| Project/Area Number |
61860009
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | Nagoya University |
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Principal Investigator |
UDAKA Shigezo Nagoya University, Professor, 農学部, 教授 (70023463)
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| Co-Investigator(Kenkyū-buntansha) |
TSUBOI Akio Nagoya Univ., Research associate, 農学部, 助手 (20163868)
YAMAGATA Hideo Nagoya Univ., Associate Professor, 農学部, 助教授 (20023468)
TSUKAGOSHI Norihiro Nagoya Univ., Associate Professor, 農学部, 助教授 (50115599)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1987: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1986: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | Secretion of Useful proteins / Bacillus brevis / Production of interleukin 2 / Production of beta-amylase / バチルス ブレビスの分泌ベクター / 蛋白質生産菌 / 有用蛋白質の高効率生産 / 分泌生産ベクター / Bacillus brevis / β-アミラーゼ |
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| Research Abstract |
Bacillus brevis was found to be a potential protein-producing bacterium. Since major exoprotein is identical to the cell surface proteins, the DNA region which controls expression and secretion of the exoprotein was inserted to a high-copy numberes plasmid to construct an expression and secretion vector, PNU 200. This vector was utilized to produce various usefull proteins in B. brevis.
1. production of human interleukin 2 (IL2). The IL2 gene was inserted to the downstream of signal sequence of B. brevis major cell surface gene in pNU 200. This plasmid was introduced into B. brevis 47. One - 2 mg/l of IL2 was produced extracellularly, but IL2 was partially degraded with 2 longer cultivation. Then, the same plasmid was transferred to B. brevis HPD 31 which has no detectable exoprotease. By optimizing culture conditions, now about 20-30 mg/l of IL2 was produced without noticiable degradation of IL2.
2. Production of bacterial beta-amylase. Beta-amylase gene of Bacillus nolymyxa was ligated to the downstream of signal sequence of pNU 200. By transforming B. brevis 47 with this plasmid, the transformant produced a large amount of beta-amylase (about 0.5 g/l) under appropriate cultural conditions. Enzymatically active beta-amylase was obtained even by deleting DNA coding for a large C-terminal region of the enzyme, revealing the necessary region of beta-amylase.
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