Abstract
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 (20–25%) 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-β-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.
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Abbreviations
- LR-CD:
-
Large-ring cyclodextrins
- CA:
-
Cycloamylose
- CD:
-
Cyclodextrin
- 4αGTase:
-
4-α-Glucanotransferase
- IPTG:
-
Isopropylthio-β-D-galactoside
- SDS-PAGE:
-
Sodium dodecylsulfate-polyacrylamide gel electrophoresis
- TLC:
-
Thin-layer chromatography
- HPAEC:
-
High performance anion exchange chromatography
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Acknowledgments
WS was financially supported by the Royal Golden Jubilee PhD Fellowship from the Thailand Research Fund. Financial support from the Ratchadapiseksomphot Endowment Fund of Chulalongkorn University to the Starch and Cyclodextrin Research Unit and from Research Institution Partnership Grant of Alexander von Humboldt Foundation are acknowledged. The authors also acknowledge the support from the Thai Government Stimulus Package 2 (TKK 2555) under the Project PERFECTA.
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Srisimarat, W., Powviriyakul, A., Kaulpiboon, J. et al. A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products. J Incl Phenom Macrocycl Chem 70, 369–375 (2011). https://doi.org/10.1007/s10847-010-9890-5
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DOI: https://doi.org/10.1007/s10847-010-9890-5