| Project/Area Number |
13440194
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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 |
Inorganic chemistry
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| Research Institution | Kanazawa University |
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Principal Investigator |
SAKURAI Takesi Kanazawa University, Faculty of Science, Prof., 理学部, 教授 (90116038)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,200,000 (Direct Cost: ¥14,200,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥10,900,000 (Direct Cost: ¥10,900,000)
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| Keywords | Multicopper Oxidase / Loccase / Bilirubin Oxidase / Dioxygen Reduction / Reaction Intermediate / Heterologous Expression / Mutant / Trinuclear Cu Cluster / 銅タンパク質 / 三核銅センター / アミノ酸配列 / ビリルビンオキシターゼ / 活性酸素 / 反応機構 |
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| Research Abstract |
The amino acid sequences of the two isozymes of Rhus vernicifera laccase were determined from their cDNA's and the amino acids to construct the four copper-binding sites and the N-type carbohydrate-binding sites were determined. Tree laccase was expressed as an inclusion body in E.coli. When Pichia pastoris was used as host, laccase was transcribed as mRNA but was not translated as protein. Therefore, laccase gene was designed and synthesized to be suitable for the heteologous expression of the tree enzyme in prokaryote and yeast. The novel heteologous expression system of bilirubin oxidase was constructed using Pichia pastoris as host. The recombinant enzyme obtained by using this overexpression system showed more than twice enzyme activity than the authentic enzyme, and also showed high thermostability, being suitable to use for the clinical test of liver. Various bilirubin oxidase mutants were formed using this novel expression system. The substitutions of the His residues for type 3 Cu's by the coordinating amino acids such as Lys and Asp gave mutants whose enzyme activities were much decreased. The mutation on the Asp residue positioned near the trinuclear center was fatal as to give the reduced Cu content. Therefore, it appears that this amino acid as a potential proton source is also indispensable to construct the trinuclear center. The mutation of Cys for type I Cu gave the mutant in which type 1 Cu site was vacant, allowing us to trap a reaction intermediate, dioxygen-reduced species, which was not detected during the reaction of the authentic enzyme.
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