Role of several key residues in the catalytic activity of sucrose isomerase from Klebsiella pneumoniae NK33-98-8

The gene encoding sucrose isomerase (palI NK33) was cloned from Klebsiella pneumoniae strain NK33-98-8. The gene was over-expressed in Escherichia coli BL21 (DE3) pLysS and its enzyme product (PalI NK33) was purified and characterized. PalI NK33 converts sucrose to 76.8% palatinose, 21.2% trehalulose and 1% each of glucose and fructose. The purified PalI NK33 showed the very high specific activity at 2362 U/mg and Km for sucrose was 42.7 ± 0.75 mM (at pH 6.0 and 30 °C). The enzyme activity was completely inhibited by 1 mM concentration of either Hg2+ or SDS. Ca2+, Li2+ and Mg2+ at 1 mM slightly enhanced enzyme activity. Mutations on Asp140, located within the conserved sequence region I to either glutamic acid, glycine or asparagine had drastically reduced enzyme activity. The change of amino acid residues in the sequence 325RLDRD329 to 325RYDRA329 reduced enzyme activity 24-fold and did not affect ratio of palatinose and trehalulose formation.