Cloning And Characterization Of Two Key Proteins Related To Carbon Catabolite From Thermoanaerobacterium Aotearoense SCUT27

Thermoanaerobacterium aotearoense SCUT27is capable of ferment a lot of cellulosicbiomass derived sugars，such as glucose,xylose, cellobiose, mannose, glucan and xylan,ect, toproduce hydrogen, ethanol and a mixture of organic acid, it is identified as the potentialbiofuel-producing species. In this study, I cloned and characterized two key proteins from T.aotearoense SCUT27during sugar utilization, which were Catabolite Control Protein A(CcpA) and β-xylosidase.We first cloned the deduced ccpA gene from T. aotearoense SCUT27. The upstream anddownstream sequences of the ccpA were amplified through genome walking technique.Sequence analysis results showed that the putative CcpA consisted of336amino acids with acalculated isoelectric point of5.7. The deduced-10sequence and-35sequence of promoterwere centered46bp and63bp upstream of the putative start codon, respectively. However, norho-independent transcription terminator was identified, indicating that the ccpA wastranscribed as a polycistronic as most of the prokaryotes.Functional complementation experiment confirmed the function of the cloned ccpA. TheCcpA from T. aotearoense SCUT27could repress the amyE gene expression in the presenceof glucose in B. subtilis. Further mixed sugar fermentation showed that the overexpression ofccpA in B. subtilis inhibited the cell growth and intensified the repression of xylose utilizationin the existence of glucose.Second, the β-xylosidase from T. aotearoense SCUT27was identified and expressed in E.coli BL21, which is an important enzyme in xylose utilization. The Kmand Vmaxvalues were48.2mM and925.9U/mg, respectively. Enzyme activity assay showed that the optimumtemperature and pH for purified β-xylosidase were65oC and6.5, respectively.