CLONING OF THE SUCROSE PHOSPHORYLASE GENE FROM LEUCONOSTOC-MESENTEROIDES AND ITS OVEREXPRESSION USING A SLEEPER BACTERIOPHAGE VECTOR

The sucrose phosphorylase gene was cloned from Leuconostoc mesenteroides ATCC 12291 and was overexpressed in Escherichia coli using a 'Sleeper' bacteriophage vector. A recombinant phage slp-spl-1, which had four copies of the sucrose phosphorylase gene, was lysogenized into E. coli 1100. After heat induction, this lysogen produced 55.7 units per ml culture of sucrose phosphorylase, i.e. 80-times higher than that in L. mesenteroides. As the amount of this recombinant enzyme was over 30% of the soluble protein in the cell, E. coli 1100 (slp-spl-1) succeeded in overcoming problems such as, inhibited enzymes, in the case of L. mesenteroides. Thus it is possible to achieve an industrial-scale production of sucrose phosphorylase. The complete nucleotide sequence showed that it coded for a 490-amino acid protein of M(r)55,749. Homology between the deduced amino acid sequences for the L. mesenteroides sucrose phosphorylase gene and Streptococcus mutants gtfA, sucrose phosphorylase gene, was 68%.