Molecular cloning of a gene encoding the sucrose phosphorylase from Leuconostoc mesenteroides B-1149 and the expression in Escherichia coli

Leuconostoc mesenteroides NRRL B-1149 sucrose phosphorylase (SPase) gene, 1149sp, was isolated and characterized. It is composed of 1479 bp nucleotides and encodes a 1149SPase of 492 amino acid residues with a calculated molecular mass of 56.1 kDa. It has unique C-terminal amino acid sequence (439DVETPSDTTIKITRKDKSGENVAVLVANAADKTFTITANGEEILANTEADKQQL492). 1149sp was expressed in Escherichia coli and the purified 1149SPase specific activity was 1.49 U/mg for sucrose. The optimum temperature and pH for SPase activities were ranged broad between 20 and 50 °C, between pH 6.0 and 7.5, respectively. The optimum temperature and pH were 37 °C at pH 6.7 and it showed Km of 6.3 mM and kcat of 1.59 s−1 for sucrose. It had a broad range of acceptor specificity and transferred the glucosyl moiety of sucrose or glucose-1-phosphate to various acceptors.