The Zymomonas mobilis gene sacB that encodes the extracellular levansucrase was cloned and expressed in Escher~chia coli. The gene product exhibited both sucrose hydrolysis activity and levan forming capability. Sub-cellular fractionation ofE. coli carrying pLSS41 revealed that about 95% of the total sucrase activity was detected in the cytoplasmic fraction. The levansucrase gene was overexpressed (about hundred fold) in E. coti under T7 polymerase expression system. Nucleotide sequence analysis of this gene revealed an open reading frame of 1269 bp long coding for a protein of 423 amino acids with a molecular mass of 46.7 KDa. The deduced amino acid sequence was identical to the N-terminal amino acids of protein A51 ofZ. mobilis ZM4. Therefore, the product ofsacB is levansucrase. This is the first extracellular enzyme of Z. mobilis sequenced which does not possess a signal sequence.This gene is located 198 bp upstream ofsacC gene encoding for the extracellular sucrase forming a gene cluster INTRODUCTION

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%).

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%).