Process for producing transglutaminase

Details Process for producing transglutaminase Process for producing transglutaminase

2001-06-28

2004-12-21

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C424S094500, C426S061000

Reexamination Certificate

active

06833258

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a high-yield process for producing high-activity, high-purity transglutaminase by refolding denatured microorganism-derived transglutaminase by incubating denatured transglutaminase in an aqueous medium under acidic conditions and reconstituting a higher-order enzymatically active transglutaminase in an aqueous medium at a neutral pH.
2. Discussion of the Background
Transglutaminase has been widely used in the production of gel-food (such as jelly, yogurt, cheese), gel-toiletries, and modification of meat, as well as other fields. Thus transglutaminase is quite a useful industrial enzyme.
Transglutaminase (EC 2. 3. 2. 13; TGase) is an enzyme that catalyzes an acyl transfer reaction between a &ggr;-carboxyamide group of a glutamine residue and a primary amine of a protein or a polypeptide chain. Transglutaminase isolated from a culture supernatant of a microorganism (variant of
Streptoverticillium mobaraense
in the present invention) is called microorganism-derived transglutaminase (MTG).
MTG is a monomer protein comprising 331 amino acids and has a molecular weight of 38,000 (Journal of Biological Chemistry, 268, 11565-11572, 1993). Culture conditions of the microorganism were optimized for mass production of this enzyme (Agricultural Biological Chemistry, 53, 2613, 1989). However, this microorganism was not widely used in the industrial production of MTG due to problems associated with the level of production and the production costs. Further, this enzyme is extracellularly secreted in an active state; however, in the host bacteria the protein sequence is susceptible to chemical modification by a deamidation reaction at four distinct sites. Accordingly, in a culture solution chemical modification proceeded during the secretory production and enzymes having reduced activity accumulated giving rise to a mixture of active-states in the recovered culture supernatant. In order to solve these problems, various methods for producing recombinant MTG using microorganisms such as
E. coli
have been studied (Bioscience & Bioindustry, 52, 554-561, 1994).
When the sequence of the enzyme was expressed in
E. coli
the yield of the enzyme was very low. High expression was successfully realized by fusing this enzyme with a fragment of a sequence derived from the T7 gene-10. Nevertheless, following restricted cleavage of the fusion protein, the resulting enzyme with the native sequence exhibited enzymatic activity which was approximately ⅕ of the native transglutaminase. This suggested that the enzyme obtained by this method was in an incomplete higher-order structure (Bioscience, Biotechnology, and Biochemistry, 61, 830-835, 1997). Further, restricted cleavage to liberate the native-like transglutaminase from the T7 gene-10 product fusion partner required a digestive enzyme or a chemical procedure. Thus, this method posed serious problems in that it was intricate and involved high production costs. It is therefore an object of the present invention to provide a low-cost, high-yield process by which transglutaminase can be produced for application in the various fields of food processing or other fields where applicable.
Solving these problems required developing a technology in which only the sequence of the transglutaminase is highly expressed directly from a microorganism, and a technology in which the higher-order structure harboring the enzymatic activity is completely reconstructed from the denatured enzyme recovered from the culture supernatant (refolding technology). With respect to refolding technology of proteins, it is impossible to predict the appropriate conditions for reconstructing the native state of the intended protein by examination of said native state. It is necessary that operation conditions be empirically derived for each intended protein (Advances in Protein Chemistry, 50, 1-59, 1997). The present applicant has previously succeeded in high intracellular expression of the sequence of this enzyme, which was impossible in the past, by replacing the codons inherently present in the DNA sequence with those more frequently utilized by the
E. coli
expression host (Japanese Patent Application No. 181,951/1998 filed Jun. 29, 1998, Japanese Patent Kokai Publication JP-A-11-75,876, published Mar. 23, 1999; hereinafter referred to as a “prior invention”). Codon optimization significantly increased the copy number of the plasmid encoding the transglutaminase gene further amplifying the expression of this enzyme. Accordingly, it is now possible to obtain the transglutaminase in a denatured state in large quantities and to investigate the refolding technology of the enzyme for the first time.
SUMMARY OF THE INVENTION
The present invention aims to develop a method of refolding recombinant microorganism expressed transglutaminase from a denatured state to reconstitute enzymatic activity. The ambition of this method is to construct a less costly industrial process for expressing the enzyme and to provide a means of producing transglutaminase having an enzymatic activity that is equal to the native transglutaminase (namely transglutaminase activity).
In order to solve the aforementioned problems and attain the aim of the present invention, the inventors have found that transglutaminase having an enzymatic activity can be produced with high efficiency by subjecting denatured transglutaminase to minimally the following steps (a) and (b):
(a) a step for forming an intermediate structure in which said enzyme in the denatured state is allowed to react in an aqueous medium under acidic conditions; and
(b) a step for forming a higher-order structure exhibiting enzymatic activity by bringing the aqueous medium having said intermediate structure to a neutral pH region.
In another embodiment of the invention, the process for producing transglutaminase having an enzymatic activity comprises:
(a) incubating a denatured transglutaminase in an acidic aqueous medium; and
(b) adjusting the pH of said aqueous medium to a neutral pH.
The enzyme having the intermediate structure obtained in step (a) has the transglutaminase enzymatic activity, which is substantially lower than that of native transglutaminase when comparing it to the enzyme present in nature. For example, the enzymatic activity is typically 15 to 25 U/mg as compared to greater than 30 U/mg observed for the native state.
Additional objects of the invention will be set forth in the Detailed Description which follows.


REFERENCES:
patent: 5827712 (1998-10-01), Yokoyama et al.
patent: 6013498 (2000-01-01), Yokoyama et al.
patent: 0 331 464 (1989-09-01), None
patent: 0 336 324 (1989-10-01), None
M. Kawai et al., “High-Level Expression of the Chemically Synthesized Gene for Microbial Transglutaminase from Streptoverticillium inEscherichia coli”, Biosci. Biotech. Biochem, 1997, vol. 61, No. 5, pp. 830-835.
D. Ejima, et al., “High Yield Refoding and Purification Process for Recombinant Human Interlukin-6 Expressed inEscherichia coli”, Biotechnology and Bioengineering, Feb. 5, 1999, vol. 62, No. 3, pp. 301-310.

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