Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae

Protein Expression and Purification
Fabiane Cristina Dos Santos, Ione Parra Barbosa-Tessmann

Abstract

Some microorganisms can produce cyclodextrin glycosyltransferases, which degrades starch by catalyzing cyclization and giving rise to cyclodextrin. Thus, to fully degrade starch, microorganisms can also synthesize cyclodextrinases, which hydrolyze cyclodextrins. In this work, a truncated gene, without the signal peptide coding sequence, encoding a cyclodextrinase from Massilia timonae was PCR amplified, cloned, and expressed in E. coli. The histidine-tagged recombinant enzyme was purified by immobilized metal ion affinity chromatography. The purified protein was found to be a tetramer of about 260 kDa, with monomers of about 65 kDa, as estimated by gel filtration and SDS-PAGE, respectively. The enzyme presented an optimum temperature of 40 °C, optimum pH of 7.0, and remained stable after 30 min of incubation at 45 °C, with a T50 of 48.45 °C. The enzyme showed a higher activity toward β-cyclodextrin compared to that for maltodextrin and starch. KM for β-cyclodextrin was 2.1 mM, Vmax was 0.084 μmol/min, kcat was 8326 min-1, and kcat/KM was 4.1 × 106 M-1min-1. Calcium acted as an activator and SDS, CTAB, several cations, and EDTA acted as strong inhibitors. The purified cyclodextrinase produced glucose and maltose as final product...Continue Reading

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Citations

Jan 15, 2021·3 Biotech·Bruna Yuki TagomoriIone Parra Barbosa-Tessmann
Jul 30, 2021·Microbiology Resource Announcements·Ariel A BradleyLoralyn M Cozy

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