| Project/Area Number |
03556011
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・発酵学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YAMASAKI Makari The University of Tokyo Faculty of Agriculture Prof., 農学部, 教授 (60011889)
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| Co-Investigator(Kenkyū-buntansha) |
KADOKURA Hiroshi The Univ. Tokyo Fac. Agric. Res. Assoc., 農学部, 助手 (70224558)
YODA Koji The University of Tokyo Fac. Agric. Assc. Prof., 農学部, 助教授 (20143406)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 1992: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1991: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Keywords | alkaline elastase / meat-tenderizer / subtilisin / protein engineering / 食肉軟化剤 / サチライシンE / エラスタ-ゼ / 枯草菌 / 大腸菌 / 食肉軟化 |
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| Research Abstract |
Alkalophilic Bacillus sp. Ya-B strain secretes alkaline elastase which has high activity in the high alkaline range. Bacillus subtilis transformed with a plasmid carrying the alkaline elastase structural gene alone does not result in the production and/or secretion of active elastase. B. subtilis transformed with a plasmid carrying the cloned elastase gene and its 5'-flanking region (about 800 bp), however, can secrete active elastase. We determined the whole nucleotide sequence of the 5'-flanking region and found an open reading frame encoding 49 amino acids. The gene named aleR seems to be necessary for the expression of elastase gene on B. subtilis. We postulate that AleR protein is an activating enzyme of pro-elastase.
On the other hand, alkaline elastase is expected as a potential meat tenderizer. It is known that the pH of raw meat is 5-6. We tested the meat tenderizing activity of alkaline elastase at pH 6.0. The enzyme still shows substantial activities toward collagen and elastin with small effects to myofibril. The enzyme, therefore, is proven to be suitable for meat tenderizing use.
Finally we studied the relationship between the structure and activity of elastase. Alkaline elastase has a sequence homology of 55% to subtilisin BPN' and subtilisin E. From the comparison of primary amino acid sequence, we introduced 4 acid amino deletion into subtilisin E, and observed a distinct alteration of subtilisin E to an elastase-like enzyme.
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