Enzymatic and computational analysis of large-ring cyclodextrin production by Corynebacterium glutamicum amylomaltase

Abstract

Amylomaltase is a well-known glucanotransferase enzyme that can produce large-ring cyclodextrins (LR-CDs) via cyclization reaction. To determine important amino acid residues involved in LR-CDs production of amylomaltase from Corynebacterium glutamicum (CgAM), mutations at CA I and CA II subdomains were introduced by site-directed mutagenesis. P228Y, E231Y, A413F and G417F CgAM mutants were constructed from CgAM recombinant plasmid. The optimum pH and temperature of WT and mutants were around pH 6.0 - 7.5 and 25 - 35°C. The mutations did not affect the pH and temperature stability. In addition, WT and all mutated enzymes have Maltotriose (G3) as the best substrate for disproportionation activity. A413F possessed much lower specific activities of transglycosylation, starch degradation, disproportionation and cyclization activities than the WT enzyme. P228Y, E231Y and G417F exhibited similar specific activities of starch transglycosylation and starch degradation to the wild-type CgAM. P228Y had much lower disproportionation activity than the wild-type enzyme, while E231Y displayed higher disproportionation activity. This suggested that P228 and E231 of CgAM may play an important role in disproportionation. At 12 h, wild-type CgAM gave the principle CD of 27, whilst the principal product of P228Y, E231Y, A413F and G417F were CD36, CD25, CD38, and CD32, respectively. Molecular dynamic (MD) simulation showed that Y418, M474, L510, F534, Y23, R458, Q423, T666, Q475, L236, Q421, E231, Q420, N419 and Y235 might interact with LR-CDs.