Recombinant Cyclomaltodextrinase from Massilia timonae: Expression, Purification and Enzymatic Characterization

Introduction: Starch degradation is essentially performed by four groups of enzymes: endo and exo-amylases acting primarily on a(1,4)-linkages, debranching enzymes attacking mainly the a(1,6)-linkages, and cyclodextrin glycosyltransferases that degrade starch by catalyzing cyclisation and giving rise to cyclodextrin. Cyclomaltodextrinase (CDase) is the enzyme responsible for hydrolyzing cyclodextrin forming linear oligosaccharides of a (1,4)-linkages. Methods: A putative CDase encoding 1800-bp gene from Massilia timonae was PCR amplified from the bacteria genomic DNA, without the signal sequence, cloned, and expressed in E.coli BL21 StarTM (DE3)®. The histidine-tagged recombinant form of the enzyme was purified by immobilized metal ion affinity chromatography, and its catalytic properties were examined with the method of reducing sugar released, using β-cyclodextrin (β-CD) as substrate. Results: The enzyme optimal temperature was 40 °C and optimal pH was 7.0. It was able to remain relatively stable between 30 °C and 45 °C, after 30 minutes at the respective temperatures. The temperature at which the M. timoniae CDase loses 50% of its activity (T50) was 49.6 °C. The best substrate was β-CD, but the enzyme could use in much lower order starch and maltodextrin, measured by color decrease of the iodine complex. The chromatography revealed the ability of the purified recombinant CDase to hydrolyze oligosaccharides as maltotetraose and maltoheptaose, releasing glucose and maltose. The KM for β-CD was 2.38 mg/mL. The enzyme Vmax was 83.3 micromol/mL.min-1. Calcium (Ca2+) acted as activator, and detergents, such as SDS, acted as strong inhibitors. EDTA also did inhibit the enzyme, probably by chelating a divalent cation that could act as an activator, as Ca2+, for instance. Conclusions: The novel characterized CDase represents an addition for the enzymatic hydrolysis of starch and might be a possibility for industrial applications.