Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium

Melanie Baudrexl,Wolfgang H. Schwarz,Wolfgang Liebl,Vladimir V. Zverlov

doi:10.1038/s41598-019-52251-0

Melanie Baudrexl, Wolfgang H. Schwarz + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-52251-0

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Abstract

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-l-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-l-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.

Highlights

The classification system of glycoside hydrolases (GH) in the Carbohydrate Active enZymes database CAZy1 is based on sequence similarity and secondary structure
The first 72 hits of a BLASTp search with the sequence of Ts_Ram106B as query sequence and all proteins listed in the non-classified Carbohydrate Active Enzymes database1 (CAZy) GH family with either over 70% query coverage (QC) or sequence identities (ID) above 20% are shown in Table S1 of the supplementary data
Three proteins from thermophilic and hyperthermophilic bacteria were selected for further characterisation

Summary

Introduction

The classification system of glycoside hydrolases (GH) in the Carbohydrate Active enZymes database CAZy1 (www.cazy.org) is based on sequence similarity and secondary structure. In GH family 2 (GH2), activities on 23 different substrates have been observed so far, which shows exemplarily that enzymes of the same family, having high sequence and structural identity, can catalyse the hydrolysis of different substrates This demonstrates the limits of bioinformatics when it comes to predicting the true enzymatic function of a new protein, which requires experimental proof. Rhamnosidases are useful for debittering, due to the less bitter taste of the de-rhamnosylated flavanones[5], for rhamnose production[6], and for the determination of the anomeric configuration in polysaccharides, glycosides and glycolipids[7] These enzymes may enhance wine aroma[8] and flavonoid bioavailability[9], or assist in the synthesis of pharmaceuticals[10]. For instance, the bitterness causing substance in grapefruit, only about 7 g/l are soluble at 55 °C, whereas at 75 °C more than 100 g/l can be dissolved in water[19]

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