Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis.
The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. The gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).
Kannan
R
R
Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, India.
Mukundan
G
G
Aït-Abdelkader
N
N
Augier-Magro
V
V
Baratti
J
J
Gunasekaran
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D013393:Sucrase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D006602:Hexosyltransferases; C021513:levansucrase; D013393:Sucrase