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

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.

Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity

“…Additionally, cation dependence, and to some extent also their temperature preference for activity (maximum activity in a 20 min assay between 82 and 93 °C), as well as thermal stability, were all similar. Like some other glycoside hydrolases (Ahmed et al, 2013;Baudrexl et al, 2019), Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A are activated by divalent metal ions. The greatest effect was observed with either MnCl 2 or CaCl 2 ions (Figure 3).…”

“…Cloning of pET24c(+) constructs was accomplished by Gibson Assembly ( Gibson et al, 2009 ) using purified PCR product and restricted vector as described above, followed by transformation of strain E. coli DH10B. Sanger sequencing at Eurofins Genomics (Ebersberg, Germany) verified sequence integrity before enzyme production in E. coli BL21 Star™, isolation and purification by immobilized metal affinity chromatography (IMAC) was carried out as described previously ( Baudrexl et al, 2019 ). The eluates from IMAC were incubated at 50°C for 15 min to denature remaining E. coli proteins followed by precipitate removal by centrifugation.…”

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

“…Samples were taken after 24 h and 48 h of incubation (enzymes in GM) or after 4 h and 24 h (enzymes in buffer). Released sugars were determined by thin layer chromatography (TLC), as described previously (Baudrexl et al 2019 ), and DNSA method (Miller 1959 ) to determine reducing ends.…”

Variimorphobacter saccharofermentans gen. nov., sp. nov., a new member of the family Lachnospiraceae, isolated from a maize-fed biogas fermenter

“…Additionally, thin-layer chromatography (TLC) was utilized to determine if the three strains release any carbon source degradation products into the culture supernatant. TLC was performed as described by Baudrexl et al [53]. The culture supernatant samples were applied on silica gel 60 plates (Merck) using acetonitrile-water 80:20 (v/v) as eluent.…”