Claims
- 1. A method for processing a protein, a non-proteinaceous amino acid polymer, or a peptide or derivatives thereof having a crosslinked structure, which comprises:
- contacting glutamine and lysine residues in a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof with a transglutaminase obtained from Bacillus subtilis to form intermolecular or intramolecular, crosslinked .epsilon.-(.gamma.-Glu)-Lys bonds between or in the molecules of the protein, non-proteinaceous amino acid polymer, peptide or derivatives thereof,
- wherein said transglutaminase has the following physicochemical properties:
- a) it is active between about pH 7 and pH 9,
- b) it is active between about 40.degree. C. and about 65.degree. C.,
- c) it is stable at about 60.degree. C. or lower,
- d) the enzymatic activity of the transglutaminase is not dependent on Ca.sup.+2 and has an activity of 50% or more in the presence of 5 mM of Ca.sup.+2,
- e) it is inhibited by N-ethylmaleimide, cystamine or ammonium sulfate,
- f) it is not inhibited by ethylenediamine-tetraacetic acid, dithiothreitol or 2-mercaptoethanol,
- g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation, and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and
- h) it catalyzes the transacylation of the .gamma.-carboxyamide group in glutamine residue(s) in a peptide chain.
- 2. A method for processing a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof having a crosslinked structure, which comprises:
- crosslinking glutamine and lysine residues in a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof with a transglutaminase fraction isolated by fractionating spores obtained by disrupting or lysing sporangia of Bacillus subtilis, to thereby form intramolecular or intermolecular, crosslinked .epsilon.-(.gamma.-Glu)-Lys bonds between or in the molecules of the protein, non-proteinaceous amino acid polymer, peptide or derivatives thereof.
- 3. A purified or isolated DNA coding for a transglutaminase obtained from Bacillus subtilis having the following physicochemical properties:
- a) it is active between pH 7 and about pH 9,
- b) it is active between about 40.degree. C. and about 65.degree. C.,
- c) it is stable at about 60.degree. C. or lower,
- d) the enzymatic activity of the transglutaminase is not dependent on Ca.sup.+2 and has an activity of 50% or more in the presence of 5 mM of Ca.sup.+2,
- e) it is inhibited by N-ethylmaleimide, cystamine or ammonium sulfate,
- f) it is not inhibited by ethylenediamine-tetraacetic acid, dithiothreitol or 2-mercaptoethanol,
- g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation, and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and
- h) it catalyzes the transacylation of the .gamma.-carboxyamide group in glutamine residue(s) in a peptide chain.
- 4. The DNA of claim 3, which has at least the sequence of from the 118.sup.th base to the 852.sup.nd base in the base sequence of SEQ ID NO:2.
- 5. A vector comprising a DNA coding for a tranglutaminase obtained from Bacillus subtilis having the following physicochemical properties:
- a) it is active between pH 7 and about pH 9,
- b) it is active between about 40.degree. C. and about 65.degree. C.,
- c) it is stable at about 60.degree. C. or lower,
- d) the enzymatic activity of the transglutaminase is not dependent on Ca.sup.+2 and has an activity of 50% or more in the presence of 5 mM of Ca.sup.+2,
- e) it is inhibited by N-ethylmaleimide, cystamine or ammonium sulfate,
- f) it is not inhibited by ethylenediamine-tetraacetic acid, dithiothreitol or 2-mercaptoethanol,
- g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation, and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and
- h) it catalyzes the transacylation of the .gamma.-carboxyamide group in glutamine residue(s) in a peptide chain.
- 6. A cell transformed with the vector of claim 5.
- 7. A method for producing a transglutaminase, which comprises:
- a) incubating a cell transformed with a vector comprising a DNA coding for a transglutaminase obtained from Bacillis subtilis in a culture medium to thereby produce and accumulate said transglutaminase in the medium or in the cells, and
- b) collecting the transglutaminase, wherein the transglutaminase has the following physicochemical properties:
- a) it is active between pH 7 and about pH 9,
- b) it is active between about 40.degree. C. and about 65.degree. C.,
- c) it is stable at about 60.degree. C. or lower,
- d) the enzymatic activity of the transglutaminase is not dependent on Ca.sup.+2 and has an activity of 50% or more in the presence of 5 mM of Ca.sup.+2,
- e) it is inhibited by N-ethylmaleimide, cystamine or ammonium sulfate,
- f) it is not inhibited by ethylenediamine-tetraacetic acid, dithiothreitol or 2-mercaptoethanol,
- g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation, and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and
- h) it catalyzes the transacylation of the .gamma.-carboxyamide group in glutamine residue(s) in a peptide chain.
Priority Claims (3)
Number |
Date |
Country |
Kind |
7-021963 |
Feb 1995 |
JPX |
|
7-226316 |
Sep 1995 |
JPX |
|
8-013072 |
Jan 1996 |
JPX |
|
Parent Case Info
This application is a Division of application Ser. No. 08/596,864, filed on Feb. 9, 1996, now U.S. Pat. No. 5,731,183.
US Referenced Citations (1)
Number |
Name |
Date |
Kind |
5736356 |
Sano et al. |
Apr 1998 |
|
Non-Patent Literature Citations (2)
Entry |
Ramanujam et al., Faseb J. 4(7):A2321, 1990. |
Brock et al., Biology of Microorganisms, 4th edition, Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 725-728, 1984. |
Divisions (1)
|
Number |
Date |
Country |
Parent |
596864 |
Feb 1996 |
|