Chemistry: molecular biology and microbiology – Vector – per se
Patent
1996-11-25
1998-03-10
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Vector, per se
536 232, 435200, C12N 1556, C12N 1563
Patent
active
057260575
DESCRIPTION:
BRIEF SUMMARY
FIELD OF TECHNOLOGY
The present invention relates to barley .beta.-amylase structural genes, to .beta.-amylase genes subjected to nucleotide substitution in order to allow amino acid replacements effective for enhancement of heat stability of .beta.-amylase, and to plasmids containing such genes.
BACKGROUND TECHNOLOGY
.beta.-Amylase (.alpha.-1,4-glucan maltohydrolase; EC 3.2.1.2) catalyzes the liberation of .beta.-maltose from the nonreducing ends of starch and related 1,4-.alpha.-glucans. The enzyme has been purified from various higher plants, e.g. barley and soybean, and microorganisms, and it is a major protein in the starchy endsperm of ungerminated barley seeds.
Barley .beta.-amylase and soybean .beta.-amylase are known as an enzyme useful in industrial production of maltose for infusion and food.
Barley malt is also known to be used as one of materials for beer or distilled liquors, and .beta.-amylase in the malts is known as one of the most important enzymes for saccharification of starch at the mashing stage.
As a structural gene of barley .beta.-amylase, a full length cDNA consisting of 1754 bases of cultivar Hiproly was reported, and it was coded for a polypeptide of 535 amini acids (Eur. J. Biochem., 169, 517 (1987)). A full length cDNA consisting of 1775 nucleotide cultivar of Haruna NIJO (two-rows) was also reported, and it was coded for a polypeptide of 535 amini acids (J. Biochem., 115, 47 (1994), Japanese Patent Kokai Hei 6-303983).
In the study of .beta.-amylase of cultivar Haruna NIJO, an expression vector (pBETA92) was constructed by deleting 5'-terminal 55 base pairs of the full length cDNA and ligating a SmaI linker to form a DNA which was then inserted into the SmaI site of a plasmid pKK 223-3 (produced by Pharmacia Biotech).
Also by transforming this expression vector into Escherichia coli JM 109 (produced by Toyoho), recombinant .beta.-amylase was produced. Recombinant .beta.-amylase consisted of 531 amino acids, and had almost the same properties as .beta.-amylase from barley (Biosci. Biotech. Biochem., 58, 1080 (1994), Japanese Patent Kokai Hei 6-303983).
However, it is not satisfactory that recombinant .beta.-amylase had almost same properties as .beta.-amylase from barley, since soybean .beta.-amylase is employed more frequently in practice than barley .beta.-amylase because of somewhat higher heat stability of the former. Accordingly, in order to enhance the utility of barley-derived recombinant .beta.-amylase, its thermostability at least equivalent to that of soybean .beta.-amylase should be needed.
As an example of .beta.-amylases whose heat stability was enhanced by protein engineering, sevenfold-mutant .beta.-amylase in which methionine at 181 position of the original recombinant .beta.-amylase is replaced with leucine, serine at 291 position with alanine, isoleucine at 293 position with valine, serine at 346 position with proline, serine at 347 position with proline, glutamine at 348 position with aspartic acid and alanine at 372 position with serine was proven to have the thermostability higher than that of the original recombinant .beta.-amylase (Japanese Patent Kokai Hei 6-233086). The original recombinant .beta.-amylase lacked four amino acids at positions 2-5 in comparison with the presumed amino acid sequence of barley .beta.-amylase. Therefore, amino acid positions described above correspond to amino acid positions higher by four in barley .beta.-amylase(Biosci. Biotech. Biochem., 58, 1080 (1994), Japanese Patent Kokai Hei 6-303983).
Barley .beta.-amylase genes are only known to be present not only on the short arm of chromosome 2 but also on the long arm of chromosome 4 (Genet. Res. Camb., 51, 13 (1988)). But no studies of the isolation and sequence of a nuclear gene encoding .beta.-amylase have been reported to data.
Furthermore, new technique of genetic manipulation will be applied in addition to conventional mating methods in the field of breeding barley cultivars, and .beta.-amylase gene is considered to be a candidate of the genes to be introduced to bar
REFERENCES:
Yoshigi, N., et al. (1995) Biosci. Biotech. Biochem. 59(10), 1991-1993.
Yoshigi, N., et al. (1995) J. Biochem. 118(3), 562-567.
Yoshigi, N., et al. (1995) J. Biochem. 117(1), 63-67.
Okada, Y., et al. (1995) Biosci. Biotech. Biochem. 59(6), 1152-1153.
Yoshigi, N., et. al. (1994) J. Biochem. 115(1), 47-51.
Okada Yukio
Yoshigi Naohiro
Patterson Jr. Charles L.
Sapporo Breweries Limited
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