Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

Abstract

Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative ␣-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was ␣,␣-1,1-trehalose, a new feature among ␣-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (␣-1,2 linkage), nigerose and turanose (␣-1,3), leucrose (␣-1,5), isomaltose and palatinose (␣-1,6), and maltose (␣-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous ␣-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the ␣-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the ␣-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the ␣-glucosidase), indicating that the ␣-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.␣-Glucosidases (EC 3.2.1.20) are a widespread group of enzymes that catalyze the hydrolysis of the ␣-glucosidic bond from the nonreducing end of a chain as well as the ␣-glucosidic bond of free disaccharides (21, 23). Many known ␣-glucosidases seem to prefer the ␣-1,4 bonds of maltose or maltooligosaccharides (21). However, the ␣-glucosidases from Thermus thermophilus strain GK24 and from Bacillus sp. strain SAM1606 preferentially hydrolyze the ␣-1,6 bond of isomaltose over other ␣ linkages (20,21,23). Trehalose is a natural disaccharide that is widespread in nature and that can serve multiple roles, namely, as a general stress protectant, a source of carbon and energy, a sensing and regulatory compound, and a structural element of the bacterial cell wall (11). The assimilation of trehalose as a carbon and energy source requires the activity of enzymes that hydrolyze the ␣-1,1 bond of this disaccharide, including trehalase (EC 3.2.1.28), trehalose phosphorylase (EC 2.4.1.64 and EC 2.4.1.231), and other ␣-glucosidases (EC 3.2.1.20) with broad specificity (14, 16). These enzymes may also play important roles in the regulation of the level of trehalose in a cell. Trehalose can also be converted into maltose by trehalose synthase (TreS; EC 5.4.99.16) and then metabolized via amylomaltase, maltose/maltodextrin phosphorylase, or other ␣-glucosidases with ␣-1,4-linkage hydrolytic activity (7,14,18). Although T. thermophilus HB27 lacks the genetic machinery for trehalose biosynthesis, this disaccharide is taken up from the medium (2, 24) but does not serve as a compatible s...

Characterization of Two Novel α-Glucosidases fromBifidobacterium breveUCC2003

“…However, the particular ␣-glucosidase activities described in this paper have not been reported for other characterized (bifido)bacterial ␣-glucosidases (1,6,9,21,28,34,54). For example, the food-borne pathogen Enterobacter sakazakii produces an ␣-glucosidase with activity toward palatinose (28), while an ␣-glucosidase isolated from Erwinia rhapontici showed strict substrate specificity toward palatinose.…”

“…For example, the food-borne pathogen Enterobacter sakazakii produces an ␣-glucosidase with activity toward palatinose (28), while an ␣-glucosidase isolated from Erwinia rhapontici showed strict substrate specificity toward palatinose. A recently identified ␣-glucosidase from Thermus thermophilus HB27 (AglH HB27 ) was shown to preferentially hydrolyze ␣,␣-trehalose (␣-1,1); however, panose, isomaltose, isomaltotriose, palatinose, turanose, nigerose, sucrose, maltose, maltotriose, and leucrose were also substrates for this enzyme (1).…”

Functional and structural characterization of a novel mannosyl‐3‐phosphoglycerate synthase from Rubrobacter xylanophilus reveals its dual substrate specificity

“…A triple mutant mpgS (A164S, A208S, F211Y) from R. xylanophilus was amplified from a mpgS ‐carrying vector (Sá‐Moura et al ., 2008) with two rounds of PCR with primers listed in Table S3 and as previously described (Alarico et al ., 2008), and the mutations confirmed by DNA sequencing (AGOWA, Germany). Cloning, overexpression, protein purification and kinetic characterization of the triple mutant MpgS were carried out as described above for the wild‐type recombinant MpgS.…”

Evaluation and directed evolution for thermostability improvement of a GH 13 thermostable α-glucosidase from Thermus thermophilus TC11

“…Industrial application of these enzymes requires stability at high temperatures as well as toward common denaturant agents, and therefore, enzymes isolated from thermophiles have gained attention over the past decade [ 7 ]. There are many thermostable α-glucosidases from different thermophilic and hyperthermophilic microorganisms such as Sulfolobus tokodaii [ 7 ], Geobacillus toebii [ 19 ], Thermus caldophilus [ 20 ], Thermoplasma acidophilum [ 21 ], Bacillus stearothermophilus [ 22 ] and Thermus thermophilus [ 23 , 24 ] have been discovered and characterized, and several mesophilic α-glucosidases have been engineered by mutagenesis to enhance enzyme thermostability [ 25 , 26 ].…”

“…Thermus thermophilus is a thermophilic bacterium with optimal growth temperatures of approximately 70–75 °C and produce several enzymes of considerable biotechnological interest, including proteases, phosphatases, catalases, DNA processing enzymes, and α-glucosidases [ 27 ]. α-Glucosidases isolated from T. thermophilus HB8, T. thermophilus HB27, and T. caldophilus GK24 have been characterised with regard to their substrate specificity [ 20 , 23 , 24 ], which are different from that of the majority of known α-glucosidases. Whereas typical enzymes favour the α-1,4 glycosidic bonds of maltose or maltooligosaccharides [ 28 ], Thermus α-glucosidases preferentially hydrolyse the α-1,6 bonds in isomaltose, α-1,2 bonds in sucrose, or α-1,1 bonds in trehalose.…”