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

Leuconostoc mesenteroides NRRLB-1149 sucrose phosphorylase(SPase) gene, 1149sp, was isolated and characterized. It is composed of 1479bp 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 ((439)DVETPSDTTIKITRKDKSGENVAVLVANAADKTFTITANGEEILANTEADKQQL(492)). 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 degrees C, between pH 6.0 and 7.5, respectively. The optimum temperature and pH were 37 degrees C at pH 6.7 and it showed K-m of 6.3 mM and k(cat) 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. (c) 2005 Elsevier Inc. All rights reserved.