Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
2001-03-09
2004-03-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S252100, C426S046000
Reexamination Certificate
active
06706492
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a koji mold having increased protease activity and peptidase activity relative to a parent strain, a method of breeding the koji mold, and a method of manufacturing a flavor enhancer using the koji mold.
BACKGROUND OF THE INVENTION
Methods for obtaining a flavor enhancer by hydrolysis of proteins can be divided into lysis methods using microorganism culture products or enzymes (hereinafter, “enzymolysis”), and chemical lysis using acids. Examples of enzymolysis methods that have been employed include traditional methods of producing flavor enhancers such as soy sauce and miso, methods in which a koji mold culture fluid which contains protein hydrolysis related enzymes are allowed to act on proteins such as gluten.
Lysis methods using acids include high temperature processing of proteins such as gluten using hydrochloric acid.
With enzymolysis, there have been cases where the palatability of the flavor enhancer has been insufficient resulting from insufficient lysis of the protein due to the type of koji mold that is used, processing conditions, etc. In such cases increasing the amount of culture product or enzyme used may be at times effective in increasing palatability, however, this was problematic from the viewpoint of costs.
For enzymolysis, the following two enzymes can be provided as examples of enzymes greatly contributing to palatability: protease, which acts to roughly cleave and dissolve proteins; and peptidase which further breaks down amino acids from the termini of peptides (exopeptidase). Koji molds produce a plurality of proteases and peptidases, however, in the lysis of proteins, alkaline protease and a type of leucine aminopeptidase are reported to be the main actors (Nakadai: Journal of Japan Soy Sauce Research Institute 11 (2), 67-79 (1985)). For this lysis method, in order to improve of the palatability of the resultant flavor enhancer, or to reduce of amount of culture product used, there is a need to breed a koji mold exhibiting high enzyme activity. To breed koji mold, a mutation processing method has been employed. However, with methods involving mutation processing, there is the problem that great efforts are required for screening.
As a method for breeding a koji mold, a method for increasing protease activity and peptidase activity at the same time by removing inhibition of the activating ability of areA due to a nitrogen source, is the subject of a patent application (WO 99/02691). However, with this method there is the problem that a factor that controls expressions of a broad range of genes is set in an activated state.
Recently, a koji mold-derived alkaline protease gene was cloned (Japanese Patent No. 2671452; Japanese Patent No. 2888955). Further, a koji mold-derived leucine aminopeptidase gene was cloned, and by transformation with this gene, a transformant with high leucine aminopeptidase activity was obtained (Japanese Patent Laid-Open Application No. 11-346777).
However, until now, no attempt had been made to obtain a koji mold having increased activity of both enzymes by transformation using both a protease gene and a peptidase gene, and no such transformant existed.
The present invention aims to provide a koji mold having increased protease activity and peptidase activity relative to a parent strain, and a breeding method therefor. Further, the present invention aims to provide a method of producing a flavor enhancer having strong palatability.
BRIEF SUMMARY OF THE INVENTION
The present inventors, as a result of deliberate study, have succeeded in breeding a koji mold transformant having increased protease activity and peptidase activity. In the case where the culture product of this koji mold is allowed to act on a protein, when compared to the case where an equivalent amount of culture product of the parent strain is used, a higher total nitrogen concentration and total amino acid concentration, i.e. a flavor enhancer having strong palatability can be obtained. The present invention was completed on the basis of these observations.
That is, the present invention relates to the following (1)-(7):
(1) An isolated koji mold having increased protease activity and peptidase activity in relation to a parent strain resulting from transformation with a protease gene and a peptidase gene.
(2) The koji mold according to (1) above, wherein the protease gene and the peptidase gene are derived from a koji mold.
(3) The koji mold according to (1) above, wherein said koji mold is a member of
Aspergillus sojae, Aspergillus oryzae
, or
Aspergillus tamarii.
(4) The koji mold according to (3) above, wherein the protease gene and the peptidase gene are derived from a koji mold.
(5) A method of breeding the koji mold according to any one of (1) to (4) above, comprising the steps of:
transforming a parent strain of koji mold with a protease gene and a peptidase gene; and,
selecting a transformant having increased protease activity and peptidase activity relative to said parent strain.
(6) A method of manufacturing a flavor enhancer which comprises allowing a culture product of the koji mold according to any one of (1) to (4) above to act on a protein.
(7) A flavor enhancer obtainable by allowing a culture product of the koji mold according to any one of (1) to (4) above, to act on a protein.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No.2000-64739, which is a priority document of the present application.
DETAILED DESCRIPTION OF THE INVENTION
Koji Mold of the Present Invention
The koji mold of the present invention is characterized by increased protease activity and increased peptidase activity relative to a parent strain due to transformation using a protease gene and a peptidase gene.
A “koji mold” refers to any strain which is phylogenetically a member of koji mold, and includes for example, strains used in the fermentation of food products, concretely,
Aspergillus sojae, Aspergillus oryzae, Aspergillus tamarii
and the like.
The term “protease gene” and “peptidase gene” include nucleic acid sequences which when introduced into a parent strain by transformation, can increase the activity of each enzyme of the koji mold. Such nucleic acid sequences include a structural gene of an enzyme, a mutant gene in which a mutation has been introduced within a structural gene to increase the activity of the enzyme itself, expression regulation sites (promoter, terminator, enhancer, etc.) of the structural gene, mutant expression regulation sites, expression regulation sites of different genes, or derivatives thereof, and a nucleic acid sequence consisting of a plurality of these sequences linked together. Further, the nucleic acid sequence can be of natural derivation or a synthetic product, and for example, genomic DNA, cDNA, PCR fragment, chemical synthetic/semi-synthetic DNA and the like can be used.
The derivation of structural genes for protease and peptidase is not particularly limited, and as long as they are capable of expressing in and being secreted from a koji mold, and act on a broad range of substrates, they can be derived from types of organisms other than a koji mold. From the viewpoints of intracellular production efficiency of the enzyme, efficiency of secretion to the outside of a cell, and public acceptance, it is preferable that the gene be of koji mold origin. For example, a koji mold-derived alkaline protease gene (Japanese Patent No. 2671452, Japanese Patent No. 2888955), a koji mold-derived amino peptidase gene (Japanese Patent Laid-Open Application No. 11-346777) can be used. Further, expression regulation sites of each gene can also be derived from another gene or from another type of organism if they are able to function in koji mold.
The koji mold of the present invention, to the extent that it possesses the characteristics described above, can have protease activity and peptidase activity that is increased by any factor relative to the parent strain. An example of the koji mold of the pr
Fukushima Yaichi
Koyama Yasuji
Sato Hiroe
Sugishita Misao
Umitsuki Genryou
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Ketter James
Kikkoman Corporation
Lambertson David A.
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