CHARACTERIZATION OF RECOMBINANT AMYLOMALTASE FROM Thermus filiformis JCM 11600 AND STABILITY IMPROVEMENT BY SITE-DIRECTED MUTAGENESIS

Abstract

Amylomaltase (EC 2.4.1.25) catalyzes the α-1,4 glycosyl transfer within or between the glucan molecules to produce cyclic large-ring cyclodextrins (LR-CDs) or linear oligosaccharides/glycosides, respectively. The amylomaltase gene from Thermus filiformis JCM11600 (TfAM) was cloned and the sequence consisted of 1,458 bps. The gene was expressed in Escherichia coli BL21(DE3), then the enzyme was purified. TfAM encoded the polypeptide of 485 amino acid residues, the shortest among Thermus amylomaltases, with a 70 % sequence identity to amylomaltases from other Thermus and a calculated molecular mass of 55.47 kDa and pI of 5.11. Highest disproportionation activity occurred with maltotriose substrate at pH 6.5 and 60 °C to produce linear oligosaccharides. However, highest cyclization activity was observed at pH 5.0 and 70 °C, resulting in large-ring cyclodextrins with CD22 as the smallest and CD24-CD29 as principle products. Although TfAM had optimum condition at high temperature, the enzyme still lost 80% of its disproportionation activity after incubation for 2 hrs at pH 9.0 or 1 hr at 90 °C. Site-directed mutagenesis at Glu27 was chosen in the attempt to improve enzyme stability. Substitution of E27 by R, Q, F and V was performed. Characterization of mutated enzymes revealed two interesting mutants, E27F with a change in substrate specificity from G3 to G5 and E27R with a higher alkaline and thermo- stability as compared to WT-TfAM. E27R forms an Arg cluster (R27-R30-R31-R34) on the enzyme surface, showed a significant increase in stability at extreme pH and temperature and a shift towards higher pH and temperature optima in cyclization reaction. Conformational change of E27R at pH 9.0 and temperature above 350 K was observed through the circular dichroism spectra and the thermal transition profiles.