Thermus aquaticus ATCC 33923 Amylomaltase Gene Cloning and Expression and Enzyme Characterization: Production of Cycloamylose

Abstract

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of theEscherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coliamylomaltase and the plant D-enzyme.

X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus . Implications for the synthesis of large cyclic glucans

“…The inactivity of T. aquaticus amylomaltase under the conditions used in this study may be due to the following two reasons. First, the thermophilic enzyme has its highest activity at pH 5.5±6.0 at 75±80 8C and the activity is strongly reduced under our soaking conditions (4 8C, pH 9.0) [30]. The unusual conformation of the side chains of Asp293 and Glu340 compared to their positions in related enzymes and in the light of their presumed catalytic function, as described above, supports this view.…”

“…Amylomaltase from Thermus aquaticus (EC 2.4.1.25) is a monomeric enzyme that produces cycloamyloses with a degree of polymerization larger than 22 [30]. It seems to be distributed in various bacterial species with different physiological functions.…”

“…In these organisms, the genes for amylomaltase are part of the glycogen operon, indicating that the enzyme might be involved in glycogen metabolism [33]. A similar enzyme, termed disproportionating enzyme (d-enzyme), is present in the plastids of plants [30,34,35]. Potato d-enzyme shares 40% sequence identity with T. aquaticus amylomaltase.…”

The unique glycoside hydrolase family 77 amylomaltase fromBorrelia burgdorferiwith only catalytic triad conserved

“…4. There are no doubts that the B. burgdorferi MalQ, which possesses the natural arginine-to-lysine substitution, was able to perform both reactions typical for a GH77 amylomaltase (Terada et al, 1999;Kaper et al, 2005;Park et al, 2007), i.e. to hydrolyse the malto-oligosaccharides (G2-G7) and to form their transglycosylation products.…”

“…The GH77 amylomaltases have been found in microorganisms (bacteria and archaeons) and plants (including algae) to be involved in the metabolism of maltooligosaccharides and glycogen or starch, respectively (Boos & Shuman, 1998;Lu & Sharkey, 2006). The name amylomaltase is used for the microbial GH77 4-a-glucanotransferases whereas the plant counterparts are usually called disproportionating enzymes (or shortly D-enzymes) (Takaha et al, 1993;Terada et al, 1999;Kaper et al, 2005).…”