| Primary alcohol dehydrogenase, AdhA, is a key enzyme of ethanol metabolic pathway in thermophilic anaerobic bacteria. When researching in the field thermophilic fermentation of cellulosic ethanol, Thermoanaerobacter ethanolicus is achieved biggish attentions, because fermentation of Thermoanaerobacter ethanolicus offers the potential to produce ethanol from lignocellulose and to separate ethanol in continuous cultures during thermophilic growth.The adhA gene encoding primary alcohol dehydrogenase from Thermoanaero-bacter ethanolicus JW200was subcloned into pHsh, resulting in pHsh-adhA and was overexpressed in E. coli DH10B. The plasmids of pTE-ori-adhA and pTE-ori-adhE-adhA were the T. ethanolicus-E.coli shuttle vectors that were constructed from pTE-ori and pTE-ori-adhE. AdhA was successfully purified by an application of ion-exchange chromatography (DEAE-Sepharose Fast Flow) and Cibacron Blue3GA Agarose affinity chromatography. The purified enzyme showed a single protein band on SDS-PAGE. Studying on the characterization of AdhA, the optimal activity of AdhA catalyzing acetaldehyde to ethanol was determined at pH7.79and60℃, but when the temperature higher than65℃or the pH bigger than8.0, it could lead to the sharp reduction of enzyme activity. The optimal activity of AdhA catalyzing ethanol to acetaldehyde was determined at pH5.0and70℃, when the temperature above85℃or the pH under4.0and the pH above6.0, the enzyme had a sharp reduction. The purified enzyme AdhA was stable over a pH range from5.8to7.2with a residual activity above80%, when the pH was higger than7.5, the enzyme was not stable. AdhA was stable at45℃and50℃for2hours with a residual activity above80%and had a half life of1hour at55℃. Zn2+could make a great effect on its activity, whereas, the presence of Cu2+, Fe+and SDS could reduce enzymatic activity.AdhA did act a typical alcohol dehydrogenase under the optimal conditions. The activity for acetaldehyde to ethanol (10.88±0.20U/mg) was much higher than that for ethanol to acetaldehyde (8.32±0.14U/mg or8.72±0.18U/mg) showed that AdhA promoted ethanol production. The ethanol concentration rising (20mmol/L to1%) led to the ability of ethanol consumption stronger (8.32±0.14U/mg to8.72±0.18U/mg). Under the physiology condition, because of numerous restrictions factors such as temperature, pH, substrate and coenzyme concentration, AdhA was highly active to catalyze ethanol to acetaldehyde and exhibited only a very weak activity for acetaldehyde to ethanol. At the same time, the activity for ethanol consumption in the high ethanol concentration (1%) level was much higher than that the low ethanol concentration level (2mmol/L), specific enzyme activity was from0.27±0.01U/mg (pH7.2) and0.17±0.0U/mg (pH6.6) to1.56±0.05U/mg (pH7.2) and11.42±0.06U/mg (pH6.6). All of these results have already indicated that AdhA catalyzes ethanol consumption under physiology condition.According to the methods of Ultrasonictransformation and chloramphenicol resistance screening, we got four recombinant bacteria T. ethanolicus JW200(pTE-ori), T. ethanolicus JW200(pTE-ori-adhE), T. ethanolicus JW200(pTE-ori-adhA) and T. ethanolicus JW200(pTE-ori-adhE-dhA). We found the results that AdhE catalyzed ethanol formation and AdhA promoted ethanol consumption through measuring four kinds of recombinant bacterials ethanol yield. When they were all in T. ethanolicus JW200, the activity for ethanol production of AdhA activity was much higher than that for ethanol consumption of AdhE activity. |
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