| Project/Area Number |
13480174
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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 |
環境保全
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KINOSHITA Shinichi Hokkaido University, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (50029253)
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| Co-Investigator(Kenkyū-buntansha) |
OOI Toshihiko Hokkaido University, Graduate School of Engineering, Associate professor, 大学院・工学研究科, 助教授 (40223713)
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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 |
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2002: ¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 2001: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | Azo Dye / Microbial Decolorisation / Azoreductase / アゾレダクターゼ / Bacillus sp. / azo dye / azoreductase / gene / enzyme / 脱色分解 / 脱色酵素 / Aeromonas sp. / Trichoderma sp. / オレンジII / コンゴーレッド |
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| Research Abstract |
Orange16 was efficiently decolorized by five kinds of bacterial mixed culture, isolated from the effluents of dyeing company, up to the 120 ppm and repeated decolorization was achieved under the oxygen limiting condition. A bacterium was isolated from this mixed culture and identified as Aeromonas sp. Aer2-l. Optimum conditions for Orange 16 decolorization were determined. Orange 16 was efficiently decolorized up to 1200 ppm and completely decolorized at 750 ppm on 50 h.
Azoreductase from isolated bacterium, identified as Bacillus sp. B29 was purified using several column chromatographies. Purified enzyme showed homodimer having MW of 68 kDa. and stable at pH 6.0 and less than 60oC. Optimal conditions for Orange II decolorization were pH 6.5 at 55oC. The enzyme was required NADH for decolozation. The enzyme showed highly activity to Orange 16 and Now cossin but no activity to diazo compounds.
Azoreductase genes from Bacillus sp. B29 were cloned. Three kinds of DNA fragment were amplified by PCR and sequenced. Predicted ORF from DNA sequences had MW of about 24 kDa. which different from purified enzyme described above. The azr genes (azr6, azr8, and azr18) were expressed using Escherichia coli expression system. Azr8 was purified and characterized. MW was calculated as 24 kDa. and the enzyme showed monomer protein. Azr8 required NADH for activity but not NADPH and FMN and FAD were stimulated the activity The enzyme had highly activity to Methyl Red and Ethyl Red but not active Orange G and Orange II. Diazo compounds such as Trypan Blue and Conge Red were decolorized slightly by the enzyme.
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