Molecular cloning and characterization of a novel GH13 saccharifying alpha-amylase AmyC from Corallococcus sp EGB

摘要

A novel amylase AmyC gene (amyC) from Corallococcus sp. EGB was cloned and expressed in Esherichia coli. Analysis of the sequence homology showed that AmyC shared 92% identity with the putative -amylase from Corallococcus coralloides DSM 2259 and formed a separate branch from other amylases. The recombinant AmyC (rAmyC) was purified and biochemically characterized. rAmyC hydrolyzes various starch and maltooligosaccharides larger than G4 to glucose to maltotetraose without production of other oligosaccharides, indicating that it is a special saccharifying -amylase. AmyC possesses weak but significant -1, 6-bond cleaving activity and transglycosylation activity. The time course of the reaction with maltotriose as a substrate evidently showed the formation of oligosaccharide of degree of polymerization (DP4) by glycosyl transfer. The substrate specificities and end products analysis showed that the hydrolytic pattern of rAmyC is unique. rAmyC exhibited maximum hydrolysis activity at 50 degrees C in 50mM sodium phosphate buffer (pH 6.0) with a specific activity of 180U/mg. Its activity towards starch was independent of calcium ions and activated by dithiothreitol. These results indicate that AmyC is a novel endo multifunctional amylase possessing distinct characteristics.