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This work aims to identify essential tryptophan residue(s) of amylomaltase from Corynebacterium glutamicum ( Cg AM) through chemical modification and site-directed mutagenesis techniques. The recombinant enzyme expressed by Escherichia coli was purified and treated with N -bromosuccinimide (NBS), a modifying agent for tryptophan. A significant decrease in enzyme activity was observed indicating that tryptophan is important for catalysis. Inactivation kinetics with NBS resulted in pseudo first-order rate constant ( k inact ) of 2.31 min −1 . Substrate protection experiment confirmed the active site localization of the NBS-modified tryptophan residue(s) in Cg AM. Site-directed mutagenesis was performed on W330, W425 and W673 to localize essential tryptophan residues. Substitution by alanine resulted in the loss of intra- and intermolecular transglucosylation activities for all mutated Cg AMs. Analysis of circular dichroism spectra showed no change in the secondary structure of W425A but a significant change for W330A and W673A from that of the WT. From these results in combination with X-ray structural data and interpretation from the binding interactions in the active site region, W425 was confirmed to be essential for catalytic activity of Cg AM. The hydrophobicity of this tryptophan was thought to be critical for substrate binding and supporting catalytic action of the three carboxylate residues at the active site.

LsRN from Bacillus licheniformis was cloned and expressed in Escherichia coli. From a 1793 bp genomic sequence, the lsRN gene was found to be composed of a single 1446 bp ORF with a putative promoter consensus boxes and a ribosome-binding site. This ORF was predicted to encode for 482 amino acid residues. The LsRN was constitutively expressed at a relatively high level without sucrose induction. The enzyme was highly purified and an apparent size of 52 kDa with an optimum temperature and pH of 50 °C and 6.0 were determined. The wide range of M(w) of levan (1-600 kDa) was synthesized in a controlled reaction with two variable parameters: temperature and ionic strength. At high temperature (50 °C), LsRN synthesized high M(w) levan (612 kDa) as a major product while at low temperature (30 °C), low M(w) levan (11 kDa) was mainly synthesized. When 0.5M NaCl was added into the reaction, the major products at both temperatures were of the size 11 kDa. Moreover we report for the first time, an enzymatic synthesis of levan nanoparticles (NPs) by a single step reaction. The LsRN synthesized levan NPs as agglomerate with average particle size of 50 nm. The encapsulation of O-acetyl-α-tocopherol was carried out to demonstrate the applicable use of levan NPs.