| Project/Area Number |
10460035
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Aomori University (1999)
The University of Tokyo (1998) |
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Principal Investigator |
MATSUZAWA Hiroshi Aomori University, Department of Bioscience and Biotechnology; Professor, 工学部, 教授 (00011966)
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| Co-Investigator(Kenkyū-buntansha) |
FUSHINOBU Shinya The University of Tokyo, Department of Biotechnology; Assistant Professor, 大学院・農学生命科学研究科, 助手 (00302589)
WAKAGI Takayoshi The University of Tokyo, Department of Biotechnology; Associate Professor, 大学院・農学生命科学研究科, 助教授 (70175058)
KAMIIE Katsuyoshi Aomori University, Department of Bioscience and Biotechnology; Professor, 工学部, 助手 (70275519)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,800,000 (Direct Cost: ¥13,800,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1998: ¥11,200,000 (Direct Cost: ¥11,200,000)
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| Keywords | Glucanotransferase / Gene expression / Codon Usage / Thermostable enzyme / Crystal structure analysis / Site-directed mutagenesis / Cycloamylose / Archaeon / 4-α-グルカン転位酵素 / 転位酵素 / Glycosyl hydrolase / 環状糖 / X線結晶解析 / 部位特異的変異 |
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| Research Abstract |
4-α-Glucanotransferase (Gtase) of a hyperthermophilic archaeon, Thermococcus litoralis, synthesizes linear and cyclic α-I ,4-glucans from oligomaltose and amylose as substrates. Following results were obtained in this research.
(1) The Gtase gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the gene in E. coli resulted in low production of the enzyme and the accumulation of inclusion bodies. However, simultaneous expression of Gtase with tRNAィイD2AGAィエD2, tRNAィイD2AGGィエD2 and GroELS affected both the production and solubility of the enzyme, and production of soluble Gtase increased about 5-fold.
(2) This enzyme reaction was carried out through the Ping-Pong BiBi mechanism, when analyzed with substrates, 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (donor) and glucose (acceptor).
(3) This Gtase belongs to the family 57 of glycosyl hydrolases. No active site residues are identified in the family 57 enzymes. Comparing the amino acid sequences, 8 carboxylic resides were conserved in the family. Analyses of the mutant enzymes constructed by site-directed mutagenesis suggested that Gln 123, Asp 145, Asp2l4, Glu216, and Asp354 are present in or around the active site.
(4) Crystals of the enzyme were obtained by using ammonium sulfate and PEG400 as precipitants. The space group was P6ィイD24ィエD222, and the unit cell dimensions were a=b= 125 Å and c=246 Å.
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