Cloning and characterisation of alternansucrase from Leuconostoc citreum ABK-1 and role of surface aromatic acids on product size

Abstract

The alternansucrase (ALT, EC 2.4.1.140) catalyses transferring of glucose from sucrose to produce a polymer with α-1,6 and α- 1,3 linkages, so-called “alternan”. The ALT-encoding gene from Leuconostoc citreum ABK-1 (WTalt) was successfully cloned and expressed in Escherichia coli BL21 (DE3). The two versions of truncated SH3-like domains, ∆3SHALT and ∆7SHALT, were successfully constructed. The WTALT, ∆3SHALT and ∆7SHALT possessed optimum temperature of 40 °C, while optimum pH of WTALT was at 5.0 but ∆3SHALT’s and ∆7SHALT’s were shifted to pH 4.0. Their activities could be enhanced by Mn²⁺. Kinetic studies of all ALTs were mainly responsible for transglycosylation activity with less detectable hydrolytic activity in the ∆3SHALT and ∆7SHALT. Deletion of SH3-like domains affected enzyme stability and size of products compared with WTALT. The ALTs’ product patterns were not significantly dissimilar judged by NMR, methylation analysis and HPAEC-PAD. However, ∆3SHALT and ∆7SHALT produced higher the amount of oligosaccharides compared to WTALT. Importantly, the polymer product harbours irregular pattern of alternating α-1,6 and α- 1,3 glycosidic linkages as confirmed by NMR, methylation analysis, and partial hydrolysis. The polymer can undergo self-assembly, forming nanoparticles in solution. At concentrations above 15% (w/v), nanoparticles disassembled and formed a high viscous solution, while a transparent film was formed once polymer was dried. Additionally, the mutation at W675 position effected on sizes of products without detectable polymer. W675A significantly revealed the loss of acceptor binding ability, resulted in the production of different product patterns compared with the WT (∆7SHALT).