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 (Pall NK33) was purified and characterized. Pall NK33 converts sucrose to 76.8% palalinose, 21.2% tehalulose and 1% each of glucose and fructose, The purified PaII NK31 showed the very high specific activity at 2162 U/mg and K-m for sucrose was 42.7 +/- 0.75 rnM (at pH 6.0 and 30 degrees 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 (RLDRD329)-R-325 to (325)RYDRA(329) reduced enzyme activity 24-fold and did not affect ratio of palatinose and trehalulose formation. (c) 2006 Elsevier Inc. All rights reserved.