2007 Fiscal Year Final Research Report Summary
Exo-β-glucosaminidase from Amycolatopsis orientalis, : Reaction mechanism and Application to industrial sugar production
| Project/Area Number |
17580085
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kinki University |
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Principal Investigator |
FUKAMIZO Tamo Kinki University, Department of Advanced Bioscience, Professor (50181243)
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| Project Period (FY) |
2005 – 2007
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| Keywords | chitosan / glucosaminidase / Reaction Specificity / catalytic residue / real-time monitoring |
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| Research Abstract |
Exo-β-glucosaminidase gene from Amycolatopsis orientalis was cloned, sequenced, and expressed in Streptomyces lividans TK24. The purified enzyme from the culture supernatant was tested for its reaction specificity. The enzyme was found to hydrolyze the β-1,4-glycosidic linkage of G1cN-G1cNAc in addition to G1cN-G1cN. Site-directed mutagenesis of Asp469 and Glu541 revealed that Asp469 is a proton donor and Glu541 a nuclephile. To more efficiently analyze the emzymatic reaction, we used continuous flow ESI-MS. The time-courses of the enzymatic hydrolysis of oligosaccharide substrates obtained by ESI-MS were found to be consistent with those obtained by HPLC. Furthermore, the amounts of the enzyme and substrate required for such a time-course determination were much lower than those required for determination by HPLC. The continuous flow ESI-MS was found to be efficient for characterizing the enzymatic reaction.
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Research Products
(14 results)
