Mechanistic Study of β- Glucosidase from Flavobacterium meningosepticum
碩士 === 國立交通大學 === 應用化學研究所 === 86 === A β-Glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in E. coli. The cloned enzyme was purified to homogeneity by following ammonium sulfate fractionation and a Hitrap SP column applying at pH6.9 and pH6.6, consecutively. The...
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| Format: | Others |
| Language: | zh-TW |
| Published: |
1997
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| Online Access: | http://ndltd.ncl.edu.tw/handle/02844049874567348033 |
| Summary: | 碩士 === 國立交通大學 === 應用化學研究所 === 86 ===
A β-Glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in E. coli. The cloned enzyme was purified to homogeneity by following ammonium sulfate fractionation and a Hitrap SP column applying at pH6.9 and pH6.6, consecutively. The concentrated enzyme (0.4mg/ml) was stable at 4 ℃ (pH 6.9, 20mM phosphate buffer) up to 30 days. The optiomal pH is at pH0.5. Glucose presents a productinhibition with Ki=4.6 mM. The enzyme displayed a restrict specificity on the glycon portion of substrate. Km value of p-nitrophenyl β-D-glucopyranoside (PNPG) for the cloned enzyme is 0.815 mM, which is similar to that of wild type (0.65mM).
By NMR spectroscopy study, the enzyme catalyzes hydrolysis of p-nitrophenyl β-D-glucopyranoside(PNPG) with the retention of anomeric configuration. A preliminary study of Bronsted relationship showed a concaveg-downward plot. These provide a strong indication for a two-step mechanism: formation (step 1)and breakdown (step 2)of glucosylenzyme intermediate. The reaction rate-limiting-step (r. l. s.) of good substrates (pKa of leaving group<7) is the breakdown of glucosy-enzyme intermediate whereas the r.l. s. of poor substrates (pKa of leaving group>7) is the formation of the intermediate. The Bronsted constant for poor substrates is β1g= -0.8. Suggest that an extensive amount of charge be developed on the oxygen atom of the leaving phenols at eh transition-state.
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