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

作者：Srisimarat Wiraya; Powviriyakul Areeya; Kaulpiboon Jarunee; Krusong Kuakarun; Zimmermann Wolfgang; Pongsawasdi Piamsook^*

来源：Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2011, 70(3-4): 369-375.

DOI：10.1007/s10847-010-9890-5

• Large-ring cyclodextrin
• Amylomaltase
• Transglucosylation
• Cyclization
• Corynebacterium

Amylomaltase catalyzes the formation of largering 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 (20-25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 degrees C for 2 h after induction with 0.4 mM isopropyl thio-beta-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-CD50, with CD27-28 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.

• 出版日期2011-8