Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation.
An open reading frame encoding a putative bi-functional beta-D-xylosidase/alpha-L-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual beta-D-xylosidases and alpha-L-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75 degrees C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like D-glucose and L-arabinose.
Morana
Alessandra
A
Istituto di Biochimica delle Proteine, CNR, Via Pietro Castellino 111, 80131, Naples, Italy.
Paris
Ornella
O
Maurelli
Luisa
L
Rossi
Mosé
M
Cannio
Raffaele
R
D001089:Arabinose / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal; D005947:Glucose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D020836:Protein Structure, Quaternary; D019642:RNA, Archaeal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D011134:Polysaccharides; D019642:RNA, Archaeal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014990:Xylans; C030080:araban; C085118:arabinoxylan; D001089:Arabinose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase; D005947:Glucose