JPWO2005075652A1 - Method for producing modified γ-glutamyl transpeptidase (modified GGT) having enhanced glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity - Google Patents

Abstract

A modified peptide with enhanced glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity using γ-glutamyl transpeptidase (GGT), an enzyme widely distributed in the biological world (ie, modified) It is an object of the present invention to produce GGT). According to the present invention, in GGT, one or more amino acid residues selected from the group consisting of 1) an amino acid residue that interacts with a γ-glutamyl compound, or 2) an amino acid residue in the region around the active center By substituting with this amino acid residue, it is possible to produce a modified GGT with enhanced glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity.

Description

  The present invention enhances glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity, characterized by substituting one or more amino acid residues of γ-glutamyltranspeptidase (GGT). The present invention relates to a modified GGT production method, a modified GGT with enhanced GL-7-ACA acylase activity, a nucleic acid molecule encoding the modified GGT, and a cell expressing the modified GGT. Furthermore, the present invention uses the modified GGT to treat GL-7-ACA to produce 7-aminocephalosporanic acid (7-ACA) which is a raw material for cephalosporins (cephems) antibiotics. Also related to the method.

  The sales of semisynthetic cephalosporin in 2002 accounted for $ 7.6 billion of the global sales of approximately $ 400 billion in that year's pharmaceutical market, making it the most sold drug among antibiotics. . Semi-synthetic cephalosporin has been produced by modifying the 3- and 7-position side chains of 7-aminocephalosporanic acid (7-ACA). Therefore, efficient production of 7-ACA is a critical concern for the pharmaceutical industry. Industrially, it is produced mainly by chemically deacylating the 7-position side chain of cephalosporin C produced by fermentation of Acremonium chrysogenum, a type of mold.

  However, in the case of chemical methods, it is necessary to go through many complicated processes, to require high-purity cephalosporin, to react under special conditions (for example, low temperature), and to produce toxic compounds. There are drawbacks such as use and large amounts of chemical substances as waste.

  Therefore, 6-aminopenicillanic acid, which is an intermediate of semisynthetic penicillin, finds a microorganism that produces cephalosporin acylase in the same manner as industrially produced by deacylating penicillin with a microorganism-derived penicillin acylase, Development of an enzymatic method for converting cephalosporin C to 7-ACA by the enzyme has been studied for a long time. An enzyme that directly deacylates cephalosporin C has also been found, but its activity is extremely low when cephalosporin C is used as a substrate. Therefore, a method of chemically converting cephalosporin C to GL-7-ACA and then deacylating to 7-ACA with cephalosporin acylase has been industrialized since 1978 (Non-patent Document 1). On the other hand, in a recently industrialized bioprocess (Non-patent Document 2), two types of enzymes, D-amino acid oxidase and cephalosporin acylase, are used. In this process, cephalosporin C is converted to ketoadipoyl-7-ACA by D-amino acid oxidase, and then non-enzymatically converted to GL-7-ACA by hydrogen peroxide produced as a by-product in this reaction. GL-7-ACA is then deacylated with cephalosporin acylase to 7-ACA. In both of these processes, cephalosporin acylase catalyzing efficient conversion is important and extensive screening has been carried out, but microorganisms with this activity have been found only in a limited variety of bacteria. Absent.

  For efficient enzymatic methods, there is a strong demand for enzymes that exist in a wide variety of organisms and catalyze the reaction of 7-ACA production in place of cephalosporin acylase.

  On the other hand, γ-glutamyl transpeptidase (GGT) exists in a wide range of biological species such as bacteria, plants, and animals, and is widely known as an enzyme that hydrolyzes the CN bond of the amide group of γ-glutamyl compounds. . For example, it catalyzes the reaction of γ-glutamyl peptide + water → glutamic acid + peptide.

However, so far, there has been no report showing that GGT has GL-7-ACA acylase activity.
Tsuzuki Katsunuma, Kenichi Komatsu, Shigeaki Ichikawa, Yuzo Shibuya. Research on manufacturing technology of 7-aminocephalosporanic acid (7-ACA) by enzymatic method. Journal of Japanese Society for Agricultural Chemistry, 63, 1847-1853 (1989). A. Schmid, F. Hollmann, JB Park, and B. Buhler.The use of enzymes in the chemical industry in Europe.Current Opinion in Biotechnology, 13, 359-366 (2002).

  The present invention relates to a method for producing a modified GGT with enhanced GL-7-ACA acylase activity, a modified GGT with enhanced GL-7-ACA acylase activity, a nucleic acid molecule encoding the modified GGT, and the modified GGT. An object is to provide cells that are expressed. Furthermore, the present invention provides a 7-aminocephalo which is a raw material for cephalosporin (cephem) antibiotics by treating GL-7-ACA with the modified GGT with enhanced GL-7-ACA acylase activity. It is a further object to provide a method for producing sporanic acid (7-ACA).

  The present inventors have conducted intensive research to develop a method for efficiently producing 7-ACA, which is a raw material for cephalosporin antibiotics, by an enzymatic method. As a result, in GGT, one or more amino acid residues selected from the group consisting of 1) an amino acid residue that interacts with a γ-glutamyl compound, or 2) an amino acid residue in the peripheral region of the active center, By substituting with an amino acid residue, we succeeded in producing a modified GGT with enhanced GL-7-ACA acylase activity.

  So far, the species in which the GGT gene has been identified and cloned include Escherichia coli K-12, Bacillus subtilis, Bacillus natto, Helicobacter pylori 26695, Pseudomonas A14, Rat, Mouse, Pig, and Human. It is 76 amino acid residues out of the 580 amino acid residues of E. coli GGT that the amino acid sequence is completely conserved among these species. Therefore, the sequence of the 76 amino acid residues was compared between GGT and class IV cephalosporin acylase. Of the 76 amino acid residues of GGT, 58 amino acid residues were classified into class IV cephalosporin acylase (Y Kim, K.-H. Yoon, Y. Khang, S. Turley, and WGJ Hol. The 2.0 A crystal structure of cephalosporin acylase. Structure, 8, 1059-1068 (2000).) all right. The 18 amino acid residues that differ between GGT and class IV cephalosporin acylase were conserved among class IV cephalosporin acylases found so far in the two strains (FIGS. 1a-1c and 2). checking). However, E. coli GGT does not have GL-7-ACA hydrolyzing activity, whereas class IV cephalosporin acylase has GL-7-ACA hydrolyzing activity. Therefore, the present inventors paid attention to amino acid residues that are conserved among species in GGT but are different from those of class IV cephalosporin acylase.

The amino acid residue (Asp-423) of human GGT corresponding to aspartic acid (Asp-433), which is the 433th residue of GGT of E. coli, interacts with the amino group at the α-position of the γ-glutamyl compound. It was suggested to stabilize (N. Taniguchi, and Y. Ikeda. Γ-Glutamyl transpeptidase: catalytic mechanism and gene expression. Ad
v. Enzymol. Rel. Areas Mol. Biol. 72, 239-278 (1998)). The amino acid residue corresponding to position 433 of this E. coli GGT was one of 18 amino acid residues that were completely conserved among GGTs but differed from class IV cephalosporin acylase. The 7-position side chain of GL-7-ACA, which is a substrate for class IV cephalosporin acylase, has a structure in which a glutaryl group is bonded to an amino group at the 7-position. The glutaryl group has a structure in which the α-position amino group of the γ-glutamyl group is deaminated.

  Accordingly, the present inventors can produce a modified GGT with enhanced GL-7-ACA acylase activity by introducing a mutation that changes the interaction with the γ-glutamyl compound into GGT. I made a hypothesis. Based on this hypothesis, the present inventors created a modified GGT in which an amino acid residue that interacts with a γ-glutamyl compound is substituted with another amino acid residue. The modified GGT has a significantly higher GL than before the modification. It had -7-ACA acylase activity. Surprisingly, this modified GGT had a lower Km value and higher substrate specificity for GL-7-ACA than the conventional cephalosporin acylase.

  In addition, the present inventors have already revealed that the amino acid residue corresponding to the 391st threonine residue of E. coli GGT is one of the amino acid residues essential for activity. Accordingly, the present inventors introduced a mutation into the region around the active center of GGT (for example, the region around the amino acid residue corresponding to the 391 th threonine residue of E. coli GGT), thereby allowing the region around the active center. We hypothesized that the formation changes and the active center approaches the amide bond next to the 7th carbon of GL-7-ACA, hydrolyzing the amide bond. Based on this hypothesis, a modified GGT with enhanced GL-7-ACA acylase activity can be obtained by introducing a mutation into the region around the active center of GGT.

That is, the present invention relates to the following matters.
Item 1.
In γ-glutamyl transpeptidase (GGT):
1) an amino acid residue that interacts with a γ-glutamyl compound, or 2) an amino acid residue in the region around the active center,
Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity is enhanced, wherein one or more amino acid residues selected from the group consisting of: Method for preparing modified GGT.
Item 2.
In GGT, residues 50-78, 93-98, 114-125, 147-188, and 209-347 of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 In a region corresponding to one or more regions selected from the group, residues 391 to 434, residues 452 to 487, and residues 508 to 569, other one or more amino acid residues The method according to Item 1, wherein the amino acid residue is substituted with an amino acid residue.
Item 3.
In GGT, the 16th, 94th, 98th, 114th, 115th, 209th, 209th, and 227th residues of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 Group, 242 residue, 263 residue, 334 residue, 342 residue, 433 residue, 452 residue, 460 residue, 461 residue, 462 residue, 468 residue Item 2. The method according to Item 1, wherein one or more amino acid residues selected from the group 484th residue are substituted with other amino acid residues.
Item 4.
Item 3. The GGT is any one of Items 1 to 3, wherein the amino acid residue corresponding to the 433 residue of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 is substituted with another amino acid residue The method described in 1.
Item 5.
Item 5. The method according to Item 4, wherein the other amino acid residue is at least one amino acid residue selected from the group consisting of asparagine, glutamine, tyrosine, tryptophan, and phenylalanine.
Item 6.
Item 6. The method according to Item 5, wherein the other amino acid residue is asparagine.
Item 7.
Item 7. A modified GGT obtained by the method according to any one of Items 1 to 6.
Item 8.
Item 8. A nucleic acid molecule encoding the modified GGT of Item 7.
Item 9.
Item 8. A cell expressing the modified GGT of Item 7.
Item 10.
Item 8. A method for producing 7-aminocephalosporanic acid (7-ACA), comprising a step of treating GL-7-ACA with the modified GGT according to Item 7.

  Hereinafter, the present invention will be described in detail.

  The GL-7-ACA acylase activity in the present invention means an activity of hydrolyzing the amide bond of the 7-position side chain of GL-7-ACA unless otherwise specified.

  The “γ-glutamyl transpeptidase (GGT)” before modification is derived from any organism and has properties other than naturally occurring GGT and non-natural GGT (artificially synthesized GGT, GL-7-ACA acylase activity) (Including GGT modified for the purpose of improving thermal stability, etc.). Further, not GGT full length but N-terminal deletion type, C-terminal deletion type, N- and C-terminal deletion type GGTs are also used in the same manner. Examples of the N-terminal deletion type GGT include GGT in which an amino acid residue corresponding to the 1st to 34th amino acid residues of Escherichia coli is deleted. Examples of the C-terminal deletion type GGT include GGT in which an amino acid residue corresponding to the 572nd to 580th amino acid residues of E. coli is deleted. Examples of the N- and C-terminal deletion types include GGT in which amino acid residues corresponding to amino acid residues 1 to 34 and 572 to 580 of Escherichia coli are deleted.

  Arbitrary organisms from which GGT is derived include bacteria (eg, E. coli, Bacillus subtilis, Bacillus natto, Pseudomonas, H. pylori, etc.), animals (eg, humans, monkeys, rats, mice, pigs, rabbits, hamsters). Etc.), insects (for example, silkworms), plants (for example, kidney beans, etc.), cyanobacteria (for example, blue-green algae, daffodil, uremo, nenjumo, etc.), yeasts (for example, genus Saccharomyces, etc.), etc. .

The “amino acid residue that interacts with the γ-glutamyl compound” may be any residue as long as it is an amino acid residue that interacts with the γ-glutamyl compound. Examples of such an amino acid residue include an α-position amino group (—NH ₂ , —NH ₃ ⁺ ), an α-position carboxyl group (—COOH group, —COO ⁻ group), and β-position of a γ-glutamyl compound. And / or amino acid residues that interact with the γ-position methylene group (CH ₂ group). The interaction is an action that can be generally recognized by those skilled in the art, such as ionic bond, hydrogen bond, electrostatic interaction, hydrophobic interaction, van der Waals force, and the like.

  The amino acid residue that interacts with the α-position amino group of the γ-glutamyl compound may be any amino acid residue as long as it can interact with the amino group, preferably aspartic acid, Examples include but are not limited to glutamic acid, serine, threonine, cysteine, glutamine, asparagine, and tyrosine.

  The amino acid residue that interacts with the α-position carboxyl group of the γ-glutamyl compound may be any amino acid residue that can interact with the carboxyl group, but is preferably arginine or lysine. And histidine, glutamic acid, aspartic acid, glutamine, asparagine, serine, cysteine, and threonine (but not limited to).

The amino acid residue that interacts with the β-position and / or γ-position methylene group (CH ₂ group) of the γ-glutamyl compound is any amino acid residue that can interact with the methylene group. However, preferably, phenylalanine, alanine, tyrosine, leucine, isoleucine, and valine can be exemplified (but not limited thereto).

  The “amino acid residue in the region around the active center” may be any amino acid residue as long as the amino acid residue is two-dimensionally and three-dimensionally close to the active center and the active center. Examples of the amino acid residues in the region around the active center include amino acid residues in the region around the amino acid residue corresponding to the 391st threonine of GGT of E. coli.

Therefore, examples of the “amino acid residue that interacts with the γ-glutamyl compound or the amino acid residue in the region around the active center” include, for example, the 50th to 78th residues of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1. 93-98 residues, 114-125 residues, 147-188 residues, 209-347 residues, 391-434 residues, 452-487 residues and 508-569 residues Examples include amino acid residues in the region of GGT corresponding to one or more regions selected from the region. An amino acid residue of GGT corresponding to Asp-433 of E. coli GGT, which is one of the amino acid residues that interact with the γ-glutamyl compound, and E. coli GGT, which is one of the amino acid residues present in the active center of GGT activity The amino acid residues of GGT corresponding to Thr-391 are present in these regions.
In one preferred embodiment of the invention, the amino acid residue that is substituted according to the invention is an amino acid residue that is conserved between species and not conserved with a class IV cephalosporin acylase in GGT. . Such amino acid residues include, for example, Escherichia coli Asp-433, Leu-16, Gly-94, Phe-98, Arg-114, Glu-115, Phe-209, Leu-227, Phe-242, Thr-263, These are amino acid residues corresponding to Tyr-334, Gly-342, Asn-452, Pro-460, Leu-461, Ser-462, Ile-468, and Gly-484.

The “other amino acid residue” after substitution may be any amino acid as long as it is different from the amino acid residue before substitution. Preferably, it is an amino acid residue having different properties (for example, acid / neutral / basic, hydrophilic / hydrophobic, bulky, etc. of the side chain) from the amino acid residue before substitution. For example, when the amino acid residue before substitution is acidic, the other amino acid residues are basic or neutral, and when the amino acid residue before substitution is basic, the other amino acid residues are acidic or medium. If the amino acid residue before substitution is neutral, the other amino acid residue is a basic or acidic amino acid residue. For example, when the amino acid before substitution is hydrophobic, the other amino acid residues are hydrophilic. When the amino acid before substitution is hydrophilic, the other amino acid residues are hydrophobic. For example, when substituting the amino acid residue of GGT corresponding to the 433rd aspartic acid of GGT of E. coli, the other amino acid residues are, for example, asparagine, glutamine, tyrosine, histidine, tryptophan, glutamic acid, cysteine, arginine, lysine , Threonine, serine, phenylalanine, leucine, isoleucine, valine (but not limited to), preferably asparagine, tyrosine, glutamine, tryptophan, phenylalanine, more preferably asparagine, tyrosine, particularly preferably asparagine. It is.
In one preferred embodiment of the invention, the other amino acid residue is an amino acid residue of a class IV cephalosporin acylase corresponding to the amino acid residue underlined in the amino acid sequence of GGT in FIGS. It is.

  “Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity was enhanced” means that the GL-7-ACA acylase activity was somewhat increased (increased) by the substitution as described above. It means). Moreover, GGT before substitution may or may not have GL-7-ACA acylase activity. The amount in which the activity is enhanced (increase amount) is not particularly limited as long as it is increased as much as possible, and is, for example, an increase amount of 0.0001 U / mg or more.

“Modified GGT” means a modified peptide and / or modified protein of GGT with enhanced GL-7-ACA acylase activity produced according to the method of the present invention.
“Nucleic acid molecule encoding modified GGT” means a DNA molecule and / or RNA molecule that is transcribed and translated into the same amino acid sequence as the modified GGT. As long as the nucleic acid molecule encoding the modified GGT correctly codes the modified GGT, any codon may be selected. Codon selection can be performed according to a conventional method. For example, it can be determined in consideration of the frequency of use of the codon of the host to be used.

  The “cell expressing the modified GGT” may be any cell as long as it is a cell expressing the modified GGT (including cells transformed with a nucleic acid molecule encoding the modified GGT). . Such cell species may be any species, but is preferably the same as the GGT-derived species.

  “Nucleic acid molecule encoding modified GGT”, “modified GGT” and “cell expressing modified GGT” as described above can be obtained according to a conventional method.

  Nucleic acid molecules encoding the modified GGT can be obtained, for example, by mutation introduction methods such as Kunkel method, Eckstein method, AlteredSite method, PCR method, Ito method and the like. In addition, the primer used when performing these mutagenesis methods can be suitably designed based on the mutagenesis method employed and the sequence information of the nucleic acid molecule encoding the modified GGT, and can be synthesized according to a conventional method.

  Isolation and purification of the obtained nucleic acid molecule can be performed by, for example, H. Suzuki, H. Kumagai, T. Echigo, and T. Tochikura. Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of γ-glutamyltranspeptidase. Biochem. Biophys. Res. Commun., 150, 33-38 (1988), separation and purification by gel electrophoresis, or molecular sieving operation.

  The nucleic acid molecule obtained as described above is sequenced as necessary, for example, according to the dideoxy method, the Maxam-Gilbert method, or using a commercially available sequence kit.

  The acquisition of “modified GGT” and “cell expressing the modified GGT” can be performed according to a conventional genetic recombination technique using the nucleic acid molecule encoding the modified GGT obtained as described above ( Sambrook, J., Frisch, EF, and Maniatis, T. Molecular Cloning, A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. 1989 Current Protocls in Molecular Biology. John Wiley Sons, Inc. 1998. Briefly, for example, by inserting a nucleic acid molecule encoding the modified GGT into a suitable expression vector, introducing it into a suitable host cell, transformation, and expressing the modified GGT under suitable conditions, Cells expressing the modified GGT can be obtained. Furthermore, the modified GGT can be recovered from the cells expressing the modified GGT. This recovered modified GGT is described in the literature (H. Suzuki, H. Kumagai, T. Echigo, and T. Tochikura. Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of γ-glutamyltranspeptidase. Biochem. Biophys. Res. Commun. , 150, 33-38 (1988)) or may be further purified according to a conventional method.

  Here, the method of introducing the expression plasmid into the host cell is not particularly limited, and a general method can be adopted. For example, electroporation, liposome method, calcium phosphate method, DEAE dextran method and the like can be used. Further, in each step, when it is necessary to select a target product, a subcloning operation or sequencing may be appropriately performed.

  Any species of prokaryotic and eukaryotic cells (including established cells) can be used as host cells for introducing the expression plasmid.

  Prokaryotic cells include, for example, cells of E. coli (eg, Escherichia coli), Bacillus subtilis (eg, Bacillus subtilis), Bacillus natto, Pseudomonas, H. pylori, or cyanobacteria (but not limited thereto). As the cells of Escherichia coli, for example, cell lines classified into K12 strain, B strain, SH641 strain, CM9 strain, and SH703 strain (but not limited thereto) are suitable. Further, as a cell of Bacillus subtilis, MH2308 strain (not limited thereto) is particularly preferable.

  Examples of eukaryotic cells include animal-derived cells (eg, human, rat, mouse-derived cells), insect-derived cells (eg, silkworm-derived cells), plant-derived cells (eg, kidney bean-derived cells), It is not limited to these. Specifically, animal-derived established cells include monkey kidney COS cells (Cell, 23: 175 (1981)), Vero cells and MK cells, Chinese hamster ovary cells CHO cells, mouse Examples include NIH3T3 cells (J. Viol., 4: 549 (1969)), which are fibroblasts, and Jurkat cells (J. Immunol., 133: 123 (1984)), which are cells derived from human T-cell lymphoma. . Yeast cells (eg, Saccharomyces spp.) Are also preferably used.

  When a prokaryotic cell is used as a host, a vector that can replicate in the host cell is used, a nucleic acid molecule that encodes the modified GGT in this vector, a promoter upstream of the nucleic acid molecule, and an SD (Shine-Dalgarno) sequence And an expression plasmid provided with an initiation codon (for example, ATG), if necessary, can be suitably used.

In general, plasmids derived from E. coli such as pBR322, pBR325, pUC18, and pUC19 are often used as the vector, but not limited thereto, and various known vectors can be used. Commercially available products of the above vectors used in expression systems utilizing E. coli include, for example, pGEX-4T, pKK223-3 (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET21, pET21 / LacI ^q (Invitrogen), pBAD / His (Invitrogen) (but not limited to).

  The promoter is not particularly limited, and when Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter, tac promoter, T7 promoter, trc promoter (in addition to these) (But not limited) can be preferably used. When the host is Bacillus, SP01 promoter, penP promoter and the like are preferable.

  When using eukaryotic cells as hosts, the expression vector usually has a promoter, an RNA splicing site, a polyadenylation site and a transcription termination sequence upstream of the nucleic acid molecule to be expressed, and further replicates as necessary. Those having a starting point are mentioned. Examples of the expression vector include, but are not limited to, pSV2dhfr (Mol. Cell. Biol., 1: 854 (1981)) carrying the SV40 early promoter. In addition to the above, various known commercially available vectors can be used. In the case of an expression system using animal cells, examples of expression vectors include pCI (Promega), pIND (Invitrogen), pcDNA3.1 / His (Invitrogen), and pEF1 / V5-His (Invitrogen). Vectors for insect cells such as animal cell vectors, pFastBac HT (GibciBRL), pAcGHLT (PharMingen), pAc5 / V5-His, pMT / V5-His, pMT / Bip / V5-his (and above, Invitrogen) Is not limited to the above).

  Preferred promoters when using animal cells as hosts include SV40-derived promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters, SRα promoters, adenovirus promoters, and elongation factor promoters. (But not limited to). As a promoter in the case of using yeast as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter (not limited thereto) can be preferably used.

  Purification of the modified GGT thus obtained was performed by a method already reported by the present inventors (H. Suzuki, H. Kumagai, T. Echigo, and T. Tochikura. Molecular cloning of Escherichia coli K-12 ggt and Rapid isolation of γ-glutamyltranspeptidase. Biochem. Biophys. Res. Commun., 150, 33-38 (1988)) or a conventional method.

  Further, GL-7-ACA can be treated with the modified GGT of the present invention to produce 7-ACA.

  For the synthesis of GL-7-ACA, for example, Y. Shibuya, K. Matsumoto, and T. Fujii. Isolation and properties of 7β- (4-carboxybutanamido) cephalosporanic acid acylase-producing bacteria. Agric. Biol. Chem., 45, 1561-1567 (1981).

  Treatment is performed using conventional methods (eg, by incubation). For example, the modified GGT and GL-7-ACA are incubated at 37 ° C. for 3 hours in Tris-HCl (eg, pH 7.6 to 9.0). By this processing, the GL-7-ACA is converted to 7-ACA.

  The measurement of GL-7-ACA and 7-ACA follows conventional methods, such as reverse phase HPLC and an amino acid analyzer, for example.

  Further, if necessary, 7-ACA is separated and purified. This separation and purification are performed by, for example, DOWEX 1X8 and reverse phase HPLC.

  According to the present invention, it is possible to produce a modified GGT with enhanced glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity. In addition, the modified GGT produced by the present invention has a smaller Km value for GL-7-ACA than the conventional cephalosporin acylase, and the modified GGT of the present invention is an excellent substrate for GL-7-ACA. Has specificity.

  Furthermore, according to the present invention, an efficient enzymatic production method of 7-ACA is provided, which is safer, simpler, lower cost and environmentally friendly than conventional chemical methods, and is a cephalosporin (cephem) antibiotic. It became possible to produce 7-ACA as a raw material.

An alignment of all sequences of GGT and class IV cephalosporin acylase is shown in FIGS. 1a-1c. Each sequence is available from SwissProt or GenBank. From above, Pseudomonas sp. SE83 cephalosporin acylase (SwissProt Accession No. P15557: SEQ ID NO: 19), Pseudomonas sp. V22 cephalosporin acylase (SwissProt Accession No. Q05053: SEQ ID NO: 21), Escherichia coli K-12 GGT (SwissProt Accession No. P18956: SEQ ID NO: 1), Bacillus subtilis 168 GGT (SwissProt Accession No. P54422: SEQ ID NO: 3), Bacillus natto GGT (GenBank Accession No. 2321184A: SEQ ID NO: 5), Helicobacter pylori 26695 GGT (GenBank Accession No. AE000618: Sequence) No. 7), Pseudomonas A14 GGT (SwissProt Accession No. P36267: SEQ ID NO: 9), Rat GGT (SwissProt Accession No. P07314: SEQ ID NO: 11), Mouse GGT (SwissProt Accession No. Q60928: SEQ ID NO: 13), Pig GGT ( SwissProt Accession No. P20735: SEQ ID NO: 15) and Human GGT1 (SwissProt Accession No. P19440: SEQ ID NO: 17). Residues in underlined italics indicate GGT sites that are conserved in all GGTs but are different in class IV cephalosporin acylases. Italic (no underlined) residues indicate residues that are conserved in all GGT and class IV cephalosporin acylases. FIG. 1a shows a part of the alignment. FIG. 1b is a continuation of the alignment shown in FIG. 1a. FIG. 1c is a continuation of the alignment shown in FIG. 1b. FIG. 2 shows an alignment around Asp-433 that was mutated this time from Thr-391, the active center of GGT of E. coli, which is one of the well-conserved regions between GGTs. Each sequence is a partial sequence of the sequence (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21) available from SwissProt or GenBank. In order from the top, Escherichia coli K-12 GGT (SEQ ID NO: 24), Bacillus subtilis 168 GGT (SEQ ID NO: 25), Bacillus natto GGT (SEQ ID NO: 26), Helicobacter pylori 26695 GGT (SEQ ID NO: 27), Pseudomonas A14 GGT (sequence) No. 28), Rat GGT (SEQ ID NO: 29), Mouse GGT (SEQ ID NO: 30), Pig GGT (SEQ ID NO: 31), Human GGT1 (SEQ ID NO: 32), Pseudomonas sp. SE83 cephalosporin acylase (SEQ ID NO: 33), Pseudomonas sp V22 is the amino acid sequence of cephalosporin acylase (SEQ ID NO: 34). Residues that are conserved in all GGTs are shown in italics (no underlining). The underlined italic letters indicate the GGT location that is conserved as an Asp residue in all GGTs, but is an Asn residue in class IV cephalosporin acylases. The numbers shown to the left and right of the alignment indicate the number of residues from the N-terminus of the left and right residues in the figure. FIG. 3 shows a HPLC chart of the enzyme reaction. The reaction was performed by incubating the reaction mixture (2 mM GL-7-ACA, 50 mM Tris-HCl (pH 8.73), 0.1 mg / ml D433N) at 37 ° C. (A) is a sample for 0 hour reaction, and (B) is a sample for 3 hour reaction. FIG. 4 shows the results of examining the optimum pH for the GL-7-ACA hydrolysis reaction of D443N. The buffer solution is potassium phosphate buffer at pH 6.5 to 7.5, Tris-HCl buffer at pH 7.5 to 8.73, and NH ₄ Cl—NH ₄ OH buffer at pH 8.73 to 10.5. Was used. FIG. 5 shows the NMR results. (A) is a commercially available GL-7-ACA, and (B) is an NMR chart of the sample obtained in this example. FIG. 6 shows various modified GGTs obtained in Examples 1-25. Modified GGT of Bacillus subtilis 168 (B. subtilis), Bacillus natto (B. natto), Helicobacter pylori 26695 (H. pylori), Pseudomonas A14 (Pseudo), rat, mouse, pig, human corresponding to the modified GGT of E. coli Indicates.

  Examples are shown below, but these are merely examples of the present invention and do not limit the present invention in any way. In the following examples, E. coli GGT is used, but the amino acid sequence of GGT is well conserved between E. coli and other biological species, and the present invention is similarly applied to various biological species other than E. coli. Can be implemented.

  Here, the abbreviations such as amino acids, proteins, base sequences, nucleic acids and the like described in the claims, the specification and the drawings of the present application are defined in the IUPAC, IUB regulations, “of specifications including base sequences or amino acid sequences”. "Guidelines for preparation" (edited by JPO) and idiomatic symbols in the field.

In addition, each sequence described in the sequence listing is as follows:
SEQ ID NO: 1: Amino acid sequence of Escherichia coli K-12 GGT SEQ ID NO: 2: Nucleic acid sequence of Escherichia coli K-12 GGT SEQ ID NO: 3: Amino acid sequence of Bacillus subtilis 168 GGT SEQ ID NO: 4: Nucleic acid sequence of Bacillus subtilis 168 GGT 5: amino acid sequence of Bacillus natto GGT SEQ ID NO: 6: nucleic acid sequence of Bacillus natto GGT SEQ ID NO: 7: amino acid sequence of Helicobacter pylori 26695 GGT SEQ ID NO: 8: nucleic acid sequence of Helicobacter pylori 26695 GGT SEQ ID NO: 9: amino acid sequence of Pseudomonas A14 GGT SEQ ID NO: 10: Pseudomonas A14 GGT nucleic acid sequence SEQ ID NO: 11: Rat GGT amino acid sequence SEQ ID NO: 12: Rat GGT nucleic acid sequence SEQ ID NO: 13: Mouse GGT amino acid sequence SEQ ID NO: 14: Mouse GGT nucleic acid sequence SEQ ID NO: 15 Pig GGT amino acid sequence SEQ ID NO: 16: Pig GGT nucleic acid sequence SEQ ID NO: 17: Human GGT1 amino acid sequence SEQ ID NO: 18: Human GGT1 nucleic acid sequence SEQ ID NO: 19: Pseudo monas sp. SE83 cephalosporin acylase amino acid sequence SEQ ID NO: 20: Pseudomonas sp. SE83 cephalosporin acylase nucleic acid sequence SEQ ID NO: 21: Pseudomonas sp. V22 cephalosporin acylase amino acid sequence SEQ ID NO: 22: Pseudomonas sp. V22 cephalosporin acylase nucleic acid sequence SEQ ID NO: 23: Nucleic acid sequence of primer used for obtaining mutant in Example 1 SEQ ID NO: 24: Partial amino acid sequence of Escherichia coli K-12 GGT shown in FIG. 2 SEQ ID NO: 25: Bacillus shown in FIG. Partial amino acid sequence of subtilis 168 GGT SEQ ID NO: 26: shown in FIG. 2 Partial amino acid sequence of Bacillus natto GGT SEQ ID NO: 27: Partial amino acid sequence of Helicobacter pylori 26695 GGT shown in FIG. 2 SEQ ID NO: 28: shown in FIG. Pseudomonas A14 GGT partial amino acid sequence SEQ ID NO: 29: shown in FIG. 2, Rat GGT partial amino acid sequence SEQ ID NO: 30: shown in FIG. Mouse GGT partial amino acid sequence SEQ ID NO: 31: shown in FIG. 2, Pig GGT partial amino acid sequence SEQ ID NO: 32: shown in FIG. 2, Human GGT1 partial amino acid sequence SEQ ID NO: 33: Pseudomonas shown in FIG. sp. SE83 cephalosporin acylase partial amino acid sequence SEQ ID NO: 34: partial amino acid sequence of Pseudomonas sp. V22 cephalosporin acylase shown in FIG.
Example 1

Method for obtaining mutant strains
1. Introduction of D433N mutation into wild type GGT First, a synthetic oligonucleotide 5′-AACCAGATGGAT A ^* AT TTCTCCCGCC-3 ′ (A ^* is designed to change Asp-433 to Asn-433 in the amino acid sequence of GGT. It is G in wild type GGT) (SEQ ID NO: 23) as a primer and pSH1248 (pSH253 (JO Claudio, H. Suzuki, H. Kumagai, and T. Tochikura) on the HindIII site of pTZ18R introduced with deoxyuracil. Excretion and rapid purification of γ-glutamyltranspeptidase from Escherichia coli K-12. J. Ferment. Bioeng., 72, 125-127 (1991))) with about 0.9 kb of HindIII-HindIII fragment) Kunkel method (TA Kunkel. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. US A, 82, 488-492 (1985).) And the DH5α strain was transformed. A plasmid was extracted from the obtained strain, DNA sequencing was performed, and it was confirmed that the target mutation was contained in two strains. One of the plasmids was designated as pCM2. An approximately 0.9 kb HindIII-HindIII fragment of pCM2 and a 5.4 kb fragment obtained by cleaving pSH253 with HindIII were ligated to obtain pCM3.

The 3.2 kb SmaI-SphI fragment of pCM3 was ligated into pBR322 cut with EcoRV and SphI. The plasmid pCM7 obtained by transforming the GGT-deficient strain SH641 was designated as CM9 (D433N) strain (H. Suzuki, H. Kumagai, T. Echigo, and T. Tochikura. Molecular cloning of Escherichia coli K-12). ggt and rapid isolation of γ-glutamyltranspeptidase. Biochem. Biophys. Res. Commun., 150, 33-38 (1988)).
Example 2

Enzyme Purification Wild type and mutant type (D433N) enzymes were purified from SH642 and CM9 strains according to the methods already reported (H. Suzuki, H. Kumagai, T. Echigo, and T. Tochikura. Molecular cloning). of Escherichia coli K-12 ggt and rapid isolation of γ-glutamyltranspeptidase. Biochem. Biophys. Res. Commun., 150, 33-38 (1988)).
Example 3

Hydrolysis activity of D433N using GL-7-ACA as a substrate
1. Synthesis Method of Substrate GL-7-ACA In carrying out the synthesis method of substrate GL-7-ACA, Y. Shibuya, K. Matsumoto, and T. Fujii. Isolation and properties of 7β- (4-carboxybutanamido) cephalosporanic acid acylase- See producing bacteria. Agric. Biol. Chem., 45, 1561-1567 (1981). The composition of the reaction solution is as shown in Table 1 below.

Ａ液をＢ液に加え、ＮａＯＨでｐＨを９．０に保ちながら、室温で３時間混合した。ロータリーエバポレーターでアセトンを蒸発させ、濃縮した。濃塩酸でｐＨを１．５に調整後、酢酸エチルを加え、分液ろ斗を用いて、酢酸エチル層を抽出した。水層に残った分を再び酢酸エチルで抽出した。ロータリーエバポレ−ターで酢酸エチルを蒸発させ、乾燥させた。再び酢酸エチルを加えて溶解し、ろ過した。ろ液をロータリーエバポレーターで乾燥させた。

Liquid A was added to liquid B and mixed at room temperature for 3 hours while maintaining the pH at 9.0 with NaOH. Acetone was evaporated on a rotary evaporator and concentrated. After adjusting the pH to 1.5 with concentrated hydrochloric acid, ethyl acetate was added, and the ethyl acetate layer was extracted using a separatory funnel. The portion remaining in the aqueous layer was extracted again with ethyl acetate. The ethyl acetate was evaporated on a rotary evaporator and dried. Ethyl acetate was added again to dissolve and filtered. The filtrate was dried on a rotary evaporator.

By this method, GL-7-ACA was obtained with a yield of 80%. The obtained GL-7-ACA preparation was confirmed to be GL-7-ACA by ¹ H-NMR.

2. Enzymatic reaction (hydrolysis reaction) conditions when GL-7-ACA was used as a substrate The reaction solution composition was 50 mM Tris-HCl buffer (pH 8.73), 0.2-2 mM GL-7-ACA, The reaction was performed at 37 ° C. using 0.04-1 mg / ml D433N enzyme. The reaction was started by adding the enzyme solution to the reaction solution. Sampling was performed over time (200 μl), the reaction was stopped by adding an equal amount of 3.5 N CH ₃ COOH and vortexing, and the mixture was filtered through a membrane filter.

3. Measurement Method of GL-7-ACA and 7-ACA The concentrations of GL-7-ACA and 7-ACA in the reaction solution were measured by reverse phase HPLC. A 50 μl sample was usually injected into an HPLC (Shimadzu Corporation, LC-10) equipped with an Inersil ODS-3 column (GL Sciences, 4.6 × 250 mm), and the absorbance at 280 nm was measured. Mobile phase A was 0.05% aqueous trifluoroacetic acid, and mobile phase B was acetonitrile containing 0.05% trifluoroacetic acid. The proportion of B is 0 to 50% in 0 to 25 minutes (linear gradient), the proportion of B is 50% in 25 to 34 minutes, the proportion of B is 50 to 0% (linear gradient) in 34 to 35 minutes, 35 to 35 minutes For 50 minutes, B was eluted at a rate of 0% and a flow rate of 1 ml / min.

For calculation of elution position and concentration of GL-7-ACA and 7-ACA, GL-7-ACA produced as described above and commercially available 7-ACA (Aldrich Chem. Co.) were used as standard products.
Example 4

HPLC Chart of Enzyme Reaction Solution HPLC chart of enzyme reaction solution is shown in FIG.
Example 5

Optimum pH of reaction (hydrolysis reaction) using GL-7-ACA as a substrate
The reaction solution composition was 50 mM buffer solution, 2 mM GL-7-ACA, 1.04 mg / ml enzyme, and the reaction was performed at 37 ° C. for 3 hours. The reaction was stopped by boiling at 100 ° C. for 1 minute, and then filtered through a membrane filter. The result of examining the optimum pH of the hydrolysis reaction is shown in FIG.
Example 6

1. Confirmation that the product produced from GL-7-ACA by reaction with D433N is 7-ACA Up to this point, the hydrolysis reaction from GL-7-ACA to 7-ACA by D433N is a retention in reverse phase HPLC. It has been carried out on the assumption that the peak whose time coincides with the commercially available 7-ACA is that of 7-ACA. However, it was not confirmed whether this peak was 7-ACA. Therefore, a large amount of those that showed a retention time consistent with commercially available 7-ACA was purified in large quantities, and was confirmed to be 7-ACA by NMR analysis, and that 7-ACA was produced from GL-7-ACA by D433N. did.

Reaction conditions 50 mM Tris-HCl buffer (pH 8.73), 2 mM GL-7-ACA, 30.9 mu / ml (with hydrolytic activity when γ-GpNA is used as a substrate) D433N enzyme, total amount Reaction was performed at 730 ml at 37 ° C.

Purification conditions for 7-ACA A 7-ACA DOWEX 1X8 column (30 ml column) was used.

<Preparation of column>
2N NaOH was flowed 5 times the column volume. Next, distilled water was flowed 5 times the column volume (until the pH of the effluent from the column was equal to the pH of the water). Further, 0.5N CH ₃ COOH was flowed 5 times the column volume. Distilled water was then flowed 5 times the column volume (until the pH of the effluent from the column was equal to the pH of the water).

<Sample application>
A sample was applied.

<Elution>
Distilled water, 0.01N CH ₃ COOH, 0.05N CH ₃ COOH, 0.1N CH ₃ COOH, 0.5N CH ₃ COOH, 1N CH ₃ COOH, 5N CH ₃ COOH in this order, 5 column volumes each Doubled the amount. 7-ACA was eluted with 0.5N CH ₃ COOH.

<Confirmation>
The elution location of the product was confirmed by the ninhydrin reaction. Specifically, 20 μl of each eluted fraction was spotted on a filter paper, dried, sprayed with a ninhydrin solution from above, dried, and then observed for ninhydrin reaction.

<Confirmation by HPLC>
The colored fraction was subjected to HPLC, and it was confirmed that it coincided with the elution position of 7-ACA as a commercial product. Yield 18.4 mg.

The fraction of the product (7-ACA) eluted by the lyophilized DOWEX 1X8 column was frozen in a deep freezer at -80 ° C. and then dried by a lyophilizer.

Purification by HPLC Lyophilized samples were dissolved in distilled water and purified by reverse phase HPLC. The column used is COSMOSIL Code No. 38150-41 Size 20 × 250 mm, and the injection amount is 500 μl. The 7-ACA fraction was lyophilized.

NMR Results The NMR results are shown in FIG. From this result, it was confirmed that the purified product was 7-ACA.

2. Specific activity, Km value, kcat value Km value when GL-7-ACA of D433N was used as a substrate was determined. The results are shown below.
Km: 0.108 (mM)
Examples 7-25

  According to Examples 1 to 6, the modified GGT described in FIG. 6 is prepared, and its GL-7-ACA acylase activity is measured. The modified GGT produced in this way can have GL-7-ACA acylase activity, like E. coli GGT (D433N).

  According to the present invention, γ-7-ACA acylase activity is enhanced by substituting an amino acid residue of GGT using γ-glutamyltranspeptidase (GGT), which is an enzyme widely present in the living world, as a raw material. It has become possible to produce peptides (ie modified GGT). Remarkably, the modified GGT of the present invention has a low Km value and excellent substrate specificity for GL-7-ACA.

  Further, according to the present invention, an efficient enzyme method for 7-ACA is provided, which is safer, simpler, lower cost, shorter in time and more environmentally friendly than conventional chemical methods, and is a cephalosporin (cephem) antibiotic. It became possible to produce 7-ACA, which is a raw material of Since cephalosporins (cephems) antibiotics are one of the drugs with great demand, the benefits of the present invention to the pharmaceutical industry are great.

  In addition, the present invention provides a new source of GL-7-ACA acylase using an enzyme widely distributed in the biological world. Based on the present invention, GGTs of a wide variety of biological species will be used in the future. The research on the enzymatic production method of antibiotics will be actively conducted.

Claims (10)

In γ-glutamyl transpeptidase (GGT):
1) an amino acid residue that interacts with a γ-glutamyl compound, or 2) an amino acid residue in the region around the active center,
Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase activity is enhanced, wherein one or more amino acid residues selected from the group consisting of: Method for preparing modified GGT.   In GGT, residues 50-78, 93-98, 114-125, 147-188, and 209-347 of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 In a region corresponding to one or more regions selected from the group, residues 391 to 434, residues 452 to 487, and residues 508 to 569, other one or more amino acid residues The method according to claim 1, wherein the amino acid residue is substituted with the amino acid residue.   In GGT, the 16th, 94th, 98th, 114th, 115th, 209th, 209th, and 227th residues of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 Group, 242 residue, 263 residue, 334 residue, 342 residue, 433 residue, 452 residue, 460 residue, 461 residue, 462 residue, 468 residue The method according to claim 1, characterized in that one or more amino acid residues selected from the group, residue 484 are substituted with other amino acid residues.   In GGT, the amino acid residue corresponding to the 433th residue of the amino acid sequence of GGT derived from E. coli represented by SEQ ID NO: 1 is substituted with another amino acid residue. The method of crab.   The method according to claim 4, wherein the other amino acid residue is at least one amino acid residue selected from the group consisting of asparagine, glutamine, tyrosine, tryptophan, and phenylalanine.   6. The method of claim 5, wherein the other amino acid residue is asparagine.   A modified GGT obtained by the method according to claim 1.   A nucleic acid molecule encoding the modified GGT of claim 7.   A cell expressing the modified GGT according to claim 7.   A method for producing 7-aminocephalosporanic acid (7-ACA), comprising a step of treating GL-7-ACA with the modified GGT according to claim 7.

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