Thermostable Amylosucrase from Calidithermus timidus DSM 17022: Insight into Its Characteristics and Tetrameric Conformation

Abstract

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0−8.0 and 30−60 °C.

Structure-based interface engineering methodology in designing a thermostable amylose-forming transglucosylase

“…Therefore, we have mined a novel ASase from Calidithermus timidus DSM 17022 (CT-ASase). CT-ASase exists as a homotetramer in solution ( 14 ), different from other reported monomeric and dimeric ASase. Although its thermostability still cannot meet industry needs to keep stable above 60 °C, its unique homotetrameric organization may become a key factor to improve its thermostability.…”

“…Expression and purification of recombinant CT-ASase and its variants were conducted as described in a previous study ( 14 ). The gene encoding CT-ASase and its variants were fused with the sequence encoding a 6× histidine tag at the 3'-terminus and cloned into a pET-22b (+) vector.…”

“…Samples containing 5 mg/ml protein were heated from 25 to 100 °C at a heating rate of 1 °C/min, and the data were analyzed using CFX Manager and DMAN software. After preliminary screening by DSF, nano-differential scanning calorimetry was used to accurately determine the T m value of mutants using the same method as described in the previous study ( 14 ). To investigate kinetic stability, CT-ASase and its variants were preincubated without substrate at 65 and 70 °C.…”

A Novel Thermostable Keratinase from Deinococcus geothermalis with Potential Application in Feather Degradation

“…Thermus and Calidithermus bacteria generally grow at medium and high temperature, and their keratinases might have high temperature range and temperature stability, such as MtaKer [11] and aqualysin I [26]. The amylosucrase from Calidithermus timidus has high thermostability with a half-life of 1.09 h at 70 • C [27]. As mentioned above, DgeKer has a long C-terminal extension, as with aqualysin-I [26] and VPR from Vibrio sp.…”

Thermophiles and carbohydrate-active enzymes (CAZymes) in biofilm microbial consortia that decompose lignocellulosic plant litters at high temperatures

“…The draft genome of Calidithermus timidus DSM17022 indicated that the bacterium harbor seven AA, 38 GH, 16 CE sequences 49 . So far, only a GH57-glycogen branching enzyme 50 and GH13 amylosucrase 51 from this bacterium have been analysed in detail. Meiothermus spp.…”