Bacillus-derived transglutaminase

• US 5,731,183 A
• Filed: 02/09/1996
• Issued: 03/24/1998
• Est. Priority Date: 02/09/1995
• Status: Expired due to Term

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Suzuki, Shunichi, Eto, Yuzuru, Yokozeki, Kenzo, Hashiguchi, Kenichi, Miwa, Kiyoshi, Kobayashi, Katsunori, Yamanaka, Shigeru, Tanita, Yuko
• Primary Examiner(s): GRIMES, ERIC BURTON
• Application Number:
  US08/596,864
• Time in Patent Office: 774 Days
• Field of Search: 435/193
• US Class Current: 435/193
• CPC Class Codes: C12N 9/1044 Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• CPC Class Codes: Y10S 435/839 Bacillus subtilis

Abstract

The present invention relates to (1) a transglutaminase (hereinafter referred to as TG) isolated from a Bacilli such as those of Bacillus subtilis, (2) a fraction having transglutaminase activity, and (3) a method for producing a protein, a non-proteinaceous amino acid polymer, a peptide or derivatives thereof having a crosslinked structure, by crosslinking the glutamine and lysine residues in the same with the TG or the fraction having TG activity to thereby form intermolecular or intramolecular, crosslinked ε-(γ-Glu)-Lys bonds. The present invention also relates to (4) a DNA coding for a TG derived from a Bacilli such as Bacillus subtilis, (5) a vector comprising said DNA coding for the TG, (6) a cell transformed with the vector, and (7) a method for producing a Bacillus-derived transglutaminase by incubating the transformant. The crosslinked polymers produced using the Bacillus-derived TG of the present invention can be used in foods such as tofu (soybean curd), pudding, yogurt, cheese, ground fish meat, boiled fish paste, sausage and other fish and meat products and also in cosmetics, etc.

Claims

1. A purified Bacillus subtilis-derived transglutaminase having the following physicochemical properties:

(a) active between about pH 7 and pH 9,

(b) active between about 40°

C. and about 65°

C.,

(c) stable at about 60°

C. or lower,

(d) enzymatic activity of the transglutaminase is independent of Ca²⁺ and has an activity of 50% or more in the presence of 5 mM of Ca²⁺,

(e) it is inhibited by N-ethylmaleimide, cystamine or ammonium sulfate,

(f) it is not inhibited by ethylenediamine-tetraacetic acid,

(g) it has a molecular weight of (i) from about 18,000 to about 22,000 as measured by gel permeation chromatography and (ii) from about 28,000 to about 30,000 as measured by SDS-PAGE, and

(h) it catalyzes the transacylation of the γ

-carboxamide group in glutamine residue(s) in a peptide chain.

2. The purified Bacillus subtilis-derived transglutaminase of claim 1, having the amino acid sequence of SEQ ID NO:

3.

3. A purified Bacillus subtilis-derived transglutaminase produced by:

incubating a cell transformed with a vector comprising a DNA coding for the transglutaminase of claim 1 in a culture medium to thereby produce and accumulate Bacillus subtilis-derived transglutaminase in the medium or in the cells, and collecting and purifying the transglutaminase.

4. A purified Bacillus subtilis-derived transglutaminase fraction isolated by fractionating spores obtained by disrupting or lysing the sporangia of bacilli comprising the transglutaminase of claim 1.