# Patent

## Title
Method of producing microbial transglutaminase

## Abstract

The present invention provides a neutral metalloprotease from actinomycetes which selectively cleaves a pro-structure part of a microbial protransglutaminase and a gene encoding said neutral metalloprotease. An active microbial transglutaminase having the pro-structure part cleaved can be obtained by culturing a microorganism into which a gene encoding the neutral metalloprotease from actinomycetes according to the present invention has been introduced, where by producing the neutral metalloprotease from actinomycetes, and reacting it on a microbial protransglutaminase.

## Background
This application is a continuation under 35 U.S.C. §120 of PCT/JP2004/002923, filed Mar. 5, 2004, the entirety of which is incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protease which efficiently cleaves the pro-structure part of pro-transglutaminase resulting in an active transglutaminase, and to a nucleic acid encoding the protease, wherein the pro-transglutaminase is produced by actinomycetes. The present invention also relates to a method of producing microbial transglutaminase in its active form using the protease. Additionally, the present invention relates to a method of producing a neutral metalloprotease.
2. Brief Description of the Related Art
Transglutaminase is an enzyme which catalyzes the acyltransfer reaction of γ-carboxylamide groups in the peptide chain of the protein. When the enzyme reacts with a protein, the formation of the cross-linkage ε-(γ-Glu)-Lys, and the replacement of Gln with Glu by deamidation can occur. Transglutaminases have been used to manufacture gelled food products such as jelly, yogurt, cheese, or gelled cosmetics and others, and to improve the quality of meat, etc. (Japanese publication of examined application (JP-Kokoku) No. 1-50382). Moreover, transglutaminase is highly useful in industry in that it has been used to manufacture materials for thermostable microcapsules, carriers for immobilized enzyme, etc.
Expression of animal transglutaminase activity is calcium-dependent, and transglutaminases from microorganisms (microbial transglutaminase(s), which is/are also referred to as “MTG(s)” hereinafter), have been previously reported to also be calcium-independent. A microbial transglutaminase from a bacterium belonging to genus Streptoverticillium has been reported. Such Streptoverticillium bacteria include, for example, Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852, Streptoverticillium mobaraense (hereinafter, S. mobaraense) IFO 13819, and others (Publication of unexamined Japanese patent application (JP-Kokai) No. 64-27471).
Because these transglutaminases, however, have been produced via purification from cultures of the microorganisms described above, problems have been reported regarding the produced amount, production efficiency, and the like. Then, in an attempt to more efficiently secrete heterologous proteins, a method was reported using a coryneform bacterium as a host, and a fused protein whereby transglutaminase was connected downstream of the signal peptide domain of the coryneform bacterium, and the transglutaminase was efficiently secreted resulting in a high yield of transglutaminase (WO 01/23591). In this study, a method is also described wherein inactive MTG is secreted as pro-transglutaminase (referred to as “pro-MTG” hereinafter) whereby a pro-structure part is connected to MTG, and then this pro-structure part is cleaved by a protease to convert it into an active transglutaminase. A further method is described wherein an active transglutaminase is directly produced in a culture medium by co-expressing SAM-P45, which is a serine protease derived from actinomycetes, in a sufficient amount in a coryneform bacterium which also produces the pro-MTG.
Although a method in which an active transglutaminase is directly produced by co-expressing pro-MTG and a protease which allows cleavage of the pro-structure part of the pro-MTG in a coryneform bacterium is assumed to be an extremely efficient method of producing transglutaminase, the substrate specificity of SAM-P45 is not very strict, and it may digest and degrade not only the pro-structure part of the pro-MTG but also the transglutaminase itself to some degree. Therefore, the handling of SAM-P45 may not be easy. When SAM-P45 is used, therefore, the production method of transglutaminase should be strictly controlled such that degradation of the transglutaminase in the culture medium will not occur.
There is still, therefore, demand for a protease which can selectively cleave only the pro-structure part of pro-MTG, with as little degradation of the transglutaminase itself as possible during the production of an active transglutaminase.
A dispase derived from Bacillus polymyxa is known (Eur. J. Biochem., vol. 257, p. 570-576 (1998)) to be an enzyme besides SAM-P45 which cleaves the pro-structure part of pro-MTG. A large amount of the enzyme, however, is required to cleave the pro-structure part, and there is a risk of degrading the transglutaminase itself. In addition, dispase is a reagent in cell culture, so it is expensive when used an enzyme for industrial use.
SUMMARY OF THE INVENTION
There remains a need for a protease which can selectively cleave solely the pro-structure part of pro-MTG, and degrade the transglutaminase itself as little as possible during the production of an active transglutaminase, as mentioned above. Additionally, if proteases which selectively cleave only the pro-structure part of pro-MTG could be used, and thereby cause as little degradation as possible of the transglutaminase itself, it would be advantageous for the production of an active transglutaminase. Furthermore, if proteases for production of transglutaminase which selectively cleave the pro-structure part of pro-MTG were able to be secreted extracellularly, it would be more preferable because active transglutaminases could be directly produced in the culture medium by co-expressing them with the pro-MTG.
It is an object of the present invention to provide a method of producing an active transglutaminase from a microbial protransglutaminase comprising contacting a neutral metalloprotease with the protransglutaminase, wherein said neutral metalloprotease is produced by culturing a microorganism into which a gene encoding the neutral metalloprotease from actinomycetes has been introduced, and recovering an active microbial transglutaminase.
It is a further object of the present invention to provide the method as described above, wherein said microorganism comprises a coryneform bacterium.
It is a further object of the present invention to provide the method as described above, wherein said neutral metalloprotease from actinomycetes comprises characteristics selected from the group consisting of a molecular weight of about 35,000, an optimum pH of pH7.0, stablity at pH of pH4-10, an optimum temperature of about 45° C., stability below about 50° C., and said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, and Streptomyces subtilisin inhibitor (SSI) from actinomycetes.
It is a further object of the present invention to provide the method as described above, wherein said neutral metalloprotease from actinomycetes comprises characteristics selected from the group consisting of a molecular weight of about 71,000, an optimum pH of 7.0, stablity at pH of 5-10, an optimum temperature of about 55° C., and said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, dithiothreitol, and Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes.
It is a further object of the present invention to provide a neutral metalloprotease from actinomycetes comprising characteristics selected from the group consisting of a molecular weight of about 35,000, an optimum pH of pH7.0, stablity at pH of pH4-10, an optimum temperature of about 45° C., stability below about 50° C., and said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, and Streptomyces subtilisin inhibitor (SSI) from actinomycetes.
It is a further object of the present invention to provide a neutral metalloprotease from actinomycetes comprising characteristics selected from the group consisting of a molecular weight of about 71,000, an optimum pH of 7.0, stablity at pH of 5-10, an optimum temperature of about 55° C., and said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, dithiothreitol, and Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes.
It is a further object of the present invention to provide a nucleic acid molecule encoding said protease described above.
It is a further object of the present invention to provide a method of producing said protease comprising culturing a coryneform bacterium into which the nucleic acid molecule as described above has been introduced, and recovering said neutral metalloprotease which has been secreted extracellularly.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph which shows the pH dependence of SVP35 and SVP70 activity.
FIG. 2 is a graph which shows the pH stability of SVP35 and SVP70.
FIG. 3 is a graph which shows the temperature dependence of SVP35 and SVP70 activity.
FIG. 4 is a graph which shows the temperature stability of SVP35.
FIG. 5 depicts the inhibitory activities of various compounds to SVP35 and SVP70 activity.
FIGS. 6 (A), (B), and (C) depict the sequential change of the conversion of pro-MTG to an active MTG by SVP35 (A), and SVP70 (B) from Streptoverticillium mobaraense, and by neutral metalloprotease SGMPII (C) from Streptomyces griseus, relative to the change in the protein amount. The lanes represents the time elapsed, as indicated by the “(h)” notation in the figure panels.
FIGS. 7 (A) and (B) are graphs which depict the time course of transglutaminase activity, if a pro-MTG is reacted with SVP70 and SAM-P45, respectively. (A): SVP70 addition, ●: additional amount of 1/200 relative to substrate, ▪: additional amount of 1/500 relative to substrate; (B): SAM-P45 addition, ♦: additional amount of 1/10 relative to substrate, ▴: additional amount of 1/50 relative to substrate.
FIGS. 8 (A) and (B) are graphs which depict the time course of the amount of MTG protein, if a pro-MTG reacts with SVP70 and SAM-P45, respectively. (A): SVP70 addition, ●: additional amount of 1/200 relative to substrate, ▪: additional amount of 1/500 relative to substrate; (B): SAM-P45 addition, ♦: additional amount of 1/10 relative to substrate, ▴: additional amount of 1/50 relative to substrate.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention describes a protease which selectively cleaves the pro-structure part of pro-MTG, but degrades the transglutaminase itself as little as possible, and the isolatation and purification of a neutral metalloprotease having such a property. The present invention also describes a DNA encoding said protease, its introduction into a host coryneform bacterium, and the successful secretory expression. In addition, the described enzyme was actually reacted with the pro-MTG to cleave the pro-structure part, and the active transglutaminase was recovered. The present invention describes the identification of neutral metalloproteases derived from microorganisms from other sources which have an equivalent function, and which have been proven similarly useful for the production of an active MTG.
Namely, the present invention is a neutral metalloprotease from actinomycetes which has high selectivity in cleaving the pro-structure part of pro-MTG, and a nucleic acid molecule encoding it.
The present invention also encompasses a method of producing an active MTG by cleaving a pro-structure part of a pro-MTG with a neutral metalloprotease.
The present invention also encompasses a method of producing said metalloprotease by introducing a nucleic acid molecule which encodes said neutral metalloprotease into a coryneform bacterium, culturing this coryneform bacterium, thereby allowing the expression of said neutral metalloprotease, and recovering the extracellularly secreted metalloprotease.
More specifically, the present invention encompasses a neutral metalloprotease SVP35 from actinomycetes having the following properties:
1) Molecular weight: about 35,000 (as measured by SDS-PAGE)
2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around 7.0
3) pH Stability: pH of 4-10
4) Optimum temperature: about 45° C.
5) Temperature stability: it is stable below about 50° C.
6) It is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, and phosphoramidon which are metalloprotease inhibitors, and by Streptomyces subtilisin inhibitor (SSI) from actinomycetes.
The present invention also encompasses a neutral metalloprotease SVP70 having the following properties:
1) Molecular weight: about 71,000 (as measured by SDS-PAGE)
2) Optimum pH: the range of 6.0-8.0, more specifically 6.5-7.5, in particular around 7.0
3) pH Stability: pH of 5-10
4) Optimum temperature: the range of about 50° C.-55° C., in particular around 55° C.
5) It undergoes a strong inhibitory action by ethylene diamine tetraacetic acid, 1,10-phenanthroline, and phosphoramidon which are metalloprotease inhibitors, dithiothreitol which is a SH-reductant, and by Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes.
The present invention also encompasses a nucleic acid molecule encoding said SVP35 or SVP70.
The present invention also encompasses a method of producing an active MTG, comprising cleaving the pro-structure part of pro-MTG by said SVP35 or SVP70.
Furthermore, the present invention is a method of producing SVP35 or SVP70, comprising introducing a nucleic acid molecule encoding said SVP35 or SVP70 into a coryneform bacterium, culturing the coryneform bacterium into which said nucleic acid molecule has been introduced, and recovering the extracellularly secreted SVP35 or SVP70.
In general, it is known that a secretory protein is translated as a prepeptide or a prepropeptide and thereafter its signal peptide (the pre-part) is cleaved resulting in a mature peptide or propeptide. The propeptide is then cleaved at the domain referred to as a pro-structure, resulting in a mature peptide. As used herein, the pro-structure part of a secretory protein may be simply referred to as “pro-structure”. In addition, as used herein, “a signal sequence” refers to the sequence which is located at the N-terminus of a secretory protein precursor and which is not present in a naturally occurring mature protein. The phrase “a signal peptide” refers to the peptide which is cleaved from the protein precursor. Generally, a signal sequence is cleaved by a protease following extracellular secretion.
As used herein, a protein which does not contain a signal peptide but does contain a pro-structure part may be referred to as a “proprotein”, for example “protransglutaminase” or “pro-MTG”. As used herein, the pro-structure part of a secretory protein may be simply referred to as “a pro-structure” or “a pro-structure part”, and these terms can herein be used interchangeably.
Among proteases which are assumed to be easily expressed in a coryneform bacterium, a protease having high specificity and selectivity for the substrate of interest was sought, i.e. a protease which selectively cleaves the pro-structure part of a pro-MTG, with as little degradation as possible of the transglutaminase itself.
When MTG is secreted extracellularly by an actinomycetes, it had been assumed to be secreted first as a pro-MTG, followed by the cleavage of the pro-structure part pro-MTG, resulting in an active MTG (Eur. J. Biochem., vol. 257, p. 570-576 (1998)). Accordingly, a protease that cleaves the pro-structure part of a pro-MTG within MTG-producing actinomycetes was expected to be present. Since this protease is originally a protease that cleaves the pro-structure part, it is expected that the protease has a high selectivity for substrates and cleaves only the pro-structure part, while acting on the MTG itself to a lesser degree.
In addition, both a structural gene of a pro-MTG of actinomycetes and a structural gene of the protease SAM-P45 can be effectively expressed in a coryneform bacterium, and secreted extracellularly. Based on this information, an investigation was conducted in order to find the protease of interest from a MTG-producing bacterium which is an actinomycetes, and as a result, it was revealed that the MTG-producing strain Streptoverticillium mobaraense had high cleavage selectivity for the pro-structure part of the pro-MTG and produces new neutral metalloproteases useful for the production of an active MTG. These neutral metalloproteases were isolated and purified, and their enzymological properties were demonstrated. Furthermore, the amino acid sequences of the N-terminal parts of these metalloproteases were determined, and the genes encoding the metalloproteases were obtained.
In addition, the enzyme gene was introduced into a coryneform bacterium, allowing the expression of it in a system using a coryneform bacterium as a host, and as a result, the enzyme was secreted extracellularly. Furthermore, the enzyme was practically contacted with a pro-MTG of a pro-structure part, resulting in the cleavage of the pro-structure part to yield an active transglutaminase. Neutral metalloproteases from microorganisms from other sources having an equivalent function were also found, and which have been found to be similarly useful for the production of an active MTG.
More specific embodiments of the present invention will be illustrated hereinafter.
The neutral metalloproteases according to the present invention can be prepared from the surfaces of a cultured actinomycetes or culture supernatant of the actinomycetes, including Streptoverticillium mobaraense, Streptomyces griseus, Streptomyces coelicolor, etc.
Next, the newly found neutral metalloproteases of Streptoverticillium mobaraense IFO13819 are described.
The cultivation of a bacterium to obtain the neutral metalloprotease according to the present invention, for example, an actinomycetes as described above, can be carried out according to the methods conventionally used for the cultivation of actinomycetes. Namely, a common medium containing conventional carbon sources, nitrogen sources, inorganic ions, and others can be used as a medium for the culture. Glucose, starch, sucrose, and others can be used as the carbon sources. Peptone, yeast extract, meat extract, malt extract, ammonium salt, and others are optionally used as the nitrogen sources, if necessary. The culture may be conducted under aerobic conditions which are appropriately controlled within the pH range of between pH 5.0 and 8.5 and the temperature range between 15° C. and 37° C. For the production of the neutral metalloproteases according to the present invention, the culture is preferably continued so that the maximum amount of the desired neutral metalloprotease may be achieved, and then it can be terminated. Although the suitable culture period depends on the temperature, pH, and the type of medium, usually the period is preferably about 1 to 12 days. After the culturing period, the culture may be separated into the cells and the culture supernatant by centrifugation and the like.
The new neutral metalloproteases according to the present invention can be obtained from the culture supernatant and/or the recovered cells, in particular, from the surface of the cells. To purify the enzyme, methods which are conventionally used for purifying an enzyme, for example, an ammonium sulfate salting-out technique, gel filtration technique, ion-exchange chromatography, hydrophobic chromatography, and the like can be adopted. The protease can be purified more efficiently using high performance liquid chromatography (HPLC) etc. The enzyme activity of the neutral metalloprotease obtained in this way can be determined by reacting the enzyme with a peptide which contains a region connecting the pro-part of a protransglutaminase and a mature transglutaminase, for example, a synthetic peptide Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) (Peptide Institute) as a substrate and the reduced amount of the substrate can be calculated.
As mentioned above, the neutral metalloprotease according to the invention purified from the recovered cells, in particular from the surface of the cells, or from the supernatant of the culture, can be analyzed for the N-terminal amino acid sequence by a gas phase protein sequencer to determine the partial amino acid sequence. Furthermore, the enzymatic properties (optimum pH, pH stability, optimum temperature, the effect of an inhibitor, etc.) of the isolated and purified neutral metalloprotease can be examined.
In one embodiment of the present invention, the neutral metalloprotease SVP35 was obtained from the surface of the cells of Streptoverticillium mobaraense and the neutral metalloprotease SVP70 can be obtained from the culture supernatant of Streptoverticillium mobaraense. 
In one embodiment of the present invention, the neutral metalloprotease according to the invention is the neutral metalloprotease SVP35 having the following properties:
1) Molecular weight: about 35,000 (as measured by SDS-PAGE)
2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around 7.0
3) pH Stability: pH of 4-10
4) Optimum temperature: about 45° C.
5) Temperature stability: it is stable below about 50° C.
6) Inhibitors: it is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline and phosphoramidon which are metalloprotease inhibitors, and by Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes.
In another embodiment of the present invention, the neutral metalloprotease according to the present invention is the neutral metalloprotease SVP70 having the following properties:
1) Molecular weight: about 71,000 (as measured by SDS-PAGE)
2) Optimum pH: 6.0-8.0, more specifically 6.5-7.5, in particular around 7.0
3) pH Stability: pH of 5-10
4) Optimum temperature: about 50° C.-55° C., in particular around 55° C.
5) Inhibitors: it is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline and phosphoramidon which are metalloprotease inhibitors, dithiothreitol which is a SH-reductant, and by Streptomyces subtilisin inhibitor (SSI) from actinomycetes.
When SVP35 or SVP70 is contacted with pro-MTG, both of them show highly selective cleavage activity on the pro-structure part of the MTG. Namely, since both of the enzymes are characterized by converting the pro-MTG into the active MTG efficiently, while the activity for degrading the resulting active MTG itself is low, both of them are suitable enzymes for producing an active MTG using pro-MTG as a raw material. The N-terminal amino acid sequences of the two new neutral metalloproteases are shown in SEQ ID NO: 1 for SVP35, and in SEQ ID NO: 2 for SVP70, which reveals the homology between these sequences. Therefore, sequences having any homology with these proteases in their N-terminal amino acid sequences were searched and a metalloprotease SGMP II (J. Biochem. Vol. 110, p. 339-344 (1991)) from Streptomyces griseus as well as the three metalloproteases (GenBank/EMBL/DDBJ CAB76000, CAB76001, CAB69762) from Streptomyces coelicolor, and the like were found. These proteases can also be used in the same manner as SVP35 and SVP70 for selective cleavage of the pro-structure part of a pro-MTG, and they can be used to produce an active MTG using a pro-MTG as the raw material.
Next, a method of producing the neutral metalloprotease according to the present invention by recombinant DNA technique will be described.
A number of examples of producing useful proteins including enzymes, physiologically active substances, and the like using recombinant DNA techniques have been known. The advantage of using recombinant DNA techniques is the ability to mass-produce useful proteins that exist in small quantities in nature.
To produce the neutral metalloprotease according to the present invention by using recombinant DNA techniques, a genetic construct is generated first which contains a promoter, a sequence encoding a proper signal peptide, a nucleic acid fragment encoding the neutral metalloprotease according to the invention, and a regulatory sequence (an operator or terminator, etc.) which is necessary to express the gene for the neutral metalloprotease in a coryneform bacterium, and properly positioned so that they can function. The neutral metalloprotease according to the invention may have a pro-structure part at the N-terminal. Vectors, which can be used for this construct, are not particularly limited and include any one which can function in a coryneform bacterium, and may be those which autonomously replicate such as plasmids or which are integrated into the chromosome of the bacterium. When a coryneform bacterium is used as a host, plasmids derived from coryneform bacteria are particularly preferable as vectors. These include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)), pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)), and modified plasmids, which possess drug-resistant genes.
Examples of Corynebacterium which can be used as a host bacterium in the present invention include mutant strains derived from wild-type strains including Brevibacterium saccharolyticum ATCC14066, Brevibacterium immariophilum ATCC14068, Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium flavum (Corynebacterium glutamicum) ATCC14067, Corynebacterium acetoacidophilum ATCC13870, Corynebacterium glutamicum ATCC13032, Corynebacterium lilium (Corynebacterium glutamicum) ATCC15990, Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC6871 and the like, or mutant strains derived from mutants strain of these wild-types.
Mutant strains which can be used in the present invention include, for example, mutant strains defective in the ability to produce glutamate, mutant strains for amino acid production, such as lysine and the like, and mutant strains modified to produce other substances such as nucleic acids, for example, inosine. Such mutant strains can be obtained by treatment with ultraviolet irradiation or a chemical mutagen such as N-methyl-N′-nitrosoguanidine and the like, and then selecting the strains which have an increased ability to secreto-produce proteins.
Especially, Corynebacterium glutamicum AJ1203 (FERM BP-734) (originally deposited on Mar. 26, 1984) (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), was isolated from the wild-type Corynebacterium glutamicum (C. glutamicum) ATCC13869 as a streptomycin-resistant mutant strain. This strain is expected to have a mutation in a functional gene associated with protein secretion, and its ability to secreto-produce heterologous proteins is extremely high—approximately 2- or 3-fold as compared with the parent (wild-type) strain—under optimum culture conditions. Therefore, this strain is suitable as a host bacterium (see WO 02/081694). In addition, it is preferable to use a strain which was obtained by modifying a host strain such that the strain no longer produces the cell surface protein, because the purification of the heterologous proteins secreted in the medium will be easier, so it is particularly preferable. Such a modification can be performed by introducing a mutation into the cell surface protein gene on the chromosome or into its expression regulatory region through mutagenesis or gene recombination techniques.
Examples of promoters from a coryneform bacterium include promoters for the genes of the cell surface proteins PS1, PS2, and SlpA, promoters for the genes in biosynthetic systems of various amino acids, for example, glutamine synthetase gene, aspartokinase gene in the lysine biosynthetic system, and the like.
The signal peptide which is used in the present invention is the signal peptide for a secretory protein from coryneform bacterium, the host, and preferably it is the signal peptide of a cell surface protein from a coryneform bacterium. The cell surface proteins of coryneform bacteria include PS1 and PS2 from C. glutamicum (JP-Kokai No. 6-502548), and SlpA from C. ammoniagenes (JP-Kokai No. 10-108675).
To produce a neutral metalloprotease whose activity for selective cleavage of a pro-structure of a pro-MTG is strong by using recombinant DNA techniques, a DNA encoding such a neutral metalloprotease is required.
In one embodiment of the present invention, the neutral metalloprotease SVP35 is produced by using recombinant DNA techniques. The DNA encoding SVP35 can be obtained as follows.
First, the amino acid sequence of the purified SVP35 is determined. The Edman method (Edman, P., Acta Chem. Scand. 4, 227 (1950)) can be used to determine the amino acid sequence. The gas-phase protein sequencer from Shimadzu Co. Ltd. Co. Ltd. and the like can also be used to determine the amino acid sequence.
For the neutral metalloprotease SVC35 according to the present invention, the sequence shown in SEQ ID NO: 1 has been found by sequencing 20 amino acid residues from the N-terminus.
This information can be used to synthesize an appropriate primer for PCR and generate a probe to obtain the neutral metalloprotease according to the present invention. For example, a protease gene from actinomycetes, which is expected to have a homology based on the search results for the homology in the N-terminal amino acid sequence, for example, a metalloprotease (GenBank/EMBL/DDBJ CAB76001) gene from Streptomyces coelicolor can be subjected to PCR using an actinomycetes DNA prepared by the method of Saito and Miura [Biochem. Biophys. Acta, 72, 619 (1963)] as a template, to amplify the fragment of the gene encoding this protease. The amplified fragment can be used as a probe.
Then, the actinomycetes DNA prepared by the method of Saito and Miura, for example, the chromosomal DNA of Streptoverticillium mobaraense IFO13819, is digested with appropriate different restriction enzymes, for example various restriction enzymes which recognize 6-base sequences. The digested actinomycetes chromosomal DNA can be analyzed by techniques well known to those skilled in the art such as the Southern blot hybridization technique described in Molecular Cloning 2nd edition [J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p9. 31 (1989)] and the like by using the 32P-labeled PCR-product obtained by the above-described PCR. For example, the nucleic acid molecule encoding the neutral metalloprotease according to the present invention or the part thereof can be cloned by recovering the fragment which has been confirmed by Southern blot to have a high homology with the chosen probe, and cloning it into an appropriate vector. The techniques necessary for such a gene cloning are well known to those skilled in the art (see for example, J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p1. 90 (1989)).
In one embodiment of the present invention, PCR is performed using the chromosomal DNA of Streptomyces coelicolor A3(2) as a template to produce a probe. Furthermore, a single band of about 8 kb which is able to hybridize with 32P-labeled probes detected in the digested product of Streptoverticillium mobaraense IFO13819 chromosomal DNA digested with SphI. Thus, the chromosomal DNA of Streptoverticillium mobaraense IFO13819 prepared by the foregoing method is digested with SphI, the fragment of about 8 kb is recovered through an agarose gel electrophoresis, the recovered fragment is introduced at the SphI site in pUC18, and then it is introduced into a competent cell of Escherichia coli JM109 to generate a library. The clones of interest can be obtained by screening the generated library using a synthetic oligonucleotide as a probe according to colony hybridization techniques described in Molecular Cloning 2nd edition (supra), and selecting the strain which harbors the plasmid containing the gene fragment of SVP35 cloned into the plasmid. The plasmid recovered from this strain is herein designated pVSV1. The nucleotide sequence of the fragment cloned into pVSV1 is analyzed, the primary amino acid sequence is deduced to confirm that the fragment encodes the previously determined N-terminal amino acid sequence. Thus, the obtained gene is confirmed to be the gene encoding SVP35.
Then, a recombinant nucleic acid molecule can be constructed to express the neutral metalloprotease according to the present invention by ligating a genetic construct containing the DNA encoding the obtained metalloprotease to an appropriate vector depending on the properties of the chosen host. The host coryneform bacterium cells are transformed with the recombinant nucleic acid molecule. The transformed cells can be cultured in a suitable medium to recover the neutral metalloprotease according to the present invention which is secreted and/or accumulates in the medium and/or in the cell.
Next, a method of producing an active MTG from pro-MTG using the neutral metalloprotease will be described.
The neutral metalloprotease used in the production of an active MTG can be reacted with a pro-MTG as a fraction containing the neutral metalloprotease prepared from the culture medium of a neutral metalloprotease producing bacterium. It can be also used as a more highly purified neutral metalloprotease with high specific activity. Furthermore, as described below, the neutral metalloprotease can be also used, wherein the neutral metalloprotease can be obtained by culturing the cell transformed with a recombinant nucleic acid molecule which may be obtained by connecting a DNA encoding a neutral metalloprotease having strong selective cleavage activity for a pro-structure part of pro-MTG.
The pro-MTG used to produce MTG may be a fraction containing the pro-MTG prepared from the culture medium a pro-MTG producing bacterium. More highly purified pro-MTG may also be used. The reaction may be performed under conditions such that the amount of a neutral metalloprotease added to the pro-MTG is from 1/10 to 1/500 by weight and is appropriately adjusted within the reaction temperature of between 15° C. and 50° C. and the pH range of between pH 5.0 and 9.
In addition, the genetic construct, which is constructed as described above and which contains the DNA encoding the neutral metalloprotease according to the present invention, may be introduced into a microorganism containing the genetic construct encoding a pro-MTG, in particular, into a coryneform bacterium, to produce in a single bacterial cell both the pro-MTG and the neutral metalloprotease according to the present invention, thereby the pro-MTG may be converted into a mature MTG under the above conditions. A more detailed method for efficiently producing a pro-MTG in coryneform cells, the genetic construct used for such a method, and a coryneform bacterium into which the genetic construct has been introduced are disclosed in, for example WO 01/23591. More specifically, for example, a coryneform bacterium which can efficiently secrete a pro-MTG protein extracellularly may be obtained by introducing a genetic construct into a coryneform bacterium, wherein the genetic construct is obtained by connecting the sequence encoding a pro-MTG, which is located downstream of the sequence encoding the signal peptide domain of a coryneform bacterium, particularly the signal peptide domain of a cell surface protein, downstream of an appropriate promoter. The signal peptide, promoter, and host which can be used for this purpose can be selected from signal peptides, promoters, and hosts which are suitable for expressing the neutral metalloproteases according to the present invention and as mentioned above. A combination of vectors that are compatible in the same cell is also well known to those skilled in the art. Therefore, the mature MTG can be obtained by introducing an appropriate genetic expression construct containing the DNA encoding the neutral metalloprotease according to the present invention as mentioned above into a coryneform bacterium producing a pro-MTG, or vice versa, by introducing an appropriate genetic expression construct encoding a pro-MTG into coryneform bacterium producing the neutral metalloprotease according to the present invention, thereby allowing the genetic constructs which can express the pro-MTG and the neutral metalloprotease according to the present invention to coexist in the same bacterium, culturing the bacterium, and maintaining the culture under appropriate conditions such that the neutral metalloprotease according to the present invention is active.
The transglutaminase produced by the method according to the present method can be isolated and purified from the reaction mixture according to methods well known to those skilled in the art. For example, the transglutaminase can be isolated and purified by removing the cells from the mixture by centrifugation, etc. and then by using known appropriate methods such as salting-out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion-exchange column chromatography, affinity chromatography, medium high-pressure liquid chromatography, reversed-phase chromatography, hydrophobic chromatography, or a combination thereof.
The present invention is further described in the following non-limiting Examples.
EXAMPLES
Example 1
Neutral Metalloprotease Produced by Streptoverticillium mobaraense IFO13819
(1) Purification of Neutral Metalloprotease (SVP70) Produced by Streptoverticillium mobaraense IFO13819
800 mL of ISP2 culture medium (4 g of Yeast Extract, 10 g of Malt Extract, 4 g of Glucose per liter of water, adjusted to pH 7.3) was placed in a 5 L Sakaguchi flask (shaking flask), and was inoculated with Streptoverticillium mobaraense IFO13819 from a plate, and cultured by shaking at 30° C. for 9 days at 120 rpm. The culture medium was centrifuged, and the supernatant of the culture was collected. It was filtered using a Depth filter (3 μm of pore size, Sartorius Co. Ltd.), followed by concentration using Sartocon Slice membrane having a pore size of 10,000 Da (Saltorius Co. Ltd.). The concentrate was diluted 10-fold with Tris-HCl buffer/5 mM calcium chloride (pH 7.5), subjected to a DEAE-Sepharose FF (2.6φ×10 cm, Amersham Pharmacia Co. Ltd.) column equilibrated with the same buffer, using FPLC (Amersham Pharmacia Co. Ltd.), and eluted using a linear concentration gradient of 0-0.5 M sodium chloride. A fraction containing the active ingredient was collected, subjected to a phenyl Sepharose HP (1.6φ×10 cm, Amersham Pharmacia Co. Ltd.) column equilibrated with 1.5 M ammonium sulfate/20 mM MES buffer/5 mM calcium chloride (pH 6.0), eluted using linear concentration gradient of 1.5-0 M ammonium sulfate, and an active fraction was collected. The resulting active fraction was dialyzed against 20 mM MES buffer/5 mM calcium chloride (pH 6.0) at 4° C. overnight to obtain a purified enzyme solution.
The measurement of the enzyme activity at each step was carried as follows:
The enzyme solution was added to 20 mM sodium phosphate buffer containing peptide GPSFRAPDS (Peptide Institute) (SEQ ID NO: 11) to yield 170 μl of total liquid volume, and it was reacted at 30° C. for 10 minutes, followed by heating at 95° C. for 5 minutes to terminate the reaction. 80 μl of this solution was analyzed by HPLC under the following conditions and its activity was calculated based on the decreased amount of the substrate.
Apparatus: HPLC L-6300 system (Hitachi Co. Ltd).
Column: YMC-PACK ODS 120A 4.6×150 mm (YMC)
Eluent: (A) 0.1% TFA (B) 80% acetonitrile/0.1% TFA
Eluting condition: linear concentration gradient of 12-16% acetonitrile (for 15 minutes)
Flow rate: 1.0 ml/min
Detection wavelength: 220 nm
Under these conditions, the peptide GPSFRAPDS (SEQ ID NO: 11) was eluted for 13 to 14 minutes of retention time, and the degraded product FRAPDS (SEQ ID NO: 12) was eluted for 7.5 to 8.5 minutes of retention time.
The amount of the enzyme that catalyzes one (1) nmol of pro-MTG degradation in a minute was defined as one (1) unit of the enzyme activity.
(2) Purification of Neutral Metalloprotease (SVP35) Produced by Streptoverticillium mobaraense IFO13819
800 mL of ISP2 culture medium was placed in a 5 L of Sakaguchi flask and was inoculated with Streptoverticillium mobaraense IFO13819 from a plate, and cultured by shaking at 30° C. for 48 hours at 120 rpm. The culture medium was centrifuged, and the supernatant of the culture was discarded to harvest cells. The cells were suspended in 20 mM Tris-HCl buffer/30 mM sodium chloride (pH 7.5), shaken on ice for 4 hours, and then the supernatant was collected by centrifugation. The supernatant obtained was filtered and sterilized using a Depth filter (0.22 μm of pore size, made by Sartorius Co. Ltd.), and then it was subjected to a CM-Sepharose FF (Amersham Pharmacia Co. Ltd.) column (1.6φ×10 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride, using FPLC (Amersham Pharmacia Co. Ltd.), eluted in the same buffer using a linear concentration gradient of 0-0.5 M sodium chloride. A fraction containing the active ingredient was collected, and was further subjected to Phenyl-Sepharose HP column (1 mL, Amersham Pharmacia Co. Ltd.) equilibrated with 20 mM Tris-HCl buffer containing 1.5 M ammonium sulfate, 5 mM calcium chloride and 0.01 mM zinc chloride, and eluted using a linear concentration gradient of 1.5-0 M ammonium sulfate. An active fraction was collected, and demineralized by 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride, using PD-10 column (Amersham Pharmacia) to give a partially purified enzyme solution.
The enzyme activity at each step was measured using the peptide GPSFRAPDS as a substrate in the same manner as in (1).
(3) Evaluation of the Properties of the Neutral Metalloprotease (SVP35) Produced by Streptoverticillium mobaraense IFO13819
i) Substrate Specificity
1 mg/ml of insulin B solution and pro-MTG solution prepared in 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride was used as a substrate, and an enzyme solution was added to the solution to react at 30° C. for 2 hours, and then peptide fragments were separated by HPLC under the following conditions:
Apparatus: L-7100/7200/7405/D-7600 (Hitachi Co. Ltd.)
Column: VYDAC C18 4.6 mm I.D.×250 mm (VYDAC)
Eluent: (A) 0.1% TFA (B) 80% acetonitrile/0.1% TFA
Eluting condition: linear concentration gradient of 4-44% acetonitrile
Flow rate: 0.5 ml min
Detection wavelength: UV 220 nm
The amino acid sequences of the obtained peptide fragments were analyzed by PPSQ-10 (Shimadzu Co. Ltd.) to characterize the sequences of the cleavage points for SVP35. As a result, it was confirmed that the peptide was cleaved before (at the N-terminal of) especially Phe, often Leu, sometimes Tyr, Trp, Ile, Val, and that SVP recognized the aromatic amino acids and hydrophobic amino acids with bulky side-chains positioned at P′ 1 of the cleavage site.
ii) Optimum pH
In 0.15 M GTA buffer (buffered by 3,3-dimethyl glutaric acid, Tris (hydroxy methyl) amino methane, 2-amino-2-methyl-1,3-propanediol) from pH 3 to pH 10, SVP35 was allowed to act on Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) as a substrate at 30° C. for 10 minutes. As a result, it was revealed that the Optimum pH of SVP35 was around 7.0, and that when the activity at pH 7.0 was defined as 100%, SVP35 had an activity of 70% or more at pH 6.0-8.0 and an activity of 80% or more at pH 6.5-7.5 (see FIG. 1).
iii) pH Stability
To 10 μl of SVP35 purified enzyme solution, 40 μl of each pH of 0.15 M GTA buffer from pH 3 to pH 10 was added, left at 4° C. overnight, followed by addition of 0.1 M sodium phosphate buffer (pH 7.0) to the liquid volume of 400 μl, and was adjusted to pH 7.0. To these enzyme solutions, Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) was added as a substrate, and reacted at pH 7.0, at 30° C. for 10 minutes. As a result, it was shown that SVP35 was stable within the range of pH 4 to pH 10 (when the activity at pH 4.0 was defined as 10000, it had an activity of 90% or more at pH 4-10) (see FIG. 2).
iv) Optimum Temperature
To the purified enzyme solution diluted with 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) was added and reacted at pH 7.0, between 5° C. and 65° C. for 10 minutes. As a result, it was shown that the Optimum temperature of SVP35 was about 45° C. and it had high activity within the range of 40° C. to 50° C. (it had an activity of 80% or more than that at 45° C.) (see FIG. 3).
v) Temperature Stability
To 10 μl of the purified enzyme solution, 40 μl of 20 mM Tris-HCl buffer (pH 7.5) containing 5 mM calcium chloride and 0.01 mM zinc chloride was added to treat at 4° C. or at from 30° C. to 70° C. for 15 minutes, and then cooled by ice, added 250 μl of 20 mM sodium phosphate buffer (pH-7.0). To this enzyme solution, Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) was added as a substrate and reacted at 30° C. for 5 minutes. When the activity treated at 4° C. was defined as 100%, the remaining activity at each temperature was calculated. As a result, it was shown that SVP35 retained 80% of activity at 50° C., but it lost its activity at 60° C. (see FIG. 4).
vi) Inhibitors
To 20 mM sodium phosphate buffer (pH 7.0) containing various compounds at the concentrations shown in FIG. 5, the purified enzyme solution was added and left for 60 minutes at room temperature. Then Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11 was added as a substrate and reacted for 10 minute at 30° C. The relative activity by adding each compound was calculated based on the Gly-Pro-Ser-Phe-Arg-Ala-Pro-Asp-Ser (SEQ ID NO: 11) cleavage activity in the absence of compounds as 100%. As a result, it was shown that SVP35 was strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline and phosphoramidon which are metalloprotease inhibitors, and by Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes (see FIG. 5).
(4) Characterization of the Properties of the Neutral Metalloprotease (SVP70) Produced by Streptoverticillium mobaraense IFO13819
i) Substrate Specificity
The substrate specificity was examined similarly as described in (3)-i). As a result, it was revealed that the substrate was cleaved before (at N-terminal side of) especially Phe, often Leu, sometimes Tyr, Trp, Ile, Val, and that SVP70 recognized the aromatic amino acids and hydrophobic amino acids with bulky side-chains positioned at P′ 1 of the cleavage site.
ii) Optimum pH
The Optimum pH of SVP70 was examined similarly as (3)-ii). As a result, it was revealed that the Optimum pH of SVP70 was around 7.0, and that if the activity at pH 7.0 is defined as 100%, SVP70 had an activity of 90% or more at pH 6.0-8.0 and an activity of 95% or more at pH 6.5-7.5 (see FIG. 1).
iii) pH Stability
The pH stability was examined similarly to (3)-iii). As a result, it was shown that SVP70 was stable within pH 5 to pH 10, but it was less stable than SVP35 at slightly alkaline (see FIG. 2). Specifically, if the activity at pH 5.0 was defined as 100%, it had an activity 90% or more in the range of pH 5 to pH 7, and it had an activity about 80% or more even in the range of pH 7 to pH 10.
iv) Optimum Temperature
The Optimum temperature of SVP70 was examined similarly as (3)-iv). As a result, it was shown that the Optimum temperature of SVP70 was within the range from about 50° C. to 55° C., especially around 55° C. (see FIG. 3).
v) Inhibitors
The inhibitory activities of various compounds to SVP70 were examined analogously to (3)-vi). As a result, SVP70 underwent strong inhibitory action by ethylene diamine tetraacetic acid, 1,10-phenanthroline, and phosphoramidon, which are metalloprotease inhibitors, and by reductant dithiothreitol, urea, and Streptomyces subtilisin inhibitor (SSI) derived from actinomycetes (see FIG. 5).
(5) Sequencing of the N-terminal Amino Acid Sequence of SVP35 and SVP70
The purified enzymes of SVP35 and SVP70 obtained in (1) and (2) above were transferred onto Polyvinilidene-difluoride (PVDF) membrane using Membrane Cartridge (Perkin Elmer Co. Ltd.) and the N-terminal amino acid sequence was analyzed using a gas-phase Protein Sequencer PPSQ-10 (Shimadzu Co. Ltd.). The amino acid sequence of SVP35 is shown in SEQ ID NO: 1, and the amino acid sequence of SVP70 is shown in SEQ ID NO: 2. A homology can be seen in these sequences.
Accordingly, those which had any homology to these proteases for N-terminal amino acid sequences were searched, and then metalloprotease SGMP II (J. Biochem., Vol. 110, p. 339-344, 1991) from Streptomyces griseus, and three metalloproteases (GenBank/EMBL/DDBJ CAB76000, the same CAB76001, and the same CAB69762), etc. from Streptomyces coelicolor were found. These proteases also can be used to cleave selectively the pro-structure part of the pro-MTG, and therefore they can be used to produce an active MTG according to the present invention.
(6) Cloning of SVP35 Gene and Its Secretory Expression in Coryneform Bacteria
The chromosomal DNA of Streptomyces coelicolor A3(2) was prepared using the method of Saito and Miura [Biochem. Biohhys. Acta, 72, 619 (1963)]. Primers shown in SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized by referring to the sequence of metalloprotease (GenBank/EMBL/DDBJ CAB76001) gene from Streptomyces coelicolor which have a homology in the N-terminal amino acid sequence. Primers shown in SEQ ID NO: 3 and SEQ ID NO: 4 were used to perform PCR using the chromosomal DNA of Streptomyces coelicolor A3(2) as a template, and the gene region in the metalloprotease gene was amplified. For the PCR reaction, Pyrobest DNA polymerase (Takarasyuzo Co. LTD.) was used and the reaction conditions followed the protocol recommended by the manufacturer. The chromosomal DNA of Streptoverticillium mobaraense IFO13819 prepared by the method of Saito and Miura was digested by various restriction enzymes which recognize 6-base sequence, the digested samples were analyzed by the Southern blot hybridization as described in Molecular Cloning 2nd edition [J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p9. 31 (1989)], using the 32P-labeled PCR product as a probe, and a single band of about 8 kb was detected by SphI cleavage. Accordingly, the chromosomal DNA of Streptoverticillium mobaraense IFO13819 which had been prepared by the foregoing method was digested with SphI, and a fragment of about 8 kb was recovered through agarose gel electrophoresis using EASYTRAP Ver. 2 (Takarasyuzo Co. LTD.). The recovered fragment was inserted into SphI site of pUC18, which was introduced into competent cells of Escherichia coli JM109 (Takarasyuzo Co. LTD.) to generate a library. The library was screened for the bacterial strain which contains the plasmid where the SVP35 gene fragment was cloned, by colony hybridization as described in Molecular Cloning 2nd edition [J. Sambrook E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p1. 90 (1989)], using the synthetic nucleotide as a probe.
The plasmid was recovered from the strain obtained above and was designated as pVSV1. The nucleotide sequence of the fragment cloned in pVSV 1 was determined. The nucleotide sequence of this cloned fragment is shown in SEQ ID NO: 5. The primary amino acid sequence encoded by this gene was deduced, which allowed the determination of the entire primary amino acid sequence of SVP35 containing the signal sequence of SVP35 including the amino acid sequence of the previously determined N-terminal portion and the region assumed to be a pro-structure part. The entire amino acid sequence of SVP35 is shown in SEQ ID NO: 6. It is presumed that amino acids nos. 1-36 of amino acid sequence described in SEQ ID NO: 6 refer to the signal sequence, amino acids nos. 37-216 refer to the pro-structure part, and amino acids nos. 217-537 correspond to the mature SVP35.
Primers shown in SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized using pVSV1 as a template by referring to the sequence of SEQ ID NO: 5, and the gene region containing the pro-structure part of SVP35 and the mature SVP35 was amplified by PCR. For the PCR reaction, Pyrobest DNA polymerase (Takarasyuzo Co. Ltd.) was used and the reaction conditions followed the protocol recommended by the manufacturer.
Next, using pPKSPTG1 described in WO 01/23591 as a template, the region including the 5′-upstream region containing the promoter region of PS2 gene which is the cell surface protein of C. glutamicum and the signal sequence of SlpA, the cell surface protein of C. ammoniagenes was amplified by PCR technique using the combination of oligonucleotides of SEQ ID NO: 9 and SEQ ID NO: 10. The primer shown in SEQ ID NO: 10 contains the sequence encoding the N-terminal amino acids of SVP35 having a pro-structure part.
Then, the gene of the heterologous fusion pre-pro SVP35 gene fragment, which was connected to the 5′-upstream region comprising the promoter region of PS2 gene and the signal sequence of SlpA, the cell surface protein, from C. ammoniagenes, was amplified by performing cross-over PCR with SEQ ID NO: 8 and SEQ ID NO: 9 using the mixture of 1 μl each of the amplified PCR solution. The amplified fragment of about 2.3 kb was detected by agarose gel electrophoresis. The PCR product was subjected to agarose gel electrophoresis to recover a fragment of about 2.3 kb, and after blunting its ends using DNA Blunting Kit (Takarasyuzo Co. Ltd.), the fragment was inserted into SmaI site of pCV7 as described in JP-Kokai No. 9-070291 to obtain pVSV1. The nucleotide sequence of the inserted fragment was determined according to a conventional method to confirm that the fusion gene was constructed as expected.
C. glutamicum ATCC13869 was transformed with the constructed pVSV1 and the strains which grew on the CM2S agar medium containing 5 mg/l of chloramphenicol (10 g of yeast extract, 10 g of tryptone, 5 g of sucrose, 5 g of NaCl, 15 g of agar per liter of distilled water) were selected. Then, the selected C. glutamicum ATCC13869 harboring pVSV1 was cultured in MMTG culture medium (60 g of glucose, 0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate, 1 g of potassium dihydrogenphosphate, 0.01 g of ferrous sulfate heptahydrate, 0.01 g of manganese sulfate pentahydrate, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate per liter of distilled water, adjusted to pH 7.5) containing 5 mg/l of chloramphenicol at 30° C. for 30 hours. 1 ml of the culture medium was centrifuged to separate to the supernatant of the culture and the bacteria. The activity of SVP35 was detected in the supernatant of the culture, and as a result of SDS-PAGE (Nature, 227, 380-685 (1970)) electrophoresis according to Laemmli's method, it was confirmed that about 200 mg/L of SVP35 was secretory-expressed.
Example 2
Conversion of Transglutaminase from Streptoverticillium mobaraense IFO13819 (pro-MTG) into an Active Form
Using pro-MTG (1 mg/ml) expressed by Corynebacterium glutamicum as a purified substrate, the neutral protease (SVP35, SVP70) from Streptoverticillium mobaraense or the neutral metalloprotease SGMP II from Streptomyces griseus was mixed in the ratio of the substrate: the enzyme=200:1, the mixture was reacted at 30° C. After 0, 1, 2, 4, 7, 20 hours, the reaction mixture was sequentially picked up, and the aliquots of the reaction mixture were mixed with SDS-PAGE sample buffer and heated at 95° C. for 3 minutes, and then subjected to SDS-PAGE according to Laemmli's method (Nature, 227, 680-685 (1970)). The result is shown in FIG. 6. As can be seen in FIG. 6, when these proteases were reacted, pro-MTGs were converted to the mature forms, and the produced MTGs were not reduced even after a long-term reaction. The transglutaminase (TG) activity of the picked up fraction was measured by the hydroxamate method, and sufficient activity was confirmed. In addition, SGMP II was purified from actinase (Kakenseiyaku Co. Ltd.) according to the reference method (J. Biocem., Vol. 110, p. 339-344, 1991).
Then, the neutral metalloprotease SVP70 from Streptoverticillium mobaraense, and serine protease SAM-P45 (Streptomyces albogriseolus) as a control, were added to the pro-MTGs with gradually increasing amounts of these enzymes, and reacted at 30° C. and pH 7.0. After 1, 4, 7, and 24 hours, the reaction mixture was picked up sequentially to determine the TG activity by the hydroxamate method (see FIG. 7). The protein concentration of TG was measured by reverse phase chromatography (see FIG. 8). As a result, it was shown that SVP could convert pro-MTG to active MTG with an amount as small as 1/500 of the substrate. It was shown that SAM-P45 generated only insufficient transglutaminase activity even at an amount of 1/50 of the substrate, and that the complete conversion to the active form was not observed. On the other hand, when SAM-P45 was added at an amount of 1/10 of the substrate, the conversion into the active MTG was observed, but a decrease in the amount and the activity of MTG-protein was observed. This suggests that over-degradation of the mature MTG occurred by SAM-P45.
The present invention provides a new protease from an actinomycetes, Streptoverticillium mobaraense, which specifically cleaves the pro-structure part of transglutaminase precursor to activate it, and the gene thereof. The new protease according to the present invention can be expressed in a large amount by a coryneform bacterium, and thereby the present invention provides a method for efficiently producing transglutaminase from microorganisms.
The advantage of using the neutral metalloproteases from actinomycetes according to the present invention for the production of an active MTG is that these enzymes have strong activities for selectively cleaving the pro-structure part of the pro-MTG, and that these enzymes can be expressed extracellularly by a coryneform bacterium.
As it is shown that the pro-MTG from actinomycetes can be efficiently expressed and secreted by a coryneform bacterium, it is possible to produce more efficiently an active MTG by a single bacterial cell by co-expressing and secreting the pro-MTG and the neutral metalloprotease. In this instance, it is sufficient to express the neutral metalloprotease in only an amount required and sufficient for cleaving the pro-structure part of the pro-MTG.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents, including the foreign priority document, JP 2003-061623, is incorporated by reference herein in its entirety.
REFERENCES


    1. JP-Kokoku No. 1-50382
    2. JP-Kokai No. 64-27471
    3. WO publication No. 01/2351
    4. JP-Kokai No. 6-502548
    5. JP-Kokai No. 10-108675
    6. Eur. J. Biochem., Vol. 257, pages 570-576, 1998,
    7. J. Biochem., Vol. 110, pages 339-344, 1991.

## Claims

1. A biochemical or cellular method of producing an active Streptoverticillium transglutaminase from a Streptoverticillium protransglutaminase comprising
contacting a neutral Streptoverticillium mobaraense IFO13819 metalloprotease with the protransglutaminase, wherein said metalloprotease is produced by culturing a microorganism into which the gene encoding the metalloprotease has been introduced, and
recovering the active transglutaminase,
wherein said metalloprotease comprises:
a molecular weight of about 35,000,
an optimum pH 6.5 to pH 7.5,
an optimum temperature of about 45° C., and
an activity to cleave the peptide of SEQ ID NO: 11
wherein, said metalloprotease is strongly inhibited by ethylene diamine tetraacetic acid, 1,10-phenanthroline, phosphoramidon, and Streptomyces subtilisin inhibitor (SSI) from actinomycetes.

2. The method according to claim 1, wherein said microorganism is a coryneform bacterium.

3. The method according to claim 1, wherein the metalloprotease comprises, as an N-terminal amino acid sequence, the sequence of SEQ ID NO: 1.

4. The method according to claim 1, wherein the metalloprotease comprises the amino acid sequence of amino acids 217-537 of SEQ ID NO:6.

5. The method of claim 1, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.

6. The method of claim 2, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.

7. The method of claim 3, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.

8. The method of claim 4, wherein a gene encoding the protransglutaminase has been introduced into the microorganism, and said protransglutaminase is produced by culturing the microorganism.