producing a polynucleotide encoding the amino acid sequence based on codon information known in the art are well known in the art. The polynucleotide may be provided as a single-stranded or double-stranded nucleic acid molecule including all DNA, cDNA and RNA sequences.

The present invention provides a recombinant expression vector comprising the polynucleotide.

In the present invention, the term "recombinant expression vector" is a vector capable of expressing a target protein or a target nucleic acid (RNA) in a suitable host cell, and a gene containing essential regulatory elements operably linked to express a polynucleotide (gene) insert It refers to the creation.

In the present invention, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest to perform a general function. , It means that a nucleic acid sequence encoding a protein or RNA (for example, a polynucleotide sequence encoding a mutant APA of the present invention) is linked in a manner that allows gene expression by an expression control sequence, for example, a promoter and The nucleic acid sequence encoding a protein or RNA must be operably linked to affect the expression of the encoded nucleic acid sequence, and the operative linkage with a recombinant vector can be prepared using gene recombination techniques well known in the art. In addition, site-specific DNA cleavage and ligation are performed using enzymes generally known in the art.

The recombinant expression vector of the present invention is characterized in that it contains a polynucleotide encoding the mutant APA. The polynucleotide sequence cloned into a vector according to the present invention may be operably linked to an appropriate expression control sequence, and the operably linked polynucleotide (gene) sequence and expression control sequence are used to select a host cell containing the vector. It may be included in an expression vector that includes a selection marker for and/or a replication origin. In addition, the expression vector includes an expression control sequence and, if necessary, a signal sequence or a leader sequence for membrane targeting or secretion, and may be prepared in various ways according to the purpose. The expression control sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, sequences regulating the termination of transcription and translation, initiation codon, stop codon, polyadenylation. Includes signals and enhancers. The promoter of the vector may be constitutive or inducible.

Signal sequences include PhoA signal sequence and OmpA signal sequence when the host is Escherichia bacteria, α-amylase signal sequence and subtilisin signal sequence when the host is Bacillus bacteria, and MFα when the host is yeast. Signal sequence, SUC2 signal sequence, etc., when the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like may be used, but are not limited thereto.

The expression vector of the present invention is not particularly limited if it is a vector commonly used in the cloning field, and includes, for example, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector, but is not limited thereto. Specifically, the plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50). , The virus may be a retrovirus, an animal virus such as an adenovirus or a vaccinia virus, an insect virus such as a baculovirus, and the like, but is not limited thereto. In one embodiment of the present invention, pBC-KS(+) was used.

The present invention provides host cells and microorganisms transformed by the expression vector.

The transformation includes any method of introducing a nucleic acid (polynucleotide encoding the mutant APA of the present invention) into an organism, cell, tissue, or organ, and as known in the art, a suitable standard technique is selected according to the host cell. You can do it. These methods include electroporation, protoplasm fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, silicon carbide whiskers, sonication, and agrobacterial mediated traits. Conversion, precipitation by PEG (polyethylenglycol), dextran sulfate, lipofectamine, heat shock, particle gun bombardment, etc. are included, but are not limited thereto.

The host cell is a prokaryotic or eukaryotic cell containing heterologous DNA introduced into the cell by any means (e.g., electric shock method, calcium phosphatase precipitation method, microinjection method, transformation method, virus infection, etc.) it means.

In the present invention, the host cells are all types of single-celled organisms commonly used in the field of cloning, such as prokaryotic microorganisms such as various bacteria (eg, Clostridia genus, E. coli, etc.), lower eukaryotic microorganisms such as yeast and insect cells, Cells derived from higher eukaryotes, including plant cells and mammals, can be used as host cells, but are not limited thereto. Since the expression level and modification of the protein differ depending on the host cell, a person skilled in the art can select and use the most suitable host cell for the purpose.

In the present invention, the host cells are Clostridia spp., such as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, or Clostridium saccharobutylicum, etc.), Escherichia spp., Acetobacter spp. , Such as Acetobacter turbidans, Acetobacter pasteurianus, etc.), Aeromonas spp., Alcaligenes spp., Aphanocladium spp., Bacillus spp., three. Cephalosporium spp., Flavobacterium spp., Kluyvera spp., Mycoplana spp., Protaminobacter spp., Pseudomonas spp. It may be any one genus microorganism selected from the group consisting of the genus Pseudomonas spp., Achromobacter spp., and the genus Xanthomonas (such as Xanthomonas citri), but is not limited thereto Does not.

The microorganism of the genus Escherichia may preferably be Escherichia coli (Escherichia coli), and in an embodiment of the present invention, Escherichia coli MC1061/pBC-ZSH3 as a transformed microorganism to express the mutant APA A strain of -1 ( Escherichia coli MC1061/pBC-ZSH3-1, accession number KCTC 13991BP) is provided. The Escherichia coli MC1061/pBC-ZSH3-1 expresses a polypeptide comprising an alpha subunit represented by SEQ ID NO: 5 and a beta subunit represented by SEQ ID NO: 10, or a polypeptide consisting of the units. . That is, the Escherichia coli MC1061/pBC-ZSH3-1 is transformed by a recombinant expression vector containing a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10.

Also seen The invention is the above-described mutant penicillin G of the present invention Acylase It provides a composition for producing (synthetic) cefazoline comprising.

As described above, the mutant APA of the present invention provides remarkable advantages in synthesizing cefazoline. Accordingly, the present invention provides a use of the mutant APAs of the present invention for the synthesis of cefazoline.

The mutant APA according to the present invention may be prepared by a protein production method known in the art with reference to the above-described sequence information, but is not limited thereto, but may be prepared by a genetic engineering method. For example, a nucleic acid encoding the mutant APA polypeptide is constructed according to a conventional method. The nucleic acid can be constructed by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (available from Biosearch or Applied Biosystems). The constructed nucleic acid is operatively linked thereto and inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that control the expression of the nucleic acid, and recombination formed therefrom. The host cell is transformed with the expression vector. The resulting transformant is cultured under a medium and conditions suitable for expression of the nucleic acid to recover the substantially pure polypeptide expressed by the nucleic acid from the culture. The recovery may be performed using a method known in the art (eg, chromatography). And it is possible to use the mutant APA of the present invention prepared as described above as it is for the production of the target product or to use it after purification. For the separation and purification of the mutant APA enzyme, the protein separation method by various chromatographic methods can be used as it is or slightly modified to suit the purpose of the experiment using the properties of APA that have been previously discovered. In addition, the mutant APA is purified by affinity chromatography using specific avidity properties such as binding strength between histidine peptide and nickel column components, cellulose binding domain (CBD), and binding strength with fibrin. It is also possible.

The term "substally pure polypeptide" as used herein means that the polypeptide according to the present invention does not contain substantially any other protein derived from a host cell. Genetic engineering methods for synthesizing the polypeptide of the present invention are known in the art.

In addition, the mutant APA of the present invention can be easily prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

The mutant APA of the present invention can be used not only in a free state but also in an immobilized state. Immobilization of the mutant APA is possible by a conventional protein immobilization method known in the art. Examples of the carrier include natural polymers such as cellulose, starch, dextran, and agarose; Synthetic polymers such as polyacrylamide, polyacrylate, polymethacylate, and Eupergit C; Alternatively, minerals such as silica, bentonite, and metal may be used. In addition, it is possible to bind mutant APA to these carriers by covalent bonding, ionic bonding, hydrophobic bonding, physical adsorption, microencapsulation, or the like. In addition, it is also possible to immobilize mutant APA by forming a covalent bond by the action of these carrier-enzyme conjugates such as glutaraldehyde or cyanogen bromide. In addition, as a more preferable method, it is also possible to use by immobilizing microbial cells containing mutant APA as it is without the need to separately purify mutant APA. In order to increase the reactivity of mutant APA contained in microorganisms during such whole cell immobilization, techniques such as drilling a hole or surface expression may be applied.

In the composition for preparing cefazoline of the present invention, the term "including mutant APA" refers to a polynucleotide encoding (coding) the mutant APA as well as a direct method including the mutant APA polypeptide itself from the beginning in the composition, the poly It means including all indirect methods of inducing the production of mutant APA by including in the composition any one or more selected from the group consisting of an expression vector containing a nucleotide and a host cell (microorganism) transformed with the expression vector. .

Specifically, the present invention,

Tetrazolylacetic acid Esters and 3-[5- methyl -1,3,4- Thiodiazole -2-yl]-7- From aminocephalosporic acid Cefazoline ( cefazolin )of Enzymatically Synthetic In a way , The mutant penicillin G of the present invention Acylase Of cefazoline, characterized in that it is used Enzymatic synthesis (manufacturing) Provides a way.

The mutant APA enzyme of the present invention provided during the process of synthesizing (manufacturing) cefazoline may be provided in the form of a composition containing the mutant APA polypeptide or a culture of the mutant APA producing strain, and the mutant APA is free ( It may be provided in a free) state or in a fixed state, but is now not limited and is understood with reference to the foregoing.

As long as the activity of the mutant APA enzyme of the present invention is not lost, the reaction conditions of each of the substrates in the above-described synthesis process are not particularly limited, but may be preferably made in an aqueous solution (water or buffer solution), and a preferred reaction mixture The pH of may be selected within the range of pH5 to pH9, the preferred reaction time within the range of 0.1 to 24 hours, and the preferred reaction temperature within the range of 3 to 30°C. The above conditions can be appropriately selected and adjusted by a person skilled in the art depending on the amount of reactive substances and enzymes used in one reaction, and the desired process speed and efficiency.

MMTD-7-ACA (3-[5-methyl-1,3,4-thiodiazol-2-yl]-7-aminocephalosporic acid) and TzAA, which are provided as substrates during the synthesis (manufacturing) process of cefazoline The concentration and ratio at which the ester (tetrazolyl acetic acid ester) is added in the reaction mixture are not particularly limited, but for example, the MMTD-7-ACA: TzAA ester ratio may be 1:1 to 1:1.5, preferably industrial and economical. Considering the advantages of the present invention in terms of, it can be used in a ratio of 1:1.

The addition amount of the mutant APA of the present invention in the reaction mixture is not particularly limited, but may be, for example, in the range of 0.1 to 100 U/ml, and preferably 2 to 10% ( w/w).

The cefazoline finally produced in the reaction process can be separated and purified from the reaction solution by a conventional compound purification method known in the art (eg, chromatography, etc.).

In addition, the present invention provides the use of the above-described mutant penicillin G acylase of the present invention for producing cefazolin.

In the present specification, the term “comprising” is used in the same meaning as “including” or “characterized by”, and in the composition or method according to the present invention, specifically mentioned It does not exclude additional components or steps of the method that have not been made. In addition, the term “consisting of” means excluding additional elements, steps, or ingredients that are not separately described. The term “essentially consisting of” means that, in the scope of a composition or method, it is possible to include a substance or step that does not substantially affect its basic properties in addition to the described substance or step.

Achromobacter sp. The penicillin G acylase variants derived from CCM 4824 are characterized by a marked increase in productivity specifically for cefazoline (Cefazolin). Specifically, the mutant APAs of the present invention are synthesized compared to their wild-type enzymes and other previously known mutant forms, while having very high synthetic activity specifically for cefazolin, compared to their degradation activity for substrates used for sefazoline synthesis The ratio (S/H ratio) is remarkably high, and the productivity of cefazoline is remarkably increased to a significant level in industrial applications.

1 shows the amino acid substitution sites of the ZSH1-13 variant, the ZSH2-5 variant, and the ZSH3-1 variant representatively among the variants of the present invention.

Figure 2 is a reaction (S) for synthesizing cefazoline from precursors (substrates) TzAAMe and TDA through an enzymatic reaction (S), a hydrolysis reaction of TzAAMe to TzAA in the precursor as a decomposition reaction (H1), and TzAAMe of the cefazoline and Hydrolysis reaction to TDA (H2) is shown.

FIG. 3 is data showing the conversion rate of cefazoline, the amount of remaining TzAAMe, and the amount of TzAA produced over time in the synthesis of cefazoline by the ZSH3-1 variant.

Hereinafter, the present invention will be described in detail.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example 1: Preparation of penicillin G acylase (APA) wild type derived from Achromobacter sp.CCM 4824

1-1. Achromobacter sp. Synthesis of a gene expressing the penicillin G acylase (APA) wild type derived from CCM 4824

Wild-type APA (penicillin G acylase derived from Achromobacter sp. ) Expression as a single-chain polypeptide linked by a peptide (SEQ ID NO: 4), and then through autocatalytic processing in cells, a mature active dimer consisting of the alpha subunit and the beta subunit It has a body shape. A gene expressing the wild-type APA precursor of SEQ ID NO: 4 (abbreviated as “apa gene”) was synthesized by requesting a company Bioneer (Daejeon, Korea).

1-2. Preparation of recombinant vector expressing wild-type APA (pBC-APA)

The apa gene obtained in Example 1-1 was inserted into the XbaI and NotI restriction enzyme recognition sites of a pBC-KS(+) vector (Stratagene, USA) to prepare a pBC-APA plasmid for expressing wild-type APA. The specific manufacturing method is as follows.

The apa gene DNA product (about 2.6 kb in size) was digested with restriction enzymes XbaI and NotI, and then purified with a purification kit (QIAquick Gel Extraction Kit; QIAGEN, Germany), and used as insertion DNA. In addition, pBC KS(+) vector (Stratagene, USA) DNA was digested with restriction enzymes XbaI and NotI, and a DNA fragment dephosphorylated with CIP was used as vector DNA. The inserted DNA and the vector DNA were ligated at 16° C. for 12 to 16 hours using T4 DNA ligase (New England Biolabs, Sweden), and then E. coli MC1061 strain was transformed. Transformants were selected by spreading the strain on LB agar medium containing chloramphenicol antibiotics at a concentration of 20 μg/mL and incubating at 30° C. overnight. By separating the plasmid from this transformant and determining the nucleotide sequence of the inserted DNA, a pBC-APA plasmid containing the apa gene was finally obtained. The pBC-APA plasmid expresses a wild type APA protein.

In order to prepare a variant having a high level of synthesizing ability with respect to Cefazolin and significantly reduced activity of hydrolyzing a reaction substrate, numerous procedures as in Examples 2 and 3 were performed, and Examples 2 and 3 below. Represents an example of a myriad of processes. The variants produced below are indicated in specific mutant forms for amino acid positions for each of the alpha subunit and beta subunit.

Example 2: Preparation and selection of a mutant having high cefazolin synthesis activity

2-1. Preparation of primary variant library by error prone PCR

To prepare mutant APA with increased cefazolin synthesis activity, random mutation was artificially induced in the nucleotide sequence of the apa gene synthesized in Example 1, and for this purpose, error prone PCR was performed. A mutant library was prepared. The manufacturing process of the specific mutation library is as described below.

Specifically, the error-prone polymerase chain reaction was performed using the Diversity PCR Random Mutagenesis kit (Clontech, USA) to cause 1-2 mutations per 1,000 bp. The composition of the PCR reaction solution was 1 ng/μL of pBC-APA plasmid as template DNA, and 10 pmol of each T3 primer (SEQ ID NO: 12) and T7 primer (SEQ ID NO: 13), 2 mM dGTP, 50X Diversity dNTP mix , 10X TITANIUM Taq buffer, TITANIUM Taq polymerase, the final volume was carried out in 50 μL. PCR reaction conditions are: The reaction mixture is pre-denatured at 96°C for 2 minutes, denaturation is performed at 96°C for 30 seconds, annealing is performed at 52°C for 30 seconds, and polymerization is performed 18 times at 68°C. Post-polymerization was performed at 68° C. for 5 minutes. The error-prone polymerase chain reaction under the above conditions was performed to amplify a mutant DNA fragment having a size of about 2.7 kb.

The mutant gene obtained through the above PCR, that is, the PCR product of 2.7 kb, was digested with restriction enzymes XbaI and NotI, purified using a purification kit (QIAquick Gel Extraction Kit; QIAGEN, Germany), and used as the insertion DNA, pBC -KS(+) vector DNA was digested with restriction enzymes XbaI and NotI, and a DNA fragment dephosphorylated with CIP was used as vector DNA. The inserted DNA and the vector DNA were conjugated using T4 DNA ligase (New England Biolabs, Sweden) at 16° C. for 16 hours, and then the E. coli MC1061 strain was transformed by electroporation using a conjugation solution. Was performed. The strain was plated on LB agar medium containing chloramphenicol antibiotics at a concentration of 20 μg/mL and cultured overnight at 30° C. to prepare a random mutant library.

The mutant APA having increased reactivity to Cefazolin synthesis from the mutant library was searched for in the following manner. The reaction quality and specific chemical reaction process for the synthesis of Cefazolin are shown in FIG. 2. The E. coli MC1061 transformant containing the mutant apa gene in which the mutation was induced was inoculated into a 96-well plate dispensed with 160 μl of LB liquid medium containing chloramphenicol antibiotics, and then 60-70 at 23°C and 165 rpm. It was incubated with shaking for hours. Thereafter, 20 μl of culture solution was taken from the 96-well plate, transferred to a new 96-well plate, and 105 μL of cell lysis solution (1.25 mg/mL lysozyme, 1.25 mM EDTA, 0.375% Triton X-100) was added. After the addition, it was left at 25° C. for 2 hours, and then at 10° C. for 1 hour. Thereafter, 125 μL of a substrate solution prepared by dissolving 10 mM TDA and 10 mM TzAAMe, which is a 1:1 substrate ratio based on 100 mM Ammonium phosphate (pH 7.5) buffer solution, was added, followed by synthesis reaction at 10° C. for 16 to 18 hours. Was performed. After performing the synthesis reaction, 40 μL of the supernatant in the reaction solution was transferred to a new 96-well plate. The enzyme reaction was stopped by adding 100 μL of the reaction stop solution 0.2 N HCl, and then the mutant with increased Cefazolin synthesis activity was searched for using HPLC. HPLC analysis conditions were 0.01 M Potassium phosphate (pH 6.8) and Methanol (7:3) as solvents, YMC-Triart C18, 250*4.6mm was used as the column, and 10 μL was injected at 0.8 mL/min. Flowed and analyzed at 270 nm for 12 minutes.

It was confirmed that cefazolin synthesis activity was increased in 7 mutants by the same method as described above. Seven variants were determined by gene sequence determination, respectively, compared to wild-type A116αT(ZEP1-9), I156αN(ZEP1-10), H173βN(ZEP1-12), Y555βC(ZEP1-13), E486βA(ZEP1-18), It was confirmed that it was mutated to N169αT (ZEP1-26), or T251βA (ZEP1-33).

2-2. Preparation of secondary variant library by DNA shuffling_Selection of ZSH1-13 improved strain

In addition, seven amino acid residues (A116αT, I156αN, N169αT, H173βN, T251βA, E486βA, Y555βC) based on the activity of the modified strain obtained from the error-induced polymerase chain reaction to improve mutant penicillin G acylase with increased cefazolin synthesis activity ) Was prepared.

Specifically, in order to prepare a library in which the A116α amino acid has been mutated, PCR was performed using wild-type APA as a template and using M13R primer (SEQ ID NO: 15) and SH116a-R primer (SEQ ID NO: 17) to perform PCR with a size of about 510 bp. The product was recovered, and wild-type APA and ZEP1-10 were used as templates to prepare a library with mutated A116α, I156α, and N169α amino acids, and SH116a-F primer (SEQ ID NO: 16) and SH169a-R primer (SEQ ID NO: 19) were used. PCR was performed to recover a PCR product of about 160 bp in size, and to prepare a library in which N169α, H173β, and T251β amino acids were mutated, wild-type APA and ZEP1-12 were used as templates, and SH169a-F primer (SEQ ID NO: 18) and SH251b PCR was performed using the -R primer (SEQ ID NO: 21) to recover a PCR having a size of about 1,100 bp. In addition, in order to mutate amino acids T251β, E486β, Y555β, PCR was performed using wild-type APA, ZEP1-18 as a template, and SH251b-F primer (SEQ ID NO: 20) and SH555b-R primer (SEQ ID NO: 23), and a size of about 910 bp. PCR was recovered, and a PCR product having a size of about 100 bp was recovered by performing PCR using wild-type APA as a template and using SH555b-F primer (SEQ ID NO: 22) and M13F primer (SEQ ID NO: 14) to mutate Y555β amino acid. I did.

The composition of the PCR reaction solution was performed by adjusting the final volume to 100 μL with each template DNA, primer, pfu-x buffer, dNTPs mix, and pfu-x polymerase. The PCR reaction conditions are: The reaction mixture was pre-denatured at 96°C for 3 minutes, denaturation was performed at 96°C for 30 seconds, annealing at 52°C for 30 seconds, and polymerization was repeated 18 times at 68°C for 2 minutes. Post-polymerization was performed at 68° C. for 5 minutes.

About 510 bp of PCR product and 160 bp of PCR product, 1,100 bp of PCR product, 910 bp of PCR product, and about 100 bp of PCR product obtained under the above conditions were mixed, and T3 primer (SEQ ID NO: 12) and T7 primer PCR was performed using (SEQ ID NO: 13) to amplify a multi-variant DNA fragment having a size of about 2.7 kb. The PCR product of about 2.7 kb thus obtained was inserted into pBC KS(+) vector DNA in the same manner as in Example 2-1, and the E. coli MC1061 strain was transformed to prepare a site-directed mutant library.

As a result of searching for a mutant having increased reactivity to cefazolin synthesis activity from the position-directed mutant library in the same manner as in Example 2-1, six mutants having increased cefazolin synthesis activity compared to wild-type APA (I156αN, N169αT, H173βN, T251βA, E486βA and Y555βC) were selected and named as ZSH1-13. ZSH1-13 expresses the alpha subunit of SEQ ID NO: 5 and the beta subunit of SEQ ID NO: 6.

2-3. Preparation of tertiary variant library by error-prone polymerase chain reaction (error prone PCR)

In order to further increase the cefazolin synthesis activity of the ZSH1-13 mutant prepared in Example 2-2, an error-prone mutation library in the same manner as in Example 2-1 using the DNA of ZSH1-13 as a template DNA Was prepared.

Subsequently, in the search for cefazolin synthesis ability, 125 μL of a substrate solution prepared by dissolving 20 mM TDA and 20 mM TzAAMe, which is a 1:1 substrate ratio based on 100 mM Ammonium phosphate (pH 7.5) buffer solution, was added. By lowering the synthesis reaction was performed at 6° C. for 17 to 18 hours. Except for the substrate concentration and ratio, and the substrate reaction temperature, all were carried out in the same manner as described in Example 2-1.

As a result of searching the constructed error-prone mutant library, it was confirmed that cefazolin synthesis activity was increased compared to ZSH1-13 in 6 mutants. Through amino acid sequencing, the six variants were T64βA (ZEP2-7), G49αF (ZEP2-9), T388βA (ZEP2-13), T201αS (ZEP2-17), M3βV (ZEP2-21), respectively, for the ZSH1-13. Or it was confirmed that it was mutated to T225βN (ZEP2-54).

2-4. Preparation of quaternary mutant library by site-directed mutation_Selection of ZSH2-5 improved strain

Based on the activity of the modified strains obtained in Example 2-3 above, in order to obtain a mutant with further enhanced Cefazolin synthesis activity, 6 amino acid residues (G49αF, T201αS, M3βV, T64βA) were used as template DNA using ZSH1-13 plasmid DNA. , T225βN, T388βA) was prepared for the location-directed mutation library. In this Example, PCR reaction conditions were performed in the same manner as in the above-described method in Example 2-2.

Specifically, in order to prepare a library in which the amino acids G49α and T201α are mutated, PCR was performed using ZSH1-13 and ZEP2-9 as templates and M13R primer (SEQ ID NO: 15) and SH201a-R primer (SEQ ID NO: 25). A PCR product having a size of 770 bp was recovered, and in order to prepare a library with mutated T201α, M3β, and T64β amino acids, using ZSH1-13 and ZEP2-21 as templates, SH201a-F primer (SEQ ID NO: 24), SH64b-R primer ( PCR was performed using SEQ ID NO: 27) to recover a PCR product having a size of about 440 bp, and to prepare a library in which amino acids T64β, T225β, and T388β were mutated, SH64b-F primers were prepared using ZSH1-13 and ZEP2-54 as templates. PCR was performed using (SEQ ID NO: 26) and SH388b-R primer (SEQ ID NO: 29) to recover a PCR product of about 970 bp in size, and to mutate the T388β amino acid, ZSH1-13 as a template was used as a SH388b-F primer ( PCR was performed using SEQ ID NO: 28) and M13F primer (SEQ ID NO: 14) to recover a PCR product having a size of about 600 bp.

A PCR product of about 770 bp obtained under the above conditions, a PCR product of 440 bp, a PCR product of 970 bp, and a PCR product of 600 bp were mixed, and T3 primer (SEQ ID NO: 12) and T7 primer (SEQ ID NO: 13) were mixed. ) Was used to perform PCR to amplify a multiple variant DNA fragment having a size of about 2.7 kb. The PCR product of about 2.7 kb thus obtained was inserted into pBC KS(+) vector DNA in the same manner as in Example 2-1, and the E. coli MC1061 strain was transformed to prepare a site-directed mutant library.

Thereafter, as a result of searching the library in the same manner as in Example 2-3, as a result of searching the library, as a mutant in which cefazolin synthesis activity was significantly increased compared to ZSH1-13, a strain in which M3βV, T64βA and T388βA were further mutated with respect to ZSH1-13 were identified. It was selected and named as ZSH2-5. The ZSH2-5 expresses the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 8.

Example 3: high Synthesis ability and At the same time Reduced , Of high efficiency cefazolin synthesis Variant Manufacturing and sorting

3-1. Preparation of 5th variant library by error-prone polymerase chain reaction (error prone PCR)

In order to increase the cefazolin synthesis activity and decrease the reaction substrate (typically, TzAAMe) degradation activity of the ZSH2-5 mutant prepared in Example 2-4, the method of Example 2-1 and the method of Example 2-1 using ZSH2-5 as a template DNA. In the same way, an error-prone mutation library was prepared. After that, the search for the ability to synthesize cefazolin was carried out in the same manner as in the method described in Example 2-3. In addition, in the search for the reactivity of the substrate TzAAMe degrading activity, 30 μL of enzyme solution was transferred to a 96-well plate, and then 5 mM TDA in 100 mM Ammonium phosphate (pH7.5) buffer solution, and based on this, a 1:1.5 ratio of 7.5 125 μL of a substrate solution prepared by dissolving mM TzAAMe together was added, and the reaction temperature was increased to 10° C. to perform a synthesis reaction for 18 to 20 hours. Except for the substrate concentration and the substrate reaction temperature, all were carried out in the same manner as described in Example 2-3.

As a result of searching for Cefazolin synthesis activity and substrate degradation activity of the prepared error-prone mutant library, it was confirmed that the increase in cefazolin synthesis activity and decrease in TzAAMe degradation activity compared to ZSH2-5 in the six variants. Through amino acid sequence analysis, the six variants were S54βG(ZEP3-2), S150βT(ZEP3-11), R478βH(ZEP3-14), N226βT (ZEP3-26), A310βD(ZEP3-33) for the ZSH2-5, respectively. ), or D541βE (ZEP3-48).

3-2. Preparation of 6th mutant library by site-directed mutation_Selection of ZSH3-1 improved strain

Based on the activity of the improved strains obtained in Example 3-1, the DNA of ZSH2-5 was used as a template DNA, and a site-directed mutation library for 6 amino acid residues (S54βG, S150βT, N226βT, A310βD, R478βH, D541βE) Was prepared. In this Example, PCR reaction conditions were performed in the same manner as in the above-described method in Example 2-2.

Specifically, in order to prepare a library in which the S54β amino acid has been mutated, a PCR product having a size of about 1,180 bp was recovered using the M13R primer (SEQ ID NO: 15) and the SH54b-R primer (SEQ ID NO: 31) using ZSH2-5 as a template, To prepare a library with mutated S54β, S150β, N226β amino acids, PCR was performed using ZSH2-5 and ZEP3-11 as templates and SH54b-F primer (SEQ ID NO: 30) and SH226b-R primer (SEQ ID NO: 33). A PCR product having a size of about 520 bp was recovered, and ZSH2-5 and ZEP3-33 were used as templates to prepare a library in which amino acids N226β, A310β, and R478β were mutated, and SH226b-F primer (SEQ ID NO: 32) and SH478b-R primer PCR was performed using (SEQ ID NO: 35) to recover a PCR product having a size of about 760 bp, and to prepare a library with mutated R478β and D541β amino acids, ZSH2-5 and ZEP3-48 were used as templates and SH478b-F primer ( SEQ ID NO: 34), PCR was performed using M13F primer (SEQ ID NO: 14) to recover a PCR product having a size of about 340 bp. The 1,180 bp PCR product, 520 bp size PCR product, 760 bp size PCR product, and 340 bp size PCR product are mixed, and PCR using T3 primer (SEQ ID NO: 12) and T7 primer (SEQ ID NO: 13) Was performed to amplify a multiple variant DNA fragment having a size of about 2.7 kb. The PCR product of about 2.7 kb thus obtained was inserted into pBC KS(+) vector DNA in the same manner as in Example 2-1, and the E. coli MC1061 strain was transformed to prepare a site-directed mutant library.

Thereafter, as a result of searching for the activity of the library in the same manner as in Example 3-1, it was found that cefazolin synthesis activity was significantly increased compared to ZSH2-5, and the substrate (TzAAMe) degrading activity was significantly reduced, as a variant, S54βG, A strain in which R478βH and D541βE were further mutated was selected and named as ZSH3-1. ZSH3-1 expresses the alpha subunit represented by SEQ ID NO: 5 and the beta subunit represented by SEQ ID NO: 10.

3-3. Strains deposit

Among the mutant enzymes produced, the E. coli MC1061 strain transformed with the pBC-ZSH3-1 plasmid containing the gene encoding the ZSH3-1 mutant showing the best activity was selected as “Escherichia coli MC1061/pBC-ZSH3-1. And deposited with the Korea Research Institute of Bioscience and Biotechnology Gene Bank on October 11, 2019 (Accession No.: KCTC 13991BP).

The comparative evaluation results of Cefazolin synthesis efficiency of each of the representative variants described in Examples 2 to 3 will be described in the following Examples.

Example 4: Comparison of cefazoline activity efficiency of APA variants

4-1. Enzyme solution preparation

In order to compare the degree of activity of the prepared APA variants, each enzyme solution was prepared as follows. After inserting the genes encoding each of the APA variants prepared in Examples 2 to 3 into the pBC-KS(+) vector in the same manner as described in Example 1-2, each of the expression vectors was E. coli MC1061 was transformed into the E. coli strain. Each E. coli transformant was inoculated in 3 mL of LB medium (1% Bacto-tryptone, 0.5% Yeast Extract, 0.5% NaCl) containing 20 μg/mL chloramphenicol, and then shaken at 28° C. and 200 rpm for 16 hours. Cultured. Thereafter, 350 μL of the culture solution was inoculated into 35 mL of fresh LB medium containing 20 μg/mL chloramphenicol, followed by shaking culture at 23° C. and 190 rpm for 48 hours. The culture solution was centrifuged (4° C., 8000 rpm, 10 minutes) to recover the cells, and then washed once with a 0.05 M ammonium phosphate (pH 7.5) buffer solution. The cells were suspended in 35 mL of the same buffer solution, crushed for 5 minutes with an ultrasonic disruptor (Vibra Cell VC750, Sonics & Materials Inc, USA) at 4°C, and centrifuged at 4°C and 13,500 rpm for 20 minutes to obtain the supernatant. Each APA variant enzyme solution was prepared by taking.

4-2. Cefazolin Measurement of synthetic activity

To measure the Cefazolin synthesis activity, a substrate solution was prepared by dissolving TDA and TzAAMe at a concentration of 10 mM each in a 0.05 M ammonium phosphate (pH 7.5) buffer solution. 500 μL of each APA variant enzyme solution of Example 4-1 was added to 500 μL of the substrate solution, and the enzyme reaction was carried out at 25°C for 10 minutes, and then 500 μL of the reaction solution was taken, and 500 μL of 0.2N HCl solution was added thereto. The reaction was stopped. After that, filtering was performed, HPLC analysis was performed under the same conditions as in Example 2-1, and quantification was performed by comparing with the quantification curve of the standard material. At this time, in the present specification, 1 unit (U) is defined as the amount of an enzyme capable of producing 1 μmole of cefazolin per minute. On the other hand, the specific activity to the substrate was expressed as an activity unit corresponding to 1 mg protein after measuring the amount of protein in the enzyme solution according to Bradford's method.

Acylase	Cefazolin synthesis		TzAAMe hydrolysis
	Specific activity (U/mg protein)	Fold	Specific activity (U/mg protein)	S/H ratio＊	Relative S/H ratio (Fold)
APA	0.2	1.0	4.2	0.05	1.0
ZSH1-13	2.5	12.5	8.6	0.3	6.1
ZSH2-5	8.6	43.0	9.1	0.9	19.8
ZSH3-1	13.1	65.5	7.5	1.7	36.7

*, Cefazolin synthesis activity / TzAAMe hydrolysis activity

Table 1 shows comparatively the Cefazolin synthesis activity between the ZSH1-13 mutant, the ZSH2-5 mutant, and the ZSH3-1 mutant and wild-type APA among the mutants of the present invention. As shown in Table 1, the yield of cefazolin synthesis was increased by continuous improvement of the mutants of the present invention compared to wild-type APA. ZSH1-13 increased the synthesis yield by about 12.5 times compared to the wild type, ZSH2-5 increased by about 43.0 times compared to the wild type APA, and the final variant ZSH3-1 increased by about 65.5 times compared to the wild type APA, significantly higher cefazolin. The production rate was shown.

4-3. Measurement of reaction substrate TzAAMe decomposition activity and S/H ratio

Similarly, in order to measure the degree of TzAAMe decomposition activity for APA variants, a substrate solution was prepared by dissolving 15 mM TzAAMe in 0.1 M Ammonium phosphate (pH 7.5) buffer solution. To 100 μl of this substrate solution, 100 μl of the enzyme solution of each of the mutants was added to perform the enzyme reaction at 25° C. for 10 minutes, and then 100 μl of 0.2 N HCl solution was added to 100 μl of the reaction solution to stop the reaction. Thereafter, filtration and HPLC analysis were performed in the same manner as in Example 4-2, to calculate the amount of hydrolyzed TzAAMe (hydrolyzed TzAAMe, that is, TzAA), and quantified by comparing with the quantification curve of the standard. At this time, 1 unit (U) is defined as the amount of enzyme capable of producing 1 μmole of TzAA per minute. The S/H ratio was measured using the values of the TzAAMe decomposition activity derived by the above method and the Cefazolin synthesis activity obtained in Example 4-2.

As shown in Table 1, it was confirmed that the relative TzAAMe decomposition activity of the mutants of the present invention decreased compared to the wild-type APA, so that the ratio of Cefazolin synthesis activity to the TzAAMe decomposition activity (S/H ratio) was remarkably increased. In particular, the S/H ratio increased in the order of ZSH1-13, ZSH2-5, and ZSH3-1, and the final variant, ZSH3-1, increased the S/H ratio by about 36.7 times compared to wild-type APA.

4-4. Measurement of cefazoline conversion from TDA and TzAAMe

In order to measure the conversion rate of Cefazolin over time, the synthesis yield of Cefazolin can be inferred by measuring the conversion rate of TDA, a precursor. TDA conversion was measured using the final variant, ZSH3-1. Specifically, the strain was cultured in the same manner as in Example 4-1, except that the amount of the LB medium containing chloramphenicol was 1 L. The cultured cell suspension was centrifuged at 8,000 rpm for 10 minutes at 4° C. and the supernatant was removed to obtain a cell pellet. After suspending in 90 ml of 0.1 M ammonium phosphate buffer, cell disruption was performed for 20 minutes using an ultrasonic disruptor. In order to separate and purify the enzyme solution, the crushed cells were allowed to stand at 10° C. for 2 hours, and then centrifuged (4° C., 10,000 rpm, 30 minutes) to obtain a supernatant. This solution was used in the TDA conversion reaction experiment.

3 U/ml of the thus prepared separation and purification solution (supernatant) was used, and 200 mM TDA and 240 mM TzAAMe were respectively added to a 0.1 M ammonium phosphate (pH 7.5) buffer solution to prepare a substrate ratio of 1:1.2 and a total of 50 mL. It was reacted with the substrate solution. While performing the cefazolin synthesis reaction at 10° C. for 2 hours, the TDA conversion rate, the residual amount of TzAAMe, and the amount of TzAA produced were analyzed by HPLC in the same manner as in Example 4-2.

As a result, as shown in FIG. 3, it was confirmed that the ZSH3-1 mutant enzyme of the present invention showed a high TDA conversion rate over time, and that TzAA was hardly produced to a degree similar to the initial stage of the cefazoline synthesis reaction. As a result, it was confirmed that the ZSH3-1 mutant enzyme of the present invention not only synthesizes Cefazolin with high efficiency, but also significantly lowers the decomposition rate of TzAAMe to TzAA and the decomposition rate of cefazoline, so that the productivity of Cefazolin is excellent.

Achromobacter sp. Penicillin G acylase variants derived from CCM 4824 have very high synthesizing activity specifically for cefazolin, compared to their wild-type enzymes and other known mutant forms, while also degrading activity against substrates used for the synthesis of cefazoline. Compared to the synthesis ratio (S/H ratio) is remarkably high, the productivity of cefazoline has been remarkably increased to a significant level for industrial application, so industrial applicability is high.

As described above, the present invention relates to a penicillin G acylase variant and its use with increased cefazoline productivity, and more particularly, to an alpha subunit represented by SEQ ID NO: 1; And in the wild-type penicillin G acylase protein (from Achromobacter sp. CCM 4824) sequence comprising the beta subunit represented by SEQ ID NO: 2, A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA, T388βA, R478βH, It relates to a mutant penicillin G acylase, characterized in that it comprises one or more mutations selected from the group consisting of E486βA, D541βE, and Y555βC, and a method for synthesizing (manufacturing) cefazoline using the same.

[Accession number]

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13991BP

Consignment Date: 20191011

Depository address: (56212) Korea Research Institute of Bioscience and Biotechnology (KRIBB) 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea

Claims (17)

1. An alpha subunit represented by SEQ ID NO: 1; And a wild-type penicillin G acylase protein sequence comprising a beta subunit represented by SEQ ID NO: 2, the group consisting of A116αT, I156αN, N169αT, M3βV, S54βG, T64βA, H173βN, T251βA, T388βA, R478βH, E486βA, D541βE and Y555βC Variant penicillin G acylase, characterized in that it comprises one or more mutations selected from.
2. The mutant penicillin G acylase according to claim 1, comprising mutations of I156αN, N169αT, H173βN, T251βA, E486βA and Y555βC in the wild-type penicillin G acylase protein sequence.
3. The method of claim 2, wherein the mutant penicillin G acylase

   An alpha subunit represented by SEQ ID NO: 5; And

   A mutant penicillin G acylase comprising the beta subunit represented by SEQ ID NO: 6.
4. The mutant penicillin G acylase of claim 2, wherein the mutant penicillin G acylase further comprises one or more mutations selected from the group consisting of G49αF, T201αS, M3βV, T64βA, T225βN and T388βA.
5. The mutant penicillin G acylase according to claim 4, wherein the mutant penicillin G acylase comprises mutations of M3βV, T64βA and T388βA.
6. The method of claim 5, wherein the mutant penicillin G acylase

   An alpha subunit represented by SEQ ID NO: 5; And

   A mutant penicillin G acylase comprising the beta subunit represented by SEQ ID NO: 8.
7. The mutant penicillin G acylase according to claim 4, wherein the mutant penicillin G acylase further comprises one or more mutations selected from the group consisting of S54βG, S150βT, N226βT, A310βD, R478βH and D541βE.
8. 8. The mutant penicillin G acylase of claim 7, wherein the mutant penicillin G acylase comprises mutations of S54βG, R478βH and D541βE.
9. The method of claim 8, wherein the mutant penicillin G acylase

   An alpha subunit represented by SEQ ID NO: 5; And

   A mutant penicillin G acylase comprising the beta subunit represented by SEQ ID NO: 10.
10. The polynucleotide encoding the mutant penicillin G acylase of any one of claims 1 to 9.
11. Recombinant expression vector comprising the polynucleotide of claim 10.
12. A host cell transformed by the expression vector of claim 11.
13. The method of claim 12, wherein the host cells are Clostridia spp., Escherichia spp., Acetobacter spp., Aeromonas spp., and alkali. Genus Alcaligenes (Alcaligenes spp.), Aphanocladium spp., Bacillus spp., Cephalosporium spp., Flavobacterium spp., Cluy Vera spp., Mycoplana spp., Protaminobacter spp., Pseudomonas spp., Achromobacter spp., and Xanthomonas spp. spp.). Transformed host cells, characterized in that any one of the genus (genus) microorganisms selected from the group consisting of.
14. The method of claim 13, wherein the microorganism of the genus Escherichia is Escherichia coli MC1061/pBC-ZSH3-1 transformed by a recombinant expression vector comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 10. (Escherichia coli MC1061/pBC-ZSH3-1, accession number KCTC 13991BP) transformed host cell, characterized in that.
15. A composition for preparing cefazolin comprising the mutant penicillin G acylase of any one of claims 1 to 9.
16. In the method for enzymatically synthesizing cefazoline from tetrazolylacetic acid ester and 3-[5-methyl-1,3,4-thiodiazol-2-yl]-7-aminocephalosporic acid, the method comprising: A method for enzymatic synthesis of cefazoline, characterized in that the mutant penicillin G acylase of any one of claims 9 to 9 is used.
17. The use of the modified penicillin G acylase of any one of claims 1 to 9 for the production of cefazolin.

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