A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20?C25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-??-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19?CCD50, with CD27?C28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.     

Production of Large-Ring Cyclodextrins by Amylomaltases

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.