Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1997-08-15
2001-01-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C536S023100, C536S023700, C536S023740
Reexamination Certificate
active
06172196
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for making bread with refrigerated dough and a process for producing ethanol.
BACKGROUND ART
Recently, in the bread manufacturing industry, a method for making bread with refrigerated dough has been widely used with the purpose of saving labor in the bread making process and meeting diverse needs of consumers. In this method, partially fermented dough is stored at a low temperature in a refrigerator and then is subjected to fermentation, proofing and baking to make bread. Such a method is usually carried out by the use of refrigeration-resistant yeast, that is, yeast which is capable of controlling fermentation during the storage of dough at a low temperature and allowing normal fermentation at temperatures for fermentation and proofing to raise the dough.
As for the breeding of refrigeration-resistant yeast, there are known methods in which yeast strains of wild type are conferred with the mutation exhibiting low-temperature-sensitive fermentability by artificial mutagenesis [e.g., Japanese Published Examined Patent Application No. 71474/95, Japanese Published Unexamined Patent Application No. 213277/95, Japanese Published Unexamined Patent Application No. 79767/95, and Appl. Environ. Microbiol., 61, 639-642 (1995)]. The yeast strains conferred with the mutation exhibiting low-temperature-sensitive fermentability are used as refrigeration-resistant yeast or as parent strains for breeding refrigeration-resistant yeast.
However, such mutagenesis induces mutation at random and thus may possibly confer the yeast with mutation relating to the basic properties of fermentation such as dough raising, in addition to the low-temperature-sensitivity mutation.
It is also known to confer baker's yeast or brewer's yeast with favorable properties such as flocculation [The 23rd European Brewery Conv. Proc., 297-304 (1991)] and flavor [Curr. Genet., 20, 453-456 (1991)] by using gene manipulation techniques.
However, a gene relating to the low-temperature-sensitivity of fermentability or a method for breeding refrigeration-resistant yeast by gene manipulation is not known.
Ethanol is produced by fermentation of sugar materials (e.g. molasses) or starch materials (e.g. corn and potato) as carbon sources. Fermentation can be generally carried out at a temperature of 30 to 43° C. Usually, the fermentation temperature is adjusted to 30 to 35° C. by cooling in order to avoid the death, insufficient growth, or decrease in fermentability of yeast caused by the rise of temperature. However, in the summer months, cooling is often insufficient, thereby causing the rise of culturing temperature to 35 to 38° C. in the course of alcohol fermentation. Thus, alcohol fermentation is usually carried out with further cooling to prevent the rise of temperature due to fermentation heat. A need exists for temperature-resistant yeast which is useful for saving cost for cooling in such process.
As for the breeding of thermotolerant yeast, there have been reports on a method in which mitochondria relating to thermotolerance is introduced [Juan Jimenez, et al.: Curr. Genet., 13, 461-469 (1988)] and a method in which heat shock protein HSP104 is expressed at a high level [Susan Lindquist, et al.: Proc. Natl. Acad. Sci. USA, 93, 5301-5306 (1996)]. However, application of these methods to alcohol fermentation has not been studied. Further, it is known that the heat-resistance of yeast is improved by heat treatment at temperatures which are not fatal to the yeast [B. G. Hall: J. Bacteriol., 156, 1363 (1983)], but this effect is not lasting, and it is difficult to apply this method to alcohol fermentation.
DISCLOSURE OF THE INVENTION
The present invention relates to a protein having the amino acid sequence represented by SEQ ID NO: 1, or a protein being capable of complementing the mutation exhibiting low-temperature-sensitive fermentability and having an amino acid sequence wherein one or more amino acid residues are added, deleted or substituted in the amino acid sequence represented by SEQ ID NO: 1; a gene which encodes said protein; and a gene which comprises DNA having the nucleotide sequence represented by SEQ ID NO: 1, or comprises DNA being capable of complementing the mutation exhibiting low-temperature-sensitive fermentability and having a nucleotide sequence wherein one or more DNAs are added, deleted or substituted in the nucleotide sequence represented by SEQ ID NO: 1. The present invention also relates to yeast belonging to the genus Saccharomyces and having low-temperature-sensitive fermentability which is characterized in that the above- mentioned gene on the chromosome is inactivated; dough containing said yeast; a process for making bread which comprises adding said yeast to dough; and a process for producing ethanol which comprises culturing said yeast in a medium, allowing ethanol to accumulate in the culture, and recovering ethanol from the culture.
The expression “having low-temperature-sensitive fermentability” as used herein means the property of having substantially no fermentability at temperatures for low temperature storage and having normal fermentability at temperatures for fermentation and proofing after the low temperature storage. For instance, in the case of baker's yeast, it means the property of having substantially no dough-raising ability at 5° C. and having normal dough- raising ability at 20 to 40° C. after the storage under refrigeration at 5° C. for 1 to 7 days, and in the case of brewer's yeast, it means the property of having substantially no alcohol fermentability at 5° C. and having normal alcohol fermentability at 20 to 40° C. after the storage under refrigeration at 5° C. for 1 to 7 days.
Isolation of a gene which complements the mutation exhibiting low-temperature-sensitive fermentability, determination of the DNA sequence of said gene, and inactivation of said gene can be carried out by using basic techniques for genetic engineering and biological engineering according to the descriptions in commercially available experiment manuals, e.g. Gene Manual, Kodansha Co., Ltd.; Methods for Experiments in Gene Manipulation, edited by Yasutaka Takagi, Kodansha Co., Ltd.; Molecular Cloning, Cold Spring Harbor Laboratory (1982); Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory (1989); Methods in Enzymology, 194 (1991); and Gene Experiments Using Yeasts (an extra number of Experimental Medicine), Yodosha Co., Ltd. (1994).
The gene which complements the mutation exhibiting low-temperature-sensitive fermentability according to the present invention (hereinafter referred to as the gene complementing low-temperature-sensitivity) can be isolated, for example, as the gene complementing the low-temperature-sensitivity of fermentability of Saccharomvces cerevisiae RZT-3 (FERM BP-3871) (hereinafter referred to as RZT-3 strain) described in Japanese Published Unexamined Patent Application No. 336872/93. That is, the gene complementing low-temperature-sensitivity can be isolated by transforming RZT-3 strain with the DNA library of the yeast carrying the gene complementing low-temperature-sensitivity, and obtaining DNA from the strain of which the mutation exhibiting low-temperature-sensitive fermentability is complemented.
The DNA library of the yeast carrying the gene complementing low-temperature-sensitivity can be prepared by cleaving the chromosomal DNA of yeast carrying a gene of wild type, e.g. Saccharomyces cerevisiae X2180-1B (hereinafter referred to as X2180-1B strain) with a restriction enzyme, and ligating each of the obtained DNA fragments with a vector capable of being maintained in yeast.
Any restriction enzymes which can cleave the chromosomal DNA can be used in the above process. Preferably, those which give DNA fragments of 20 Kbp or less are used. The chromosomal DNA may be completely digested or partially digested with the restriction enzyme.
Examples of the vectors capable of being maintained in yeast are YCp vectors, YEp vectors,
Kawasaki Hideki
Kikuchi Yasuhiro
Ouchi Kozo
Tokai Masaya
Achutamurthy Ponnathapu
Fitzpatrick ,Cella, Harper & Scinto
Kyowa Hakko Kogyo Co. Ltd.
Tung Peter P.
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