CN115397979A - Penicillin G acylase mutants with increased cefazolin productivity and uses thereof - Google Patents

Penicillin G acylase mutants with increased cefazolin productivity and uses thereof Download PDF

Info

Publication number
CN115397979A
CN115397979A CN202080093452.2A CN202080093452A CN115397979A CN 115397979 A CN115397979 A CN 115397979A CN 202080093452 A CN202080093452 A CN 202080093452A CN 115397979 A CN115397979 A CN 115397979A
Authority
CN
China
Prior art keywords
ala
gly
arg
asp
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202080093452.2A
Other languages
Chinese (zh)
Inventor
慎镛哲
朴哲
王垠善
尹荣晟
金娜俐
姜美淑
李红仙
林秀禛
尹智勋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amicogen Inc
Original Assignee
Amicogen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amicogen Inc filed Critical Amicogen Inc
Publication of CN115397979A publication Critical patent/CN115397979A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/04—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by acylation of the substituent in the 7 position
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01011—Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to penicillin G acylase mutants with increased productivity of cefazolin and their use, wherein the achromobacter sp CCM 4824-derived penicillin G acylase mutants with the unique mutated forms disclosed in the present invention have a very high synthetic activity specifically towards cefazolin compared to their wild-type enzymes and other known mutant forms and surprisingly high synthesis ratio (S/H ratio) compared to the degradation activity of the substrate for the synthesis of cefazolin and thus the productivity of cefazolin is surprisingly increased to a significant level in industrial applications.

Description

Penicillin G acylase mutants with increased cefazolin productivity and uses thereof
Technical Field
This application claims priority from korean patent application No. 10-2019-0147086, filed on 15/11/2019, and the entire specification of which is incorporated herein by reference in its entirety.
The present invention relates to penicillin G acylase mutants having increased cefazolin productivity and uses thereof, and more particularly, to mutant penicillin G acylase comprising at least one mutation selected from a116 α T, I156 α N, N169 α T, M3 β V, S54 β G, T64 β a, H173 β N, T251 β a, T388 β a, R478 β H, E486 β a, D541 β E and Y555 β C in the sequence of a wild-type penicillin G acylase protein (derived from Achromobacter (Achromobacter) species CCM 4824) comprising an α subunit defined by SEQ ID NO: 1; and the beta subunit defined by SEQ ID NO 2.
Background
In a method for synthesizing a semisynthetic β -lactam compound, a compound having a β -lactam core structure and a compound (acyl donor) bonded thereto to become a side chain are used as substrates. When penicillin G acylase is added to the substrate mixture, an enzymatic coupling reaction takes place between the activated side chain and the β -lactam nucleus, and penicillin G acylase catalyzes the transfer (acylation) of an acyl group from the activated side chain (i.e. acyl donor) to the β -lactam nucleus. On the other hand, the enzyme also has an activated side chain serving as an acyl donor and an activity to hydrolyze a synthesized β -lactam compound, which poses a problem in productivity of a semi-synthesized β -lactam compound. In particular, considering that the synthesis reaction with a substrate is industrially and economically performed at a ratio of 1.
In particular, in the conventional enzymatic synthesis of cefazolin, ester compounds in which groups such as methyl, ethyl, propyl and TzAA are bonded have been used as reaction materials. In the case of the synthesis of cefazolin from an ester compound of TzAA and MMTD-7-ACA according to conventional methods and means, the synthesis proceeds by the enzymatic activity of penicillin G amidase (penicillin G acylase) and at the same time the TzAA of the ester compound is naturally hydrolyzed, i.e. penicillin G amidase (penicillin G acylase) used as biocatalyst undergoes hydrolysis on the one hand to competitively take place the decomposition reaction of tetrazole-1-acetic acid and the corresponding alcohol. A specific example of this reaction is shown in figure 2. As shown in fig. 2, for example, tetrazolylacetic acid methyl ester (abbreviated as "TzAAMe" in the present specification) and 7-amino-3- (2-methyl-1, 3, 4-thiadiazol-5-yl) -thiomethyl-3-cephem-4-carboxylic acid (abbreviated as "TDA" in the present specification) may be used as substrates for synthesizing (S) cefazolin by an enzymatic reaction. The substrate TzAAMe is sometimes hydrolyzed enzymatically to convert to tetrazolyl acetic acid (abbreviated herein as "TzAA") (H1), and the synthetic cefazolin is also hydrolyzed enzymatically (H2) to produce TzAA. Therefore, in order to increase the productivity of cefazolin, it is important to increase the synthesis reaction (S) and decrease the decomposition reactions (H1, H2).
Therefore, it is possible to reduce the amount of the TzAA ester compound by reducing the hydrolysis rate as much as possible. In order to reduce the hydrolysis rate as described above, it is known that it is desirable to carry out the enzymatic reaction at as low a pH as possible, but this method is limited by the optimum conditions for the cefazolin synthesis reaction and the solubility problem of MMTD-7-ACA. Therefore, until now, when cefazolin was synthesized, the reaction was carried out by adding TzAAMe twice or more at a ratio equivalent to MMTD-7-ACA.
In addition, even if the same enzyme is used, significant differences in synthetic yield may occur depending on the specific type of antibiotic. Antibiotics with a benzyl ring structure, such as cephalexin, cefaclor and cephradine, and those with a phenol ring structure, such as amoxicillin, cefadroxil and cefprozil, are structurally similar, but in the case of cefazolin, they have a tetrazole ring and are structurally different from the antibiotics mentioned above. Therefore, even if the same enzyme is used, a case occurs in which the efficiency of production of cefazolin is relatively low. For example, in the case of the enzyme disclosed in the document KR101985911 (patent document 1), it is known to have excellent productivity by having highly efficient multiple-synthesis ability for semisynthetic β -lactam antibiotics of cephalexin, cefprozil, cefaclor, cephradine, cefadroxil and amoxicillin, but for cefazolin, it cannot significantly increase the production efficiency to the extent that it has utility at an industrial level. Enzymes with high cefazolin production rates high enough for industrial-level applications are not yet known.
[ Prior art documents ]
[ patent document ]
(patent reference 1) KR101985911B
Disclosure of Invention
Technical problem
Accordingly, when the present inventors are studying a method for increasing cefazolin productivity, the present invention has been completed by confirming that: the penicillin G acylase mutant having the unique mutation disclosed in the present invention not only significantly increases the ability to specifically synthesize cefazolin, but also improves the S/H ratio to a significantly higher level, compared to the wild type (derived from achromobacter sp CCM4824 strain), and thus it has high productivity with respect to cefazolin.
Accordingly, it is an object of the present invention to provide a mutant penicillin G acylase comprising at least one mutation selected from the group consisting of A116. Alpha.T, I156. Alpha.N, N169. Alpha.T, M3. Beta.V, S54. Beta.G, T64. Beta.A, H173. Beta.N, T251. Beta.A, T388. Beta.A, R478. Beta.H, E486. Beta.A, D541. Beta.E and Y555. Beta.C in the wild type penicillin G acylase protein sequence comprising an alpha subunit defined by SEQ ID NO. 1 and a beta subunit defined by SEQ ID NO. 2.
It is another object of the present invention to provide a polynucleotide encoding a mutant penicillin G acylase.
Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide.
It is another object of the present invention to provide a host cell transformed with the expression vector.
It is another object of the present invention to provide a composition for the preparation of cefazolin comprising the mutant penicillin G acylase of the present invention.
In addition, a composition for the preparation of cefazolin is provided, which consists of the mutant penicillin G acylase of the invention.
In addition, a composition for the preparation of cefazolin is provided, consisting essentially of the mutant penicillin G acylase of the invention.
Another object of the invention is a process for the enzymatic synthesis of cefazolin from tetrazolyl acetate and 3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid, which comprises using a mutant penicillin G acylase of the invention.
It is another object of the present invention to provide the use of the mutant penicillin G acylase of the present invention for the preparation of cefazolin.
Technical solution
In order to achieve the above object, the present invention provides a mutant penicillin G acylase comprising at least one mutation selected from the group consisting of A116. Alpha.T, I156. Alpha.N, N169. Alpha.T, M3. Beta.V, S54. Beta.G, T64. Beta.A, H173. Beta.N, T251. Beta.A, T388. Beta.A, R478. Beta.H, E486. Beta.A, D541. Beta.E and Y555. Beta.C in a wild type penicillin G acylase protein sequence comprising an alpha subunit defined by SEQ ID NO. 1 and a beta subunit defined by SEQ ID NO. 2.
To achieve another object of the present invention, the present invention provides a polynucleotide encoding a 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.
To achieve another object of the present invention, the present invention provides a composition for preparing cefazolin, which comprises the mutant penicillin G acylase of the present invention.
In addition, the present invention provides a composition for the preparation of cefazolin, which consists of the mutant penicillin G acylase of the present invention.
In addition, the present invention provides a composition for the preparation of cefazolin consisting essentially of the mutant penicillin G acylase of the invention.
To achieve another object of the present invention, the present invention provides a method for enzymatically synthesizing (preparing) cefazolin from tetrazolyl acetate and 3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid, which comprises using the mutant penicillin G acylase of the present invention.
To achieve another object of the invention, the invention provides the use of the mutant penicillin G acylase of the invention for the preparation of cefazolin.
Hereinafter, the present invention will be described in detail.
As used herein, the term "protein" is used interchangeably with "polypeptide" or "peptide", for example, to refer to a polymer of amino acid residues as commonly found in proteins in their native state.
As used herein, "nucleic acid" or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides in either single-or double-stranded form. Unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
As used herein, the term "expression" refers to the production of a protein or nucleic acid in a cell.
As used herein, according to standard abbreviation rules in the field of biochemistry, one letter (three letters) of an amino acid refers to the following amino acid: a (Ala): alanine; c (Cys): (ii) cysteine; d (Asp): aspartic acid; e (Glu): glutamic acid; f (Phe): phenylalanine; g (Gly): glycine; h (His): (ii) histidine; i (IIe): isoleucine; k (Lys): lysine; l (Leu): (ii) leucine; m (Met): methionine; n (Asn): asparagine; p (Pro): (ii) proline; q (Gln): (ii) glutamine; r (Arg): arginine; s (Ser): serine; t (Thr): threonine; v (Val): valine; w (Trp): tryptophan; y (Tyr): tyrosine.
As used herein, "(amino acid one letter) (amino acid position) (amino acid one letter)" means that the amino acid labeled before at the corresponding amino acid position in the native (wild-type) polypeptide is replaced with the amino acid labeled after. For example, I156. Alpha.N indicates that the isoleucine corresponding to position 156 of the native polypeptide alpha subunit (in the present invention, the wild-type APA. Alpha. Subunit of SEQ ID NO: 1) is replaced with asparagine and M3. Beta.V means that the third methionine of the native polypeptide beta subunit (in the present invention, the wild-type APA. Beta. Subunit of SEQ ID NO: 2) is replaced with valine.
As used herein, the term MMTD-7-ACA refers to the reaction product of MMTD (mercapto-5-methyl-1, 3, 4-thiadiazole) and 7-ACA (7-aminocephalosporanic acid), and it is also denoted as "3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid" and the like. It is used as a substrate in a reaction for synthesizing a cefazolin antibiotic and is a compound having a beta-lactam core structure. The reaction products of MMTD and 7-ACA are known in the art in various representations, but in the present invention it is intended to include all materials which are considered in the art to be substantially equivalent. Although not limited thereto, TDA (7-amino-3- (2-methyl-1, 3, 4-thiadiazol-5-yl) -thiomethyl-3-cephem-4-carboxylic acid) may be preferably used as an example in the present invention.
As used herein, the term "TzAA ester" compound refers to "tetrazolyl acetate", and refers to an ester compound in which a group such as methyl, ethyl or propyl is bonded to TzAA, which may also be referred to as "tetrazole-1-acetate" and the like in this specification. It acts as a substrate and as an acyl donor in the cefazolin synthesis reaction. 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.
The present invention provides achromobacter species CCM 4824-derived penicillin G acylase mutants with increased productivity for cefazolin.
As used herein, the achromobacter species derived from CCM4824 penicillin G acylase protein is referred to as "APA" and the polynucleotide or gene sequence encoding it is denoted "APA".
The Achromobacter species CCM 4824-derived penicillin G acylase (APA) wild type comprises the alpha subunit defined by SEQ ID NO 1 and the beta subunit defined by SEQ ID NO 2. Specifically, wild-type APA is expressed as a precursor type of single-chain polypeptide (e.g., SEQ ID NO: 4) consisting of an alpha subunit defined by SEQ ID NO:1 and a beta subunit defined by SEQ ID NO:2 linked by a spacer peptide (e.g., SEQ ID NO: 3), which is thereafter removed by autocatalytic processing in the cell to form a mature active dimer consisting of the alpha and beta subunits. For example, the precursor wild-type APA defined by SEQ ID NO. 4 may be encoded by a polynucleotide such as the nucleotide sequence designated NCBI genbank AY919310.1, but is not limited thereto.
Penicillin G acylases derived from the achromobacter species CCM4824 strain (APA wild-type) have been reported to have superior advantages over isozymes derived from other known organisms. For example, it is reported that thermal stability is excellent compared to an enzyme derived from Achromobacter xylosoxidans (Achromobacter xylosoxidans), and catalytic activity for synthesis of β -lactam antibiotics is superior to that of an enzyme derived from Escherichia coli (E.coli) at a low substrate concentration (Stanislav Becka et al, penicillin G acyl ase from Achromobacter sp.CCM 4824, apple Microbiol Biotechnol DOI 10.1007/s 00253-013-4945-3). However, penicillin G acylase (APA) wild-type protein derived from achromobacter sp CCM4824 strain also has a problem that significant levels of substrate and product degradation activity occur compared to the synthetic activity as described in the examples of the present specification. On the other hand, attempts have been made to prepare mutants of penicillin G acylase derived from various microorganisms and to use them industrially, but these have limitations that only the conversion rate is reported without considering the decomposition reaction and S/H, or that the development is focused only on each specific reaction, for example, only the synthesis reaction. In practice, they still have insufficient effects for application at the industrial level, for example, although some of the amino acid mutants disclosed in the patent CN105483105A disclose that the substrate turnover rate of a small amount of antibiotic such as amoxicillin is substantially 99% after 90 to 120 minutes, the synthesizing ability of the mutant is improved only about two-fold compared with the wild type, and the S/H value of such synthesizing ability is only about 3.9-fold (based on amoxicillin). This degree of improvement has a limitation in process efficiency when applied to a practical industrial unit. That is, in improving the enzyme, in order to determine the comparative advantages thereof, it is necessary to evaluate not only the increase in the synthetic activity but also the decrease in the decomposition activity and the increase in the S/H value.
In contrast, as a representative example of the mutant of the present invention, the ZSH1-13 mutant not only significantly increased the cefazolin synthesis activity by 12.5 times as compared with the wild type, but also showed 6.1 times S/H ratio as compared with the wild type in addition to this synthesis activity. In addition, as another example, in the ZSH2-5 mutant (developed based on the ZSH1-13 mutant), the synthetic activity of cefazolin was significantly increased by 43 times compared to the wild type, and in addition to this synthetic activity, a S/H ratio of 19.8 times compared to the wild type was also shown. In particular, among the various mutants generated through innumerable steps, in the case of the ZSH3-1 mutant finally selected (developed based on the ZSH2-5 variant), the synthetic activity of cefazolin was significantly increased 65-fold compared to the wild type, and in addition to this synthetic activity, a 36.7-fold S/H ratio compared to the wild type was also shown. Accordingly, cefazolin can be produced with high efficiency according to the mutated APA provided in the present invention. This is a good improvement that can be used in the production process and it means a significant technical advance compared to the prior art.
Thus, achromobacter species CCM 4824-derived penicillin G acylase mutants having the unique mutant (especially substituted) forms disclosed in the present invention are characterized by having a high efficiency of synthesis with respect to cefazolin. In the present invention, the term "high efficiency" means that the production and speed of the synthesized product are increased and the S/H ratio is high.
Accordingly, the mutant forms of APA disclosed for the first time in the present invention are as follows. The mutant penicillin G acylase of the invention (hereinafter referred to as mutant APA in the present specification) is characterized in that it comprises a mutation wherein one or more amino acid residues selected from the group consisting of A116. Alpha.T, I156. Alpha.N, N169. Alpha.T, M3. Beta.V, S54. Beta.G, T64. Beta.A, H173. Beta.N, T251. Beta.A, T388. Beta.A, R478. Beta.H, E486. Beta.A, D541. Beta.E and Y555. Beta.C are substituted with other amino acids in the wild-type penicillin G acylase protein sequence comprising an. Alpha.subunit defined by SEQ ID NO. 1 and a. Beta.subunit defined by SEQ ID NO. 2.
In particular, the mutant penicillin G acylase comprises at least one mutation selected from the group consisting of A116. Alpha.T, I156. Alpha.N, N169. Alpha.T, M3. Beta.V, S54. Beta.G, T64. Beta.A, H173. Beta.N, T251. Beta.A, T388. Beta.A, R478. Beta.H, E486. Beta.A, D541. Beta.E and Y555. Beta.C in the wild type penicillin G acylase protein sequence comprising the. Alpha.subunit defined by SEQ ID No. 1 and the. Beta.subunit defined by SEQ ID No. 2.
For example, it may be a polypeptide having one mutation site in the wild-type penicillin G acylase protein sequence comprising the alpha subunit defined by SEQ ID NO. 1 and the beta subunit defined by SEQ ID NO. 2, such as a polypeptide comprising an A116 alpha T substitution, a polypeptide comprising an I156 alpha N substitution, a polypeptide comprising an N169 alpha T substitution, a polypeptide comprising an H173 beta N substitution, a polypeptide comprising a T251 beta A substitution, a polypeptide comprising an E486 beta A substitution or a polypeptide comprising a Y555 beta C substitution, or
For example, it may be a polypeptide having multiple mutation sites in the wild type penicillin G acylase protein sequence comprising the alpha subunit defined by SEQ ID NO. 1 and the beta subunit defined by SEQ ID NO. 2, such as, but not limited to, a polypeptide comprising 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 mutation sites, optionally selected from A116 alpha T, I156 alpha N, N169 alpha T, M3 beta V, S54 beta G, T64 beta A, H173 beta N, T251 beta A, T388 beta A, R478 beta H, E486 beta A, D541 beta E and Y555 beta C.
Preferably, the mutant penicillin G acylase (mutant APA) of the present invention may comprise 6 mutation sites of I156. Alpha.N, N169. Alpha.T, H173. Beta.N, T251. Beta.A, E486. Beta.A and Y555. Beta.C in the wild-type penicillin G acylase protein sequence (see ZSH 1-13). More preferably, the mutant penicillin G acylase of the present invention may comprise an alpha subunit defined by SEQ ID NO. 5 and a beta subunit defined by SEQ ID NO. 6. As a preferred providing form thereof, i) it may be provided as a precursor protein in the form of a single-chain polypeptide (e.g., SEQ ID NO: 7) consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:6 linked by a spacer peptide (e.g., SEQ ID NO: 3), or ii) it may be provided as a mature protein consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:6, but is not limited thereto.
In addition, the invention may include modification of additional amino acid residues in the mutated APA. The term "modification" refers to deletion, substitution or addition of amino acid residues. Modification of additional amino acid residues is performed for the purpose of increasing the cefazolin synthesis activity of the mutant APA of the present invention and decreasing the degradation activity of the substrate for synthesis, and the modification form (type) and position of the amino acid residue are not particularly limited as long as the above-mentioned purpose can be achieved.
Preferably, one or more mutations selected from G49 α F, T201 α S, M3 β V, T64 β a, T225 β N and T388 β a may be further included in the mutant APA sequence comprising 6 mutation sites of I156 α N, N169 α T, H173 β N, T251 β a, E486 β a and Y555 β C compared to the wild type. More preferably, one or more mutations selected from the group consisting of G49. Alpha.F, T201. Alpha.S, M3. Beta.V, T64. Beta.A, T225. Beta.N and T388. Beta.A may be further included in the mutant APA sequence comprising the alpha subunit defined by SEQ ID NO. 5 and the beta subunit defined by SEQ ID NO. 6.
For example, in a mutant APA sequence comprising an alpha subunit defined by SEQ ID No. 5 and a beta subunit defined by SEQ ID No. 6, a mutation site may additionally be included, such as a polypeptide comprising: a polypeptide comprising a G49 α F substitution, a polypeptide comprising a T201 α S substitution, a polypeptide comprising an M3 β V substitution, a polypeptide comprising a T64 β a substitution, a polypeptide comprising a T225 β N substitution or a T388 β a substitution, or
For example, it may be a polypeptide additionally including multiple mutation sites in a mutant APA sequence comprising an alpha subunit defined by SEQ ID No. 5 and a beta subunit defined by SEQ ID No. 6, such as a polypeptide comprising 2 mutations, 3 mutations, 4 mutations, 5 mutations, or 6 mutations, optionally selected from G49 alphaf, T201 alphas, M3 betav, T64 betaa, T225 betan, and T388 betaa, but not limited thereto.
Most preferably, the mutant APA of the present invention may further comprise three mutation (substitution) sites (including a total of 9 substitution sites compared to the wild type, see ZSH 2-5) of M3 β V, T64 β a and T388 β a in the mutant APA comprising an α subunit defined by SEQ ID No. 5 and a β subunit defined by SEQ ID No. 6. More preferably, the mutant APA of the invention may comprise an alpha subunit defined by SEQ ID NO. 5 and a beta subunit defined by SEQ ID NO. 8. As a preferred providing form thereof, i) it may be provided as a precursor protein in the form of a single-chain polypeptide (e.g., SEQ ID NO: 9) consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:8 linked by a spacer peptide (e.g., SEQ ID NO: 3), or ii) it may be provided as a mature protein consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:8, but not limited thereto.
The invention also provides polypeptides comprising a mutation further added to the mutant APA. Preferably, one or more mutations selected from the group consisting of S54 β G, S150 β T, N226 β T, a310 β D, R478 β H and D541 β E may be further included in the mutant APA sequence comprising 9 mutation sites of I156 α N, N169 α T, M3 β V, T64 β a, H173 β N, T251 β a, T388 β a, E486 β a and Y555 β C compared to the wild type. More preferably, one or more mutations selected from the group consisting of S54 β G, S150 β T, N226 β T, A310 β D, R478 β H and D541 β E may be further included in the mutant APA sequence comprising the α subunit defined by SEQ ID NO. 5 and the β subunit defined by SEQ ID NO. 8.
For example, in a mutant APA sequence comprising an alpha subunit defined by SEQ ID No. 5 and a beta subunit defined by SEQ ID No. 8, a mutation site may additionally be included, such as a polypeptide including: a polypeptide comprising an S54 β G substitution, a polypeptide comprising an S150 β T substitution, a polypeptide comprising an N226 β T substitution, a polypeptide comprising an a310 β D substitution, a polypeptide comprising an R478 β H substitution or a T388 β a substitution, a polypeptide comprising an R478 β H substitution, or
For example, it may be a polypeptide comprising multiple mutation sites in addition to a mutant APA sequence comprising an alpha subunit defined by SEQ ID No. 5 and a beta subunit defined by SEQ ID No. 8, such as a polypeptide comprising 2 mutations, 3 mutations, 4 mutations, 5 mutations, or 6 mutations optionally selected from S54 β G, S150 β T, N226 β T, a310 β D, R478 β H, and D541 β E, but not limited thereto.
Most preferably, the mutant APA of the present invention may further comprise three mutation (substitution) sites (including a total of 12 substitution sites compared to the wild type, see ZSH 3-1) of S54 β G, R478 β H and D541 β E in the mutant APA comprising an α subunit defined by SEQ ID No. 5 and a β subunit defined by SEQ ID No. 8. More preferably, the mutant APA of the invention may comprise an alpha subunit defined by SEQ ID NO. 5 and a beta subunit defined by SEQ ID NO. 10. As a preferred providing form thereof, i) it may be provided as a precursor protein in the form of a single-chain polypeptide (e.g., SEQ ID NO: 11) consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:10 linked by a spacer peptide (e.g., SEQ ID NO: 3), or ii) it may be provided as a mature protein consisting of an alpha subunit defined by SEQ ID NO:5 and a beta subunit defined by SEQ ID NO:10, but is not limited thereto.
Also, the present invention provides polynucleotides encoding a mutant penicillin G acylase (mutant APA).
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 the amino acid sequence is known, techniques for preparing polynucleotides encoding the amino acid sequence based on codon information known in the art are well known in the art. Polynucleotides may be provided as single-stranded or double-stranded nucleic acid molecules, including all DNA, cDNA, and RNA sequences.
The present invention provides recombinant expression vectors comprising the polynucleotides.
As used herein, the term "recombinant expression vector" refers to a vector capable of expressing a target protein or target nucleic acid (RNA) in an appropriate host cell, and refers to a genetic construct comprising the necessary regulatory elements operably linked such that a polynucleotide (gene) insert is expressed.
As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence that encodes a desired protein or RNA to perform a general function. That is, this means that the nucleic acid sequence encoding the protein or RNA (e.g., the polynucleotide sequence encoding the mutant APA of the invention) is linked in such a way that gene expression can occur through the expression control sequences, e.g., the promoter and the nucleic acid sequence encoding the protein or RNA must be operably linked to achieve expression of the encoding nucleic acid sequence. Operable linkage to a recombinant vector can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation using enzymes generally known in the art, and the like.
The recombinant expression vector of the invention is characterized in that it comprises a polynucleotide encoding a mutant APA. The polynucleotide sequence cloned into the vector according to the invention may be operably linked to appropriate expression control sequences, and the operably linked polynucleotide (gene) sequence and expression control sequences may be comprised in an expression vector comprising a selection marker and/or an origin of replication for the selection of a host cell containing the vector. 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 can be prepared in various ways according to the purpose. Expression control sequences refer to DNA sequences that control the expression of an operably linked polynucleotide sequence in a particular host cell. Such regulatory sequences include a promoter for effecting transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, sequences which control termination of transcription and translation, an initiation codon, a stop codon, polyadenylation signals and enhancers, and the like. The promoter of the vector may be constitutive or inducible.
As the signal sequence, a PhoA signal sequence, an OmpA signal sequence, etc. may be used when the host is Escherichia (Escherichia), an α -amylase signal sequence, a subtilisin signal sequence, etc. when the host is Bacillus (Bacillus), an MF α signal sequence, an SUC2 signal sequence, etc. when the host is yeast, an insulin signal sequence, an α -interferon signal sequence, an antibody molecule signal sequence, etc. may be used when the host is an animal cell, but not limited thereto.
The expression vector of the present invention is not particularly limited as long as it is a vector commonly used in the cloning field, and examples include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Specifically, the plasmids include Escherichia coli-derived plasmids (pBR 322, pBR325, pUC118 and pUC119, pET-22b (+)), bacillus subtilis-derived plasmids (pUB 110 and pTP 5), yeast-derived plasmids (YEp 13, YEp24 and YCp 50), and the like, and the viruses may be animal viruses such as retroviruses, adenoviruses or vaccinia viruses, may be insect viruses such as baculovirus, and the like, but are not limited thereto. In one embodiment of the present invention, pBC-KS (+) is used.
The present invention provides host cells and microorganisms transformed with the expression vector.
Transformation comprises transforming a nucleic acid (encoding the invention)The mutant APA of (a) into an organism, cell, tissue or organ, which can be carried out by appropriate known techniques, as known in the art, depending on the host cell chosen. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) 4 ) Precipitate, calcium chloride (CaCl) 2 ) Precipitation, silicon carbide whiskers, sonication, agrobacterium-mediated transformation, precipitation by polyethylene glycol (PEG), dextran sulfate, lipofectamine, heat shock methods, particle gun bombardment, and the like.
A host cell refers to a prokaryotic or eukaryotic cell containing heterologous DNA introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, etc.).
In the present invention, as the host cell, any type of unicellular organism commonly used in the cloning field, for example, prokaryotic microorganisms such as various bacteria (e.g., clostridium (Clostridium), escherichia coli, etc.), lower eukaryotic microorganisms such as yeast, and cells derived from higher eukaryotes including insect cells, plant cells, mammals, etc. can be used as the host cell, but not limited thereto. Since the expression level and modification of a protein are differently shown depending on host cells, those skilled in the art can select and use the most suitable host cell for the intended purpose.
In the present invention, the host cell may be a microorganism of any one genus selected from the group consisting of: clostridium species (e.g., clostridium acetobutylicum (Clostridium acetobutylicum), clostridium beijerinckii (Clostridium beijerinckii), clostridium glycoacetate (Clostridium saccharoperbutylacetonicum) or Clostridium saccharobutanoate (Clostridium saccharochromobuticum), etc.), escherichia species, acetobacter (Acetobacter) species (e.g., acetobacter sphaeroides, acetobacter pasteurianus (Acetobacter pasteurianus), etc.), aeromonas (Aeromonas) species, alcaligenes (Alcaligenes) species, cryptospira (Aphanocandium) species, bacillus species, cephalosporium (Celosporium) species, flavobacterium (Flavobacterium) species, kluyveromyces (Kluyveromyces) species, pseudomonas chromogenes (Xanthomonas) species, such as Xanthomonas species, and the like.
The microorganism of the genus Escherichia may preferably be Escherichia coli (Escherichia coli), and in one embodiment of the present invention, escherichia coli strain MC1061/pBC-ZSH3-1 (Escherichia coli MC1061/pBC-ZSH3-1, accession number KCTC13991 BP) is provided as the microorganism transformed to express the mutant APA. Coli MC1061/pBC-ZSH3-1 expresses a polypeptide comprising or consisting of an alpha subunit defined by SEQ ID NO. 5 and a beta subunit defined by SEQ ID NO. 10. That is, E.coli MC1061/pBC-ZSH3-1 was transformed by a recombinant expression vector comprising a polynucleotide encoding the amino acid sequences of SEQ ID NO. 5 and SEQ ID NO. 10.
The present invention also provides a composition for the preparation of cefazolin (for synthesis) comprising the above-described mutant penicillin G acylase of the invention.
As mentioned above, the mutant APAs of the present invention provide significant advantages in the synthesis of cefazolin. Accordingly, the present invention provides the use of a mutant APA of the invention for cefazolin synthesis.
The mutant APA of the present invention can be prepared by protein production methods well known in the art with reference to the above sequence information, but is not limited thereto, and can be constructed by, for example, genetic engineering methods. For example, nucleic acids encoding mutant APA polypeptides are constructed according to conventional methods. Nucleic acids 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 automated DNA synthesizer (sold by Biosearch or Applied Biosystems). The constructed nucleic acid is inserted into a vector comprising one or more expression control sequences (expression control sequences, e.g., promoters, enhancers, etc.) operably linked thereto to control the expression of the nucleic acid, and the host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are cultured in a medium and under conditions suitable for expression of the nucleic acid to recover from the culture a substantially pure polypeptide expressed by the nucleic acid. Recovery can be carried out using methods known in the art (e.g., chromatography). In addition, it is possible to use the mutant APA of the present invention prepared as described above, either directly or by purification for use in the preparation of the target product. The isolation and purification of mutant APA enzymes can be used as is, by using protein separation methods, by using various chromatographic methods of the APA characteristics previously disclosed, or with slight modifications thereto for experimental purposes. In addition, it is also possible to purify the mutated APA by affinity chromatography using specific binding properties, such as binding force between histidine peptides and nickel column components or Cellulose Binding Domains (CBDs), binding force to cellulose, and the like.
As used herein, the term "substantially pure polypeptide" means that the polypeptide according to the invention is substantially free of any other proteins derived from the host cell. Genetic engineering methods for synthesizing the polypeptides of the invention are known in the art.
In addition, the mutant APAs of the present invention can be readily prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, solution or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.
The mutant APA of the present invention can be used in an immobilized state or in a free state. The mutant APA may be immobilized by a conventional protein immobilization method known in the art, and as the carrier, natural polymers such as cellulose, starch, dextran, and agarose; synthetic polymers such as polyacrylamide, polyacrylate, polymethacrylate, and Eupergit C; or minerals such as silica, bentonite or metals. In addition, it is possible to bind the mutant APAs to these carriers by covalent bonding, ionic bonding, hydrophobic bonding, physisorption, microencapsulation, and the like. In addition, it is possible to immobilize the mutant APA by forming a covalent bond between these carrier-enzyme conjugates by the action of glutaraldehyde, cyanogen bromide, or the like. In addition, as a more preferable method, it is possible to immobilize and use the mutant APA-containing microbial cells as they are without separately purifying the mutant APA. In such whole cell immobilization, it is also possible to puncture cells or apply techniques such as surface expression in order to increase the reactivity of mutant APAs contained in microorganisms.
In the composition for preparing cefazolin of the present invention, the term "comprising a mutant APA" means that the composition comprises at least one selected from the group consisting of a polynucleotide encoding (coding) a mutant APA, an expression vector containing the polynucleotide, and a host cell (microorganism) transformed with the expression vector, and a direct means of including the mutant APA polypeptide itself in the composition from the beginning. Thus, it is ultimately intended to include all indirect ways of inducing the production of mutant APAs.
In particular, the present invention provides a process for the enzymatic synthesis (preparation) of cefazolin from tetrazolyl acetate and 3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid, which comprises using the mutant penicillin G acylase of the present invention.
The mutant APA enzyme of the present invention provided during cefazolin synthesis (preparation) may be provided in the form of a composition comprising the mutant APA polypeptide or a culture of a mutant APA-producing strain, and the mutant APA may be provided in free or immobilized form, although the invention is not limited thereto and will be understood with reference to the foregoing.
If the activity of the mutant APA enzyme of the invention is not lost, the reaction conditions for each substrate are not particularly limited, but may preferably be prepared in an aqueous solution (water or buffer) in the above-mentioned synthetic method, and a preferable pH of the reaction mixture may be selected in the range of pH5 to pH9, a preferable reaction time in the range of 0.1 to 24 hours, and a preferable reaction temperature in the range of 3 to 30 ℃. The above conditions may be appropriately selected and controlled by those skilled in the art according to the amounts of reaction substrates and enzymes used in a reaction, and the desired speed and efficiency of the process.
The concentration and ratio of MMTD-7-ACA (3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid) and TzAA ester (tetrazolacetate) provided as substrates to the reaction mixture during the cefazolin synthesis (preparation) are not particularly limited, but the ratio of MMTD-7-ACA: tzAA ester may be, for example, 1 to 1.5, and preferably, it may be used in a ratio of 1.
The amount of the mutant APA of the present invention added to the reaction mixture is not particularly limited, but may be, for example, in the range of 0.1 to 100U/ml, and preferably, it may be included at a ratio of 2 to 10% (w/w) with respect to the total amount of the substrate compound included in the reaction mixture.
The cefazolin finally produced during the reaction can be isolated and purified from the reaction solution by a conventional compound purification method known in the art (e.g., chromatography, etc.).
The invention also provides the application of the mutant penicillin G acylase in the invention in preparing cefazolin.
In the present invention, the term 'comprising' is used synonymously with 'comprising' or 'characterized in' and does not exclude additional component elements or method steps not mentioned in the composition or method. The term 'consisting of 8230% \8230composition' is intended to exclude additional elements, steps or components not otherwise specified. Within the scope of the compositions or methods, the term 'consisting essentially of 8230 \8230: \8230compositional' is intended to include the recited component elements or steps, as well as component elements or steps that do not substantially affect their essential characteristics.
Advantageous effects
The mutant of penicillin G acylase derived from achromobacter species CCM4824 having the unique mutant form disclosed in the present invention is characterized by a significantly improved productivity specifically for cefazolin. In particular, the mutant APA of the present invention has a very high synthetic activity for cefazolin, in particular, compared to its wild-type enzyme and other known mutant forms, while the synthesis ratio (S/H ratio) is significantly high compared to the degradation activity of the substrate for cefazolin synthesis, and therefore it is characterized by a significant increase in cefazolin productivity to a significant level for industrial applications.
Drawings
FIG. 1 shows the amino acid substitution sites of representative ZSH1-13, ZSH2-5 and ZSH3-1 mutants among the mutants of the present invention.
Fig. 2 shows a reaction (S) of synthesizing cefazolin from TzAAMe and TDA as precursors (substrates), a hydrolysis reaction (H1) of TzAAMe to TzAA in the precursors, and a hydrolysis reaction (H2) of cefazolin to TzAAMe and TDA as decomposition reactions, by enzymatic reactions.
FIG. 3 is data showing cefazolin conversion rate, residual amount of TzAAMe and production amount of TzAA with time in cefazolin synthesis reaction by ZSH3-1 mutant.
Detailed Description
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 Achromobacter sp CCM 4824-derived penicillin G acylase (APA) wild type
1-1 Synthesis of Gene expressing wild type penicillin G acylase (APA) derived from Achromobacter species CCM4824
Wild-type APA (achromobacter species CCM 4824-derived penicillin G acylase) is a precursor type and is expressed as a single-chain polypeptide (SEQ ID NO: 4) in which the alpha subunit defined by SEQ ID NO:1 and the beta subunit defined by SEQ ID NO:2 are linked by a spacer peptide defined by SEQ ID NO:3, and thereafter through autocatalytic processing in the cell it has a mature active dimeric form consisting of the alpha and beta subunits. The gene expressing the wild-type APA precursor of SEQ ID NO. 4 (abbreviated as "APA gene") was synthesized by requesting Bioneer (Daejeon, korea) Co.
1-2 preparation of recombinant vector (pBC-APA) expressing wild-type APA
The APA gene obtained in example 1-1 was inserted into XbaI and NotI restriction enzyme recognition sites of pBC-KS (+) vector (Stratagene, USA) to prepare pBC-APA plasmid for expressing wild-type APA. The preparation method is as follows.
The apa gene DNA product (about 2.6kb 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 an insert DNA. In addition, pBC KS (+) vector (Stratagene, USA) DNA digested with restriction enzymes XbaI and NotI and a DNA fragment dephosphorylated with CIP were used as vector DNA. After the insert DNA and the vector DNA have been ligated using T4 DNA ligase (New England Biolabs, sweden) at 16 ℃ for 12 to 16 hours, the E.coli MC1061 strain is transformed by electroporation using a solution of the conjugate. Transformants were selected by smearing the strain on LB agar medium containing chloramphenicol antibiotic at a concentration of 20. Mu.g/mL, and culturing overnight at 30 ℃. A pBC-APA plasmid containing the APA gene was finally obtained by isolating the plasmid from the transformant and determining the base sequence of the inserted DNA. The pBC-APA plasmid expresses the wild-type APA protein.
Numerous procedures such as examples 2 and 3 below, representing numerous examples of which, were carried out in order to prepare mutants exhibiting a high level of synthesis capacity with respect to cefazolin and at the same time having a significantly reduced activity of hydrolyzing the reaction substrates. The mutants prepared below are indicated in the form of specific mutations with respect to the respective amino acid positions of the alpha and beta subunits.
Example 2: preparation and selection of variants with high cefazolin synthesis activity
2-1 preparation of Primary mutant libraries by error-prone polymerase chain reaction (error-prone PCR)
In order to prepare mutant APAs having increased cefazolin synthesis activity, random mutations were artificially induced in the nucleotide sequence of the APA gene synthesized in example 1, for which a mutant library was prepared by performing error-prone polymerase chain reaction (error-prone PCR). The specific mutant library was prepared as follows.
Specifically, the Diversity PCR Random Mutagenesis kit (Clontech, USA) was used to perform the misinduced polymerase chain reaction to cause 1-2 mutations/1,000bp. The PCR reaction solution was composed using 1 ng/. Mu.L of pBC-APA plasmid as a template DNA, 10pmol each of T3 primer (SEQ ID NO: 12) and T7 primer (SEQ ID NO: 13), 2mM dGTP,50X Diversity dNTP mix, 10 XTANIUM Taq buffer and TITANIUM Taq polymerase, and used in a final volume of 50. Mu.L. The PCR reaction conditions were pre-denaturing the reaction mixture at 96 ℃ for 2 minutes, repeating the reaction of denaturation at 96 ℃ for 30 seconds, annealing at 52 ℃ for 30 seconds, and polymerization at 68 ℃ for 3 minutes 18 times, followed by post-polymerization at 68 ℃ for 5 minutes. By performing the error-induced polymerase chain reaction under the above conditions, a mutant DNA fragment of about 2.7kb in size was amplified.
After the mutant gene obtained by PCR, i.e., the 2.7kb PCR product, was digested with restriction enzymes XbaI and NotI, purified using a purification Kit (QIAquick Gel Extraction Kit; QIAGEN, germany), and used as an insert DNA, pBC-KS (+) vector DNA was digested with restriction enzymes XbaI and NotI, and a DNA fragment dephosphorylated with CIP was used as a vector DNA.
After conjugating the above insert DNA with vector DNA using T4 DNA ligase (New England Biolabs, sweden) at 16 ℃ for 16 hours, transformation of E.coli MC1061 strain by electroporation was performed using the conjugate solution. The random mutant library was prepared by smearing the strain on LB agar medium containing chloramphenicol antibiotic at a concentration of 20. Mu.g/mL, and culturing overnight at 30 ℃.
From the library of mutants, mutant APAs with increased reactivity towards cefazolin synthesis were searched for according to the following procedure. The reaction substrates and the specific chemical reaction processes for the synthesis of cefazolin are shown in fig. 2. After inoculating the E.coli MC1061 transformant containing the mutated apa gene into a 96-well plate in which 160. Mu.l of LB broth containing a chloramphenicol antibiotic was dispensed, it was then incubated at 23 ℃ and 165rpm for 60-70 hours with shaking. After that, 20. Mu.l of the culture broth was taken out from the 96-well plate and transferred to a new 96-well plate, and 105. Mu.l of cell lysate (1.25 mg/mL lysozyme, 1.25mM EDTA, 0.375% Triton X-100) was added thereto, left at 25 ℃ for 2 hours, and left at 10 ℃ for 1 hour. After that, 125. Mu.L of a substrate solution prepared by dissolving 10mM TDA and 10mM TzAAMe (which is 1. After the synthesis reaction was performed, 40. Mu.L of the supernatant in the reaction solution was transferred to a new 96-well plate. After terminating the enzymatic reaction by adding 100. Mu.L of 0.2N HCl as a reaction terminator, the mutants having increased cefazolin synthesis activity were searched for using HPLC. Regarding HPLC analysis conditions, 0.01M potassium phosphate (pH 6.8) and methanol (7.
It was confirmed that the cefazolin synthesis activity in the 7 mutants was increased in the same manner as described above. By sequencing the genes, it was confirmed that the 7 mutants were mutated to A116. Alpha.T (ZEP 1-9), I156. Alpha.N (ZEP 1-10), H173. Beta.N (ZEP 1-12), Y555. Beta.C (ZEP 1-13), E486. Beta.A (ZEP 1-18), N169. Alpha.T (ZEP 1-26) or T251. Beta.A (ZEP 1-33), respectively, as compared with the wild type.
2-2. Secondary mutant library _ ZSH1-13 prepared by site-directed mutagenesis (DNA shuffling) improves strain selection
In addition, in order to improve the activity of the mutant penicillin G acylase having increased cefazolin synthesis activity, a site-directed mutagenesis library of 7 amino acid residues (A116. Alpha.T, I156. Alpha.N, N169. Alpha.T, H173. Beta.N, T251. Beta.A, E486. Beta.A, Y555. Beta.C) was prepared based on the activity of the improved strain obtained by error-prone polymerase chain reaction.
Specifically, for the preparation of a library in which the A116. Alpha. Amino acid was 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 recover a PCR product having a size of about 510bp, for the preparation of a library in which the A116. Alpha., I156. Alpha., and N169. Alpha. Amino acids were mutated, wild-type APA and ZEP1-10 were used as templates and SH116a-F primer (SEQ ID NO: 16) and SH169a-R primer (SEQ ID NO: 19) were used to recover a PCR product having a size of about 160bp, and for the preparation of a library in which the N. Alpha., H173. Beta., and T251. Beta. Amino acids were mutated, PCR was performed using wild-type APA and ZEP1-12 as templates and SH169a-F primer (SEQ ID NO: 18) and SH251b-R primer (SEQ ID NO: 21) to recover a PCR product having a size of about 1,100bp. In addition, PCR was performed using wild-type APA and ZEP1-18 as templates to mutate T251. Beta., E486. Beta., and Y555. Beta. Amino acids, SH251b-F primer (SEQ ID NO: 20) and SH555b-R primer (SEQ ID NO: 23) to recover a PCR product having a size of about 910bp, wild-type APA as a template to mutate Y555. Beta. Amino acids, and SH555b-F primer (SEQ ID NO: 22) and M13F primer (SEQ ID NO: 14) to recover a PCR product having a size of about 100 bp.
The composition of the PCR reaction solution was carried out by adjusting the final volume to 100. Mu.L with each template DNA, primers, pfu-x buffer, dNTP mix and pfu-x polymerase. The PCR reaction conditions were pre-denaturation of the reaction mixture at 96 ℃ for 3 minutes, and the reaction of denaturation at 96 ℃ for 30 seconds, annealing at 52 ℃ for 30 seconds, and polymerization at 68 ℃ for 2 minutes was repeated 18 times, followed by post-polymerization at 68 ℃ for 5 minutes.
The PCR product of about 510bp, the PCR product of 160bp, the PCR product of 1,100bp, the PCR product of 910bp and the PCR product of about 100bp obtained under the above conditions were mixed, and PCR was performed using the T3 primer (SEQ ID NO: 12) and the T7 primer (SEQ ID NO: 13) herein to amplify a multiple mutant DNA fragment of about 2.7kb in size. The PCR product of about 2.7kb thus obtained was inserted into pBC KS (+) vector DNA in the same manner as in example 2-1, and transformation of the E.coli MC1061 strain was performed to prepare a site-directed mutant library.
As a result of searching for mutants having increased reactivity to cefazolin synthesis activity from a site-directed mutant library in the same manner as in example 2-1, 6-fold mutants (I156. Alpha.N, N169. Alpha.T, H173. Beta.N, T251. Beta.A, E486. Beta.A and Y555. Beta.C) having increased cefazolin synthesis activity as compared with wild-type APA were selected and named ZSH1-13.ZSH1-13 expresses alpha subunit of SEQ ID NO. 5 and beta subunit of SEQ ID NO. 6.
2-3 preparation of three-stage mutant libraries by error-prone polymerase chain reaction (error-prone PCR)
To further increase the cefazolin synthesis activity of the ZSH1-13 mutants prepared in example 2-2, an error-causing mutant library was prepared in the same manner as in example 2-1 using the DNA of ZSH1-13 as a template DNA.
After that, in the search for the synthesis ability of cefazolin, after adding 125 μ L of a substrate solution prepared by dissolving 20mM TDA and 20mM TzAAMe in a substrate ratio of 1 in 100mM ammonium phosphate (pH 7.5) buffer solution, the synthesis reaction was performed by lowering the reaction temperature at 6 ℃ for 17 to 18 hours. All the procedures were carried out in the same manner as in example 2-1 except for the substrate concentration and ratio and the substrate reaction temperature.
As a result of searching the prepared error-causing mutant library, it was confirmed that the cefazolin synthesis activity was increased in comparison with ZSH1-13 among six mutants. Through amino acid sequencing of ZSH1-13, it was confirmed that each of the 6 mutants was mutated to T64. Beta. A (ZEP 2-7), G49. Alpha. F (ZEP 2-9), T388. Beta. A (ZEP 2-13), T201. Alpha. S (ZEP 2-17), M3. Beta. V (ZEP 2-21), or T225. Beta. N (ZEP 2-54).
2-4. Quaternary mutant library-ZSH 2-5 prepared by site-directed mutagenesis improves strain selection
To obtain mutants with improved cefazolin synthesis activity based on the activity of the improved strains obtained in examples 2-3, a site-directed mutagenesis library of 6 amino acid residues (G49. Alpha.F, T201. Alpha.S, M3. Beta.V, T64. Beta.A, T225. Beta.N, T388. Beta.A) was prepared using ZSH1-13 plasmid DNA as template DNA. In this example, PCR reaction conditions were performed in the same manner as described in example 2-2 above.
Specifically, to prepare a library in which G49 α and T201 α amino acids were mutated, ZSH1-13 and ZEP2-9 were used as templates, and PCR was performed using M13R primer (SEQ ID NO: 15) and SH201a-R primer (SEQ ID NO: 25) to recover PCR products having a size of about 770bp, to prepare a library in which T201 α, M3 β and T64 β amino acids were mutated, ZSH1-13 and ZEP2-21 were used as templates, and PCR was performed using SH201a-F primer (SEQ ID NO: 24) and SH64b-R primer (SEQ ID NO: 27) to recover PCR products having a size of about 440bp, to prepare a library in which T64 β, T225 β and T388 β amino acids were mutated, ZSH1-13 and ZEP2-54 were used as templates, and PCR was performed using SH64b-F primer (SEQ ID NO: 26) and SH b-R primer (SEQ ID NO: 29) to perform PCR, and PCR was performed using SH 1b-F primer (SEQ ID NO: 970) as templates to recover PCR products having a size of about 600bp, and SH 13 b-F (SEQ ID NO:14 bp) and SH388b-R primer were used to recover PCR products having a size of about 16 bp.
The PCR product of about 770bp obtained under the above conditions and the PCR product of 440bp, the PCR product of 970bp and the PCR product of 600bp were mixed, and PCR was performed using T3 primer (SEQ ID NO: 12) and T7 primer (SEQ ID NO: 13) herein to amplify a multiple mutant DNA fragment of about 2.7kb in size. The PCR product of about 2.7kb thus obtained was inserted into pBC KS (+) vector DNA in the same manner as in example 2-1, and transformation of the E.coli MC1061 strain was performed 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 mutant having a significantly increased cefazolin synthesis activity as compared to ZSH1-13, a strain in which M3 β V, T64 β a and T388 β a were additionally mutated for ZSH1-13 was selected and named ZSH2-5.ZSH2-5 expresses an alpha subunit defined by SEQ ID NO. 5 and a beta subunit defined by SEQ ID NO. 8.
Example 3: preparation and selection of highly efficient cefazolin synthetic mutants with reduced substrate decomposition activity combined with high synthesis capacity
3-1 preparation of 5 th Generation mutant libraries by error-prone polymerase chain reaction (error-prone PCR)
In order to increase the cefazolin synthesis activity of the ZSH2-5 mutants prepared in examples 2-4 and decrease the reaction substrate (generally TzAAMe) decomposition activity, an error-causing mutation library was prepared using ZSH2-5 as a template DNA in the same manner as in example 2-1. Thereafter, a search was conducted for cefazolin synthesis ability in the same manner as in examples 2 to 3. In addition, in the reactivity search for the substrate TzAAMe, after 30 μ L of the enzyme solution was transferred to a 96-well plate, 125 μ L of the substrate solution prepared by dissolving 5mM TDA and 7.5mM TzAAMe at a ratio of 1.5 on this basis was added to 100mM ammonium phosphate (ph 7.5) buffer, the reaction temperature was raised to 10 ℃, and the synthesis reaction was carried out for 18 to 20 hours. All the procedures were carried out in the same manner as in examples 2 to 3 except for the substrate concentration and the substrate reaction temperature.
As a result of exploring the cefazolin synthesis activity and substrate degradation activity of the thus prepared error-causing mutant library, it was confirmed that in the six mutants, the cefazolin synthesis activity was increased and the TzAAMe decomposition activity was decreased as compared with ZSH2-5. Through amino acid sequence analysis of ZSH2-5, it was confirmed that each of the 6 mutants was mutated to S54 β G (ZEP 3-2), S150 β T (ZEP 3-11), R478 β H (ZEP 3-14), N226 β T (ZEP 3-26), A310 β D (ZEP 3-33), or D541 β E (ZEP 3-48).
3-2 Generation of mutant library 6-ZSH 3-1 by site-directed mutagenesis improves Strain selection
Based on the activity of the improved strain obtained in example 3-1, a site-directed mutagenesis library of 6 amino acid residues (S54. Beta.G, S150. Beta.T, N226. Beta.T, A310. Beta.D, R478. Beta.H, D541. Beta.E) was prepared using DNA of ZSH2-5 as a template DNA. In this example, PCR reaction conditions were performed in the same manner as described in example 2-2 above.
Specifically, in order to prepare a library in which the S54. Beta. Amino acid was mutated, PCR was performed using M13R primer (SEQ ID NO: 15) and SH54b-R primer (SEQ ID NO: 31) and ZSH2-5 as templates to recover a PCR product having a size of about 1,180bp, and in order to prepare a library in which the S54. Beta., S150. Beta. And N226. Beta. Amino acids were mutated, PCR was performed using SH54b-F primer (SEQ ID NO: 30) and SH226b-R primer (SEQ ID NO: 33) and ZSH2-5 and ZEP3-11 as templates to recover a PCR product having a size of about 520 bp. In order to prepare a library in which N226. Beta., A310. Beta. And R478. Beta. Amino acids were mutated, PCR was performed using SH226b-F primers (SEQ ID NO: 32) and SH478b-R primers (SEQ ID NO: 35) and ZSH2-5 and ZEP3-33 as templates to recover PCR products having a size of about 760bp, and in order to prepare a library in which R478. Beta. And D541. Beta. Amino acids were mutated, PCR was performed using SH478b-F primers (SEQ ID NO: 34) and M13F primers (SEQ ID NO: 14) and ZSH2-5 and ZEP3-48 as templates to recover PCR products having a size of about 340 bp. The PCR product of 1,180bp in size and the PCR product of 520bp in size, the PCR product of 760bp in size and the PCR product of 340bp in size were mixed, and PCR was performed using a T3 primer (SEQ ID NO: 12) and a T7 primer (SEQ ID NO: 13) to amplify a multiple mutant DNA fragment of about 2.7kb in size. The PCR product of about 2.7kb thus obtained was inserted into pBC KS (+) vector DNA in the same manner as in example 2-1, and transformation of the E.coli MC1061 strain was performed to prepare a site-directed mutant library.
After that, as a result of searching for library activities in the same manner as in example 3-1, as a mutant in which cefazolin synthesis activity was significantly increased and substrate (TzAAMe) decomposition activity was significantly decreased as compared to ZSH2-5, a strain in which S54 β G, R478 β H, and D541 β E were further mutated was selected and designated ZSH3-1.ZSH3-1 expresses the alpha subunit defined by SEQ ID NO. 5 and the beta subunit defined by SEQ ID NO. 10.
3-3. Preservation of strains
Among the prepared mutant enzymes, the E.coli MC1061 strain transformed with the pBC-ZSH3-1 plasmid containing the Gene encoding the ZSH3-1 mutant enzyme exhibiting the optimal activity by 11/10/2019 was named "E.coli MC1061/pBC-ZSH3-1" and deposited in the Korean Institute of Life engineering (Gene Bank of the Korea Research Institute of Bioscience and Biotechnology) (accession No. KCTC13991 BP).
The results of comparative evaluation of cefazolin synthesis efficiency of each of the representative mutants described in examples 2 to 3 are described in the following examples.
Example 4: comparison of efficiency of cefazolin Activity of APA mutants
4-1 preparation of enzyme solution
To compare the degree of activity of the prepared APA mutants, each enzyme solution was prepared as follows. After the gene encoding each of the APA mutants prepared in examples 2 to 3 was inserted into the pBC-KS (+) vector in the same manner as described in example 1-2, each expression vector was transformed into a strain of E.coli MC 1061. After inoculating each of the E.coli transformants into 3mL of LB medium (1% Bacto-peptone, 0.5% yeast extract, 0.5% NaCl) containing 20. Mu.g/mL of chloramphenicol, shaking culture was performed at 28 ℃ and 200rpm for 16 hours. After that, 350. Mu.L of the medium was inoculated into 35mL of fresh LB medium containing 20. Mu.g/mL of chloramphenicol, and shaking culture was performed at 23 ℃ and 190rpm for 48 hours. The medium was centrifuged (4 ℃,8000rpm,10 minutes) to recover the cells, and then washed once with 0.05M ammonium phosphate (pH 7.5) buffer. Each APA mutant enzyme solution was prepared by suspending the cells in 35mL of the same buffer, disrupting it with a sonicator (Vibra Cell VC750, sonic & Materials Inc, USA) at 4 ℃ for 5 minutes, and then centrifuging it at 4 ℃ and 13,500rpm for 20 minutes to obtain a supernatant.
4-2 measurement of Cefazolin Synthesis Activity
To measure cefazolin synthesis activity, a substrate solution was prepared by dissolving TDA and TzAAMe each at a concentration of 10mM in 0.05M ammonium phosphate (pH 7.5) buffer. After 500. Mu.L of each of the APA mutant enzyme solutions of example 4-1 was added to 500. Mu.L of the substrate solution, the enzyme reaction was carried out at 25 ℃ for 10 minutes, and then 500. Mu.L of the reaction solution was taken, and 500. Mu.L of a 0.2N HCl solution was added thereto to stop the reaction. Thereafter, it was filtered under the same conditions as in example 2-1 and analyzed by HPLC, and quantified by comparison with a quantitative curve of a standard material. In this case, 1 unit (unit, U) is defined as an amount of enzyme capable of producing 1. Mu. Mole of cefazolin per minute in the present specification. On the other hand, after measuring the amount of protein in the enzyme solution according to the Bradford method, the non-activity on the substrate was expressed as an activity unit corresponding to 1mg of protein.
[ TABLE 1 ]
Figure BDA0003748684890000181
* Cefazolin synthesis activity/TzAAMe hydrolysis activity
Table 1 comparatively shows the cefazolin synthesis activity between the ZSH1-13 mutants, the ZSH2-5 mutants and the ZSH3-1 mutants and wild-type APA among the mutants of the present invention. As shown in table 1, the mutants of the present invention increased the synthetic yield of cefazolin by successive improvements compared to wild-type APA. ZSH1-13 increased the synthesis yield by about 12.5-fold compared to wild-type, ZSH2-5 by about 43.0-fold compared to wild-type APA, and final mutant ZSH3-1 by about 65.5-fold compared to wild-type APA, indicating significantly higher cefazolin productivity.
4-3 measurement of substrate TzAAMe decomposition Activity and S/H ratio
Similarly, to measure the extent of degradation activity of TzAAMe against the APA mutants, substrate solutions were prepared by dissolving 15mM TzAAMe in 0.1M ammonium phosphate (pH 7.5) buffer. Mu.l of each of the above mutants was added to 100. Mu.l of this substrate solution, and the enzyme reaction was carried out at 25 ℃ for 10 minutes, and then the reaction was terminated by adding 100. Mu.l of 0.2N HCl solution to 100. Mu.l of the reaction solution. Thereafter, the hydrolysis amount of TzAAMe (i.e., tzAA) was calculated and quantified by comparison with a quantitative curve of a standard material through filtration and HPLC analysis in the same manner as in example 4-2. In this case, 1 unit (U) is defined as the amount of enzyme capable of producing 1. Mu. Mole of TzAA per minute. The S/H ratio was measured using the value of TzAAMe decomposition activity derived by the above method and cefazolin synthesis activity obtained in example 4-2.
As shown in table 1 above, it was confirmed that the mutants of the present invention decreased the relative TzAAMe decomposition activity compared to the wild-type APA, and the ratio of the TzAAMe decomposition activity to the cefazolin synthesis activity (S/H ratio) was significantly increased. Specifically, the S/H ratio increased in the order of ZSH1-13, ZSH2-5, and ZSH3-1, and the final mutant ZSH3-1 increased the S/H ratio by approximately 36.7-fold compared to wild-type APA.
4-4 determination of the conversion ratio of cefazolin from TDA and TzAAMe
To measure the turnover of cefazolin over a period of time, the synthesis yield of cefazolin can be inferred by measuring the turnover of the precursor TDA. The 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 LB medium containing chloramphenicol was 1L. The cultured cell suspension was centrifuged at 8,000rpm for 10 minutes at 4 ℃ and the supernatant was removed to obtain a cell pellet. After suspension in 90ml of 0.1M ammonium phosphate buffer, the cells were disrupted for 20 minutes using a sonicator. For the purpose of isolation and purification of the enzyme solution, the disrupted cells were allowed to stand at 10 ℃ for 2 hours, and then centrifuged (4 ℃,10,000rpm,30 minutes) to obtain a supernatant. This solution was used for TDA conversion experiments.
3U/mL of the isolated and purified solution (supernatant) prepared in this way was used, and 200mM TDA and 240mM TzAAMe were added to 0.1M ammonium phosphate (pH 7.5) buffer, respectively, and reacted with a total of 50mL of a substrate solution prepared at a substrate ratio of 1.2. During the cefazolin synthesis reaction at 10 ℃ 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, the ZSH3-1 mutant enzyme of the present invention showed a high TDA conversion rate over a period of time, confirming that TzAA is difficult to be produced to a degree almost similar to the initial stage of the cefazolin synthesis reaction. As a result, the ZSH3-1 mutant enzyme of the present invention not only synthesized cefazolin with high efficiency, but also significantly reduced the degradation rate of TzAAMe to TzAA and the degradation rate of cefazolin, confirming that cefazolin had excellent productivity.
INDUSTRIAL APPLICABILITY
As described above, the present invention relates to penicillin G acylase mutants having increased cefazolin productivity and uses thereof, and more particularly, to mutant penicillin G acylase enzymes comprising at least one mutation selected from a116 α T, I156 α N, N169 α T, M3 β V, S54 β G, T64 β a, H173 β N, T251 β a, T388 β a, R478 β H, E541 β a, D541 β E and Y555 β C in the sequence of a wild-type penicillin G acylase protein (from achromobacter sp. CCM 4824) comprising an α subunit defined by SEQ ID NO: 1; and the beta subunit defined by SEQ ID NO 2.
Compared to its wild-type enzyme and other known mutants, the achromobacter species CCM4824 derived penicillin G acylase mutant having the unique mutant form disclosed in the present invention has a very high synthetic activity for cefazolin specifically and a surprisingly high synthesis ratio (S/H ratio) and a surprisingly increased production rate of cefazolin to a significant level in industrial applications, thus being highly applicable industrially.
[ deposit No. ]
The name of the depository institution: korean institute of Life engineering
The preservation number is as follows: KCTC13991BP
The preservation date is as follows: 20191011
The preservation address is as follows: (56212) Korean institute of Life engineering (KRIBB), 181, ipsin-gil, jeongeup-si, jeollabuk-do, republic of Korea
Receipt of original deposit
So that: inc.
Amicogen.Inc.
14-10，Worasan-ro 950beon-gil，Munsan-euo，Jinju-si，Gyeongsangnam-do
Korea
Figure BDA0003748684890000201
Sequence listing
<110> Aimeike jianzhu corporation
<120> penicillin G acylase mutant having increased cefazolin productivity and use thereof
<130> OP21-0155/PCT/CN
<150> KR 10-2019-0147086
<151> 2019-11-15
<150> PCT/KR 2020/016010
<151> 2020-11-13
<160> 35
<170> KoPatentIn 3.0
<210> 1
<211> 247
<212> PRT
<213> Artificial sequence
<220>
<223> subunit alpha (amino acid sequence) of wild-type penicillin G acylase (APA) protein from Achromobacter sp. CCM4824
<400> 1
Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val Ala
1 5 10 15
Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp Ala
20 25 30
Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe Tyr
35 40 45
Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met
50 55 60
Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala Ser
65 70 75 80
Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu Arg
85 90 95
Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val Leu
100 105 110
Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg Ala
115 120 125
Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe Ala
130 135 140
Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Ile Phe Val Gly Thr
145 150 155 160
Met Ala Asn Arg Phe Ser Asp Ala Asn Ser Glu Ile Asp Asn Leu Ala
165 170 175
Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met Arg
180 185 190
Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr Thr
195 200 205
Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro Asp
210 215 220
Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr Pro
225 230 235 240
Pro Met Leu Glu Arg Val Val
245
<210> 2
<211> 557
<212> PRT
<213> Artificial sequence
<220>
<223> subunit beta (amino acid sequence) of wild-type penicillin G acylase (APA) protein from Achromobacter sp. CCM4824
<400> 2
Ser Asn Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser
1 5 10 15
Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr
20 25 30
Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr
35 40 45
Pro Phe Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Thr
50 55 60
Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu
65 70 75 80
Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp
85 90 95
Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala
100 105 110
Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys
115 120 125
Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu
130 135 140
Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser
145 150 155 160
Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg His Ala Leu Thr
165 170 175
Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His
180 185 190
Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro
195 200 205
Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser
210 215 220
Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp
225 230 235 240
Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Thr Asp Leu Phe Ala Ile
245 250 255
Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys
260 265 270
Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp
275 280 285
Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu
290 295 300
Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg
305 310 315 320
Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser
325 330 335
Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp
340 345 350
Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro
355 360 365
Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln
370 375 380
Ala Pro Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu
385 390 395 400
Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp
405 410 415
Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp
420 425 430
Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala
435 440 445
Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu
450 455 460
Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr
465 470 475 480
Gln Asn Arg Gly Thr Glu Asn Asn Met Thr Val Phe Asp Gly Lys Ser
485 490 495
Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala
500 505 510
Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr
515 520 525
Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg
530 535 540
Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Tyr Pro Arg
545 550 555
<210> 3
<211> 38
<212> PRT
<213> Artificial sequence
<220>
<223> spacer peptide
<400> 3
Arg Asp Pro Ala Thr Arg Gly Val Val Asp Gly Ala Pro Ala Thr Leu
1 5 10 15
Arg Ala Gln Leu Ala Ala Gln Tyr Ala Gln Ser Gly Gln Pro Gly Ile
20 25 30
Ala Gly Phe Pro Thr Thr
35
<210> 4
<211> 843
<212> PRT
<213> Artificial sequence
<220>
<223> wild-type penicillin G acylase (APA) protein (amino acid sequence, precursor, all) from Achromobacter sp. CCM4824
<400> 4
Met Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val
1 5 10 15
Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp
20 25 30
Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe
35 40 45
Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu
50 55 60
Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala
65 70 75 80
Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu
85 90 95
Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val
100 105 110
Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg
115 120 125
Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe
130 135 140
Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Ile Phe Val Gly
145 150 155 160
Thr Met Ala Asn Arg Phe Ser Asp Ala Asn Ser Glu Ile Asp Asn Leu
165 170 175
Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met
180 185 190
Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr
195 200 205
Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro
210 215 220
Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr
225 230 235 240
Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala Thr Arg Gly Val
245 250 255
Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu Ala Ala Gln Tyr
260 265 270
Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro Thr Thr Ser Asn
275 280 285
Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser Ile Leu
290 295 300
Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr Tyr Gly
305 310 315 320
Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr Pro Phe
325 330 335
Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Thr Trp Gly
340 345 350
Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu Lys Leu
355 360 365
Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp Lys Thr
370 375 380
Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala Pro Val
385 390 395 400
Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys Phe Asp
405 410 415
Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu Gly Tyr
420 425 430
Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser Ala Asn
435 440 445
Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg His Ala Leu Thr Ile Asn
450 455 460
Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His Thr Gly
465 470 475 480
Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro Val Pro
485 490 495
Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser Thr Asn
500 505 510
Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp Asn Asn
515 520 525
Gln Pro Met Arg Gly Tyr Pro Ser Thr Asp Leu Phe Ala Ile Val Trp
530 535 540
Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys Ala Met
545 550 555 560
Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp Leu Ile
565 570 575
Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu Pro Phe
580 585 590
Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg Val Arg
595 600 605
Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser Glu Arg
610 615 620
Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp Ala Trp
625 630 635 640
Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro Ala Asp
645 650 655
Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln Ala Pro
660 665 670
Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu Phe Asn
675 680 685
Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp Phe Phe
690 695 700
Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp Asp Ala
705 710 715 720
Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala Trp Lys
725 730 735
Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu Gly Val
740 745 750
Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr Gln Asn
755 760 765
Arg Gly Thr Glu Asn Asn Met Thr Val Phe Asp Gly Lys Ser Val Arg
770 775 780
Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala Pro Asp
785 790 795 800
Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr Asn Thr
805 810 815
Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg Arg Asn
820 825 830
Ala Thr Ser Glu Glu Thr Leu Arg Tyr Pro Arg
835 840
<210> 5
<211> 247
<212> PRT
<213> Artificial sequence
<220>
<223> alpha subunits of mutants ZSH1-13, ZSH2-5 and ZSH3-1
<400> 5
Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val Ala
1 5 10 15
Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp Ala
20 25 30
Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe Tyr
35 40 45
Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu Met
50 55 60
Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala Ser
65 70 75 80
Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu Arg
85 90 95
Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val Leu
100 105 110
Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg Ala
115 120 125
Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe Ala
130 135 140
Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Asn Phe Val Gly Thr
145 150 155 160
Met Ala Asn Arg Phe Ser Asp Ala Thr Ser Glu Ile Asp Asn Leu Ala
165 170 175
Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met Arg
180 185 190
Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr Thr
195 200 205
Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro Asp
210 215 220
Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr Pro
225 230 235 240
Pro Met Leu Glu Arg Val Val
245
<210> 6
<211> 557
<212> PRT
<213> Artificial sequence
<220>
<223> beta subunit of ZSH1-13 mutant
<400> 6
Ser Asn Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser
1 5 10 15
Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr
20 25 30
Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr
35 40 45
Pro Phe Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Thr
50 55 60
Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu
65 70 75 80
Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp
85 90 95
Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala
100 105 110
Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys
115 120 125
Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu
130 135 140
Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser
145 150 155 160
Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr
165 170 175
Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His
180 185 190
Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro
195 200 205
Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser
210 215 220
Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp
225 230 235 240
Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile
245 250 255
Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys
260 265 270
Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp
275 280 285
Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu
290 295 300
Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg
305 310 315 320
Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser
325 330 335
Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp
340 345 350
Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro
355 360 365
Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln
370 375 380
Ala Pro Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu
385 390 395 400
Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp
405 410 415
Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp
420 425 430
Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala
435 440 445
Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu
450 455 460
Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr
465 470 475 480
Gln Asn Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser
485 490 495
Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala
500 505 510
Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr
515 520 525
Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg
530 535 540
Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
545 550 555
<210> 7
<211> 843
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence of ZSH1-13 mutant (precursor, full)
<400> 7
Met Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val
1 5 10 15
Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp
20 25 30
Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe
35 40 45
Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu
50 55 60
Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala
65 70 75 80
Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu
85 90 95
Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val
100 105 110
Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg
115 120 125
Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe
130 135 140
Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Asn Phe Val Gly
145 150 155 160
Thr Met Ala Asn Arg Phe Ser Asp Ala Thr Ser Glu Ile Asp Asn Leu
165 170 175
Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met
180 185 190
Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr
195 200 205
Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro
210 215 220
Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr
225 230 235 240
Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala Thr Arg Gly Val
245 250 255
Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu Ala Ala Gln Tyr
260 265 270
Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro Thr Thr Ser Asn
275 280 285
Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser Ile Leu
290 295 300
Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr Tyr Gly
305 310 315 320
Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr Pro Phe
325 330 335
Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Thr Trp Gly
340 345 350
Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu Lys Leu
355 360 365
Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp Lys Thr
370 375 380
Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala Pro Val
385 390 395 400
Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys Phe Asp
405 410 415
Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu Gly Tyr
420 425 430
Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser Ala Asn
435 440 445
Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr Ile Asn
450 455 460
Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His Thr Gly
465 470 475 480
Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro Val Pro
485 490 495
Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser Thr Asn
500 505 510
Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp Asn Asn
515 520 525
Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile Val Trp
530 535 540
Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys Ala Met
545 550 555 560
Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp Leu Ile
565 570 575
Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu Pro Phe
580 585 590
Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg Val Arg
595 600 605
Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser Glu Arg
610 615 620
Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp Ala Trp
625 630 635 640
Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro Ala Asp
645 650 655
Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln Ala Pro
660 665 670
Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu Phe Asn
675 680 685
Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp Phe Phe
690 695 700
Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp Asp Ala
705 710 715 720
Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala Trp Lys
725 730 735
Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu Gly Val
740 745 750
Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr Gln Asn
755 760 765
Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser Val Arg
770 775 780
Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala Pro Asp
785 790 795 800
Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr Asn Thr
805 810 815
Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg Arg Asn
820 825 830
Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
835 840
<210> 8
<211> 557
<212> PRT
<213> Artificial sequence
<220>
<223> beta subunit of ZSH2-5 mutant
<400> 8
Ser Asn Val Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser
1 5 10 15
Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr
20 25 30
Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr
35 40 45
Pro Phe Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Ala
50 55 60
Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu
65 70 75 80
Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp
85 90 95
Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala
100 105 110
Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys
115 120 125
Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu
130 135 140
Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser
145 150 155 160
Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr
165 170 175
Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His
180 185 190
Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro
195 200 205
Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser
210 215 220
Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp
225 230 235 240
Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile
245 250 255
Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys
260 265 270
Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp
275 280 285
Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu
290 295 300
Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg
305 310 315 320
Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser
325 330 335
Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp
340 345 350
Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro
355 360 365
Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln
370 375 380
Ala Pro Ala Ala Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu
385 390 395 400
Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp
405 410 415
Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp
420 425 430
Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala
435 440 445
Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu
450 455 460
Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr
465 470 475 480
Gln Asn Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser
485 490 495
Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala
500 505 510
Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr
515 520 525
Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg
530 535 540
Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
545 550 555
<210> 9
<211> 843
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence of ZSH2-5 mutant (precursor, full)
<400> 9
Met Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val
1 5 10 15
Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp
20 25 30
Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe
35 40 45
Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu
50 55 60
Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala
65 70 75 80
Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu
85 90 95
Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val
100 105 110
Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg
115 120 125
Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe
130 135 140
Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Asn Phe Val Gly
145 150 155 160
Thr Met Ala Asn Arg Phe Ser Asp Ala Thr Ser Glu Ile Asp Asn Leu
165 170 175
Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met
180 185 190
Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr
195 200 205
Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro
210 215 220
Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr
225 230 235 240
Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala Thr Arg Gly Val
245 250 255
Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu Ala Ala Gln Tyr
260 265 270
Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro Thr Thr Ser Asn
275 280 285
Val Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser Ile Leu
290 295 300
Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr Tyr Gly
305 310 315 320
Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr Pro Phe
325 330 335
Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His Val Ala Trp Gly
340 345 350
Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu Lys Leu
355 360 365
Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp Lys Thr
370 375 380
Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala Pro Val
385 390 395 400
Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys Phe Asp
405 410 415
Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu Gly Tyr
420 425 430
Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser Ala Asn
435 440 445
Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr Ile Asn
450 455 460
Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His Thr Gly
465 470 475 480
Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro Val Pro
485 490 495
Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser Thr Asn
500 505 510
Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp Asn Asn
515 520 525
Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile Val Trp
530 535 540
Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys Ala Met
545 550 555 560
Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp Leu Ile
565 570 575
Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu Pro Phe
580 585 590
Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg Val Arg
595 600 605
Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser Glu Arg
610 615 620
Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp Ala Trp
625 630 635 640
Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro Ala Asp
645 650 655
Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln Ala Pro
660 665 670
Ala Ala Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu Phe Asn
675 680 685
Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp Phe Phe
690 695 700
Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp Asp Ala
705 710 715 720
Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala Trp Lys
725 730 735
Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu Gly Val
740 745 750
Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg Ala Thr Gln Asn
755 760 765
Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser Val Arg
770 775 780
Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala Pro Asp
785 790 795 800
Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr Asn Thr
805 810 815
Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu Val Arg Arg Asn
820 825 830
Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
835 840
<210> 10
<211> 557
<212> PRT
<213> Artificial sequence
<220>
<223> beta subunit (amino acid sequence) of mutant ZSH3-1
<400> 10
Ser Asn Val Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser
1 5 10 15
Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr
20 25 30
Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr
35 40 45
Pro Phe Ala Tyr Pro Gly Ile Leu Phe Gly His Asn Ala His Val Ala
50 55 60
Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu
65 70 75 80
Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp
85 90 95
Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala
100 105 110
Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys
115 120 125
Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu
130 135 140
Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser
145 150 155 160
Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr
165 170 175
Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His
180 185 190
Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro
195 200 205
Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser
210 215 220
Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp
225 230 235 240
Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile
245 250 255
Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys
260 265 270
Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp
275 280 285
Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu
290 295 300
Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg
305 310 315 320
Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser
325 330 335
Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp
340 345 350
Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro
355 360 365
Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln
370 375 380
Ala Pro Ala Ala Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu
385 390 395 400
Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp
405 410 415
Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp
420 425 430
Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala
435 440 445
Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu
450 455 460
Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr His Ala Thr
465 470 475 480
Gln Asn Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser
485 490 495
Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala
500 505 510
Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr
515 520 525
Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Glu Glu Val Arg
530 535 540
Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
545 550 555
<210> 11
<211> 843
<212> PRT
<213> Artificial sequence
<220>
<223> amino acid sequence (pro, full) of ZSH3-1 mutant
<400> 11
Met Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr Ser Glu Ala Val
1 5 10 15
Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr Ile Arg Arg Asp
20 25 30
Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val Tyr Gly Ile Phe
35 40 45
Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu Phe Gln Met Glu
50 55 60
Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu Val Leu Gly Ala
65 70 75 80
Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn Phe Ser Pro Glu
85 90 95
Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala Asp Arg Gln Val
100 105 110
Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu Ala Arg Val Arg
115 120 125
Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn Asp Leu Gly Phe
130 135 140
Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met Asn Phe Val Gly
145 150 155 160
Thr Met Ala Asn Arg Phe Ser Asp Ala Thr Ser Glu Ile Asp Asn Leu
165 170 175
Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala Ala Asp Ala Met
180 185 190
Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser Arg Ala Pro Thr
195 200 205
Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro Val Phe Gln Pro
210 215 220
Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg Tyr Asp Gly Thr
225 230 235 240
Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala Thr Arg Gly Val
245 250 255
Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu Ala Ala Gln Tyr
260 265 270
Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro Thr Thr Ser Asn
275 280 285
Val Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala Arg Ser Ile Leu
290 295 300
Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala Tyr Thr Tyr Gly
305 310 315 320
Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly Asn Thr Pro Phe
325 330 335
Ala Tyr Pro Gly Ile Leu Phe Gly His Asn Ala His Val Ala Trp Gly
340 345 350
Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe Ala Glu Lys Leu
355 360 365
Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly Gln Trp Lys Thr
370 375 380
Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp Ala Ala Pro Val
385 390 395 400
Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile Val Lys Phe Asp
405 410 415
Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala Trp Glu Gly Tyr
420 425 430
Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr Gln Ser Ala Asn
435 440 445
Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg Asn Ala Leu Thr Ile Asn
450 455 460
Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr Ala His Thr Gly
465 470 475 480
Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg Leu Pro Val Pro
485 490 495
Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro Phe Ser Thr Asn
500 505 510
Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala Asn Trp Asn Asn
515 520 525
Gln Pro Met Arg Gly Tyr Pro Ser Ala Asp Leu Phe Ala Ile Val Trp
530 535 540
Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg Leu Lys Ala Met
545 550 555 560
Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met Trp Asp Leu Ile
565 570 575
Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His Phe Leu Pro Phe
580 585 590
Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp Pro Arg Val Arg
595 600 605
Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met Thr Ser Glu Arg
610 615 620
Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val Met Asp Ala Trp
625 630 635 640
Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu Met Pro Ala Asp
645 650 655
Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr Pro Gln Ala Pro
660 665 670
Ala Ala Gly Ser Leu Asn Leu Thr Thr Gly Val Lys Val Leu Phe Asn
675 680 685
Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg Tyr Asp Phe Phe
690 695 700
Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala Leu Asp Asp Ala
705 710 715 720
Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro Ala Ala Trp Lys
725 730 735
Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn Phe Leu Gly Val
740 745 750
Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr His Ala Thr Gln Asn
755 760 765
Arg Gly Thr Ala Asn Asn Met Thr Val Phe Asp Gly Lys Ser Val Arg
770 775 780
Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe Val Ala Pro Asp
785 790 795 800
Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp Leu Tyr Asn Thr
805 810 815
Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Glu Glu Val Arg Arg Asn
820 825 830
Ala Thr Ser Glu Glu Thr Leu Arg Cys Pro Arg
835 840
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> T3 primer
<400> 12
aattaaccct cactaaaggg a 21
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> T7 primer
<400> 13
taatacgact cactataggg 20
<210> 14
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> M13F primer
<400> 14
cgccagggtt ttcccagtca cga 23
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> M13R primer
<400> 15
agcggataac aatttcacac agga 24
<210> 16
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH116a-F
<400> 16
gttctggacg gttacrctgc tggcatgaac g 31
<210> 17
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH116a-R
<400> 17
cgttcatgcc agcagygtaa ccgtccagaa c 31
<210> 18
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> SH169a-F
<400> 18
cgtttcagcg atgcaamctc cgagatcgac aac 33
<210> 19
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> SH169a-R
<400> 19
gttgtcgatc tcggagkttg catcgctgaa acg 33
<210> 20
<211> 34
<212> DNA
<213> Artificial sequence
<220>
<223> SH251b-F
<400> 20
gcgcggttac ccaagcrctg atctgttcgc gatc 34
<210> 21
<211> 34
<212> DNA
<213> Artificial sequence
<220>
<223> SH251b-R
<400> 21
gatcgcgaac agatcagygc ttgggtaacc gcgc 34
<210> 22
<211> 34
<212> DNA
<213> Artificial sequence
<220>
<223> SH555b-F
<400> 22
gaggagacgc tgcgttrccc gcgttaataa aagc 34
<210> 23
<211> 34
<212> DNA
<213> Artificial sequence
<220>
<223> SH555b-R
<400> 23
gcttttatta acgcgggyaa cgcagcgtct cctc 34
<210> 24
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> SH201a-F
<400> 24
gctgcgctgg ctgascgatt ctcgtgctc 29
<210> 25
<211> 29
<212> DNA
<213> Artificial sequence
<220>
<223> SH201a-R
<400> 25
gagcacgaga atcgstcagc cagcgcagc 29
<210> 26
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH64b-F
<400> 26
cataacgctc acgtarcctg gggctctact g 31
<210> 27
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH64b-R
<400> 27
cagtagagcc ccaggytacg tgagcgttat g 31
<210> 28
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> SH388b-F
<400> 28
cgcaggcacc ggcgrctggt tccctgaacc 30
<210> 29
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> SH388b-R
<400> 29
ggttcaggga accagycgcc ggtgcctgcg 30
<210> 30
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> SH54b-F
<400> 30
cgttcgctta tccgrgyatt ctgtttggcc 30
<210> 31
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> SH54b-R
<400> 31
ggccaaacag aatrcycgga taagcgaacg 30
<210> 32
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> SH226b-F
<400> 32
ctgccgttca gcaccamccc acaggtgtac aac 33
<210> 33
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> SH226b-R
<400> 33
gttgtacacc tgtgggktgg tgctgaacgg cag 33
<210> 34
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH478b-F
<400> 34
cggttctgtg ttaccrtgcc acccagaatc g 31
<210> 35
<211> 31
<212> DNA
<213> Artificial sequence
<220>
<223> SH478b-R
<400> 35
cgattctggg tggcayggta acacagaacc g 31

Claims (17)

1. A mutant penicillin G acylase comprising at least one mutation selected from the group consisting of a116 α T, I156 α N, N169 α T, M3 β V, S54 β G, T64 β a, H173 β N, T251 β a, T388 β a, R478 β H, E486 β a, D541 β E and Y555 β C in a wild type penicillin G acylase protein sequence comprising an α subunit defined by SEQ ID No. 1 and a β subunit defined by SEQ ID No. 2.
2. The mutant penicillin G acylase according to claim 1, wherein the wild-type penicillin G acylase protein sequence comprises mutations in I156 α N, N169 α T, H173 β N, T251 β a, E486 β a and Y555 β C.
3. The mutant penicillin G acylase according to claim 2 wherein said mutant penicillin G acylase comprises an alpha subunit defined by SEQ ID NO 5; and the beta subunit defined by SEQ ID NO 6.
4. The mutant penicillin G acylase according to claim 2, wherein said mutant penicillin G acylase further comprises at least one mutation selected from the group consisting of G49 α F, T201 α S, M3 β V, T64 β A, T225 β N and T388 β A.
5. The mutant penicillin G acylase according to claim 4 wherein said mutant penicillin G acylase comprises mutations in M3 β V, T64 β A and T388 β A.
6. The mutant penicillin G acylase according to claim 5 wherein said mutant penicillin G acylase comprises an alpha subunit defined by SEQ ID NO 5; and the beta subunit defined by SEQ ID NO 8.
7. Mutant penicillin G acylase according to claim 4 wherein the mutant penicillin G acylase further comprises at least one mutation selected from the group consisting of S54 β G, S150 β T, N226 β T, A310 β D, R478 β H and D541 β E.
8. Mutant penicillin G acylase according to claim 7 wherein the mutant penicillin G acylase comprises mutations of S54 β G, R478 β H and D541 β E.
9. The mutant penicillin G acylase according to claim 8 wherein said mutant penicillin G acylase comprises an alpha subunit defined by SEQ ID NO 5; and the beta subunit defined by SEQ ID NO 10.
10. A polynucleotide encoding the mutant penicillin G acylase of any of claims 1 to 9.
11. A recombinant expression vector comprising the polynucleotide of claim 10.
12. A host cell transformed with the expression vector of claim 11.
13. The transformed host cell according to claim 12, wherein the host cell is a microorganism of any one of the genera selected from the group consisting of: clostridium species (clostridium spp.), escherichia species (Escherichia spp.), acetobacter species (Acetobacter spp.), aeromonas species (Aeromonas spp.), alcaligenes species (Alcaligenes spp.), cryptomonas species (aphanococcus spp.), bacillus species (Bacillus spp.), cephalosporium species (Cephalosporium spp.), flavobacterium species (Flavobacterium spp.), kluyveromyces species (Kluyvera spp.), cladosporium species (Mycoplana spp.), protromyces species (protamellar spp.), protromobacter species (Protaminobacter spp.), pseudomonas species (Pseudomonas spp.), achromobacter species (Achromobacter spp.), and Xanthomonas species (Achromobacter spp.).
14. The transformed host cell according to claim 13, wherein the microorganism of the genus escherichia is escherichia coli MC1061/pBC-ZSH3-1 (escherichia coli MC1061/pBC-ZSH3-1, deposit No. KCTC13991 BP) transformed with a recombinant expression vector comprising a polynucleotide encoding the amino acid sequences of SEQ ID No. 5 and SEQ ID No. 10.
15. A composition for the preparation of cefazolin comprising the mutant penicillin G acylase of any of claims 1 to 9.
16. A process for the enzymatic synthesis of cefazolin from tetrazolyl acetate and 3- [ 5-methyl-1, 3, 4-thiadiazol-2-yl ] -7-aminocephalosporanic acid, said process comprising the use of a mutant penicillin G acylase according to any of claims 1 to 9.
17. Use of a mutant penicillin G acylase of any of claims 1 to 9 for the preparation of cefazolin.
CN202080093452.2A 2019-11-15 2020-11-13 Penicillin G acylase mutants with increased cefazolin productivity and uses thereof Pending CN115397979A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR1020190147086A KR102363768B1 (en) 2019-11-15 2019-11-15 Mutants of penicillin G acylase with increased production of cefazolin, and uses thereof
KR10-2019-0147086 2019-11-15
PCT/KR2020/016010 WO2021096298A1 (en) 2019-11-15 2020-11-13 Penicillin g acylase mutant having increased cefazoline productivity and use thereof

Publications (1)

Publication Number Publication Date
CN115397979A true CN115397979A (en) 2022-11-25

Family

ID=75913074

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202080093452.2A Pending CN115397979A (en) 2019-11-15 2020-11-13 Penicillin G acylase mutants with increased cefazolin productivity and uses thereof

Country Status (3)

Country Link
KR (1) KR102363768B1 (en)
CN (1) CN115397979A (en)
WO (1) WO2021096298A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000053041A (en) * 1996-11-05 2000-08-25 스티븐 비. 데이비스 Mutant Penicillin G Acylase
KR20110099137A (en) * 2008-12-23 2011-09-06 디에스엠 아이피 어셋츠 비.브이. Mutant penicillin g acylases
CN103805671A (en) * 2013-11-11 2014-05-21 华北制药河北华民药业有限责任公司 Method for preparing cefalexin
US20160326508A1 (en) * 2015-05-07 2016-11-10 Codexis, Inc. Penicillin-g acylases
WO2017143944A1 (en) * 2016-02-23 2017-08-31 上海星维生物技术有限公司 Penicillin g acylase mutant
KR101985911B1 (en) * 2017-12-28 2019-06-04 아미코젠주식회사 Mutants of penicillin G acylase from Achromobacter sp. CCM 4824, and uses thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100530299B1 (en) * 2003-08-11 2005-11-22 산도즈 게엠베하 Cephalosporin c acylase mutant and method for preparing 7-aca using same
CN110506113B (en) * 2017-01-05 2024-05-03 科德克希思公司 Penicillin G acylase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000053041A (en) * 1996-11-05 2000-08-25 스티븐 비. 데이비스 Mutant Penicillin G Acylase
KR20110099137A (en) * 2008-12-23 2011-09-06 디에스엠 아이피 어셋츠 비.브이. Mutant penicillin g acylases
CN103805671A (en) * 2013-11-11 2014-05-21 华北制药河北华民药业有限责任公司 Method for preparing cefalexin
US20160326508A1 (en) * 2015-05-07 2016-11-10 Codexis, Inc. Penicillin-g acylases
WO2017143944A1 (en) * 2016-02-23 2017-08-31 上海星维生物技术有限公司 Penicillin g acylase mutant
KR101985911B1 (en) * 2017-12-28 2019-06-04 아미코젠주식회사 Mutants of penicillin G acylase from Achromobacter sp. CCM 4824, and uses thereof

Also Published As

Publication number Publication date
KR20210059533A (en) 2021-05-25
WO2021096298A1 (en) 2021-05-20
KR102363768B1 (en) 2022-02-16

Similar Documents

Publication Publication Date Title
CN109971743B (en) Penicillin G acylase mutant derived from achromobacter CCM4824 and application thereof
AU718648B2 (en) Mutant penicillin G acylases
CN106929521B (en) Aldehyde ketone reductase gene recombination co-expression vector, engineering bacterium and application thereof
EP1656450B2 (en) Cephalosporin c acylase mutant and method for preparing 7-aca using same
KR20140094150A (en) A mutant enzyme for production of cephalosporin antibiotics
Yang et al. Expression and purification of extracellular penicillin G acylase in Bacillus subtilis
CN111172142B (en) Cephalosporin C acylase mutant with high thermal stability
JP2005198655A (en) Cephalosporin c acylase
CN1062903C (en) A new cephalosporin C acylase
CN109072215B (en) Cephalosporin C acylase mutant and application thereof
WO2001085951A1 (en) A modified expandase and uses thereof
CN115397979A (en) Penicillin G acylase mutants with increased cefazolin productivity and uses thereof
RU2388826C2 (en) RECOMBINANT DNA, WHICH CODES FUNCTIONALLY ACTIVE HYBRID PROTEIN GL7ACA-ACYLASE WITH CHITIN-BINDING DOMAIN (BrdGL7ACA-cbd) AND RECOMBINANT PLASMID pSVH0108, PROVIDING FOR ITS SYNTHESIS IN CELLS Escherichia coli, RECOMBINANT STRAIN Escherichia coli BL21(DE3)/pSVH0108-PRODUCER BrdGL7ACA-cbd
Liu et al. Environmentally safe production of 7-ACA by recombinant Acremonium chrysogenum
CN108949736B (en) High-selectivity cefradine synthetase mutant and encoding gene thereof
KR101677755B1 (en) Mutant alpha-amino acid ester hydrolase with enhanced productivity of amoxicillin
CN111484986B (en) Short-chain dehydrogenase and application thereof
EP4202044A2 (en) Polypeptide having cephalosporin c acylase activity and use thereof
CN112852912B (en) Method for synthesizing 7-aminodesacetoxycephalosporanic acid
Isogai et al. Direct production of 7-aminocephalosporanic acid and 7-aminodeacetylcephalosporanic acid by recombinant Acremonium chrysogenum
RU2499830C1 (en) RECOMBINANT STRAIN Escherichia coli-PRODUCER OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS FROM Xanthomonas rubrilineans, METHOD FOR MICROBIOLOGICAL SYNTHESIS OF HYDROLASE OF ALPHA-AMINO ACIDS ESTERS BASED ON ABOVE SAID STRAIN
CN115491361A (en) Application of expandase and mutant thereof in production of G-7-ADCA
CN117947137A (en) Penicillin amidase for synthesizing cefaclor
CZ2015586A3 (en) DNA sequences encoding hydrolases of esters of α-amino acids and the recombinant microorganisms carrying this sequence
JPWO2002086127A1 (en) Acylase gene

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination