Properties and gene structure of the Thermotoga maritima α-amylase amyA, a putative lipoprotein of a hyperthermophilic bacterium

Wolfgang Liebl, Ilse Stemplinger, Peter Ruile

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Thermotoga maritima MSB8 has a chromosomal α-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-α-glucanotransferase of the same strain and to α-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can he translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima α-amylase appears to be the first known example of a lipoprotein α-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90°C. Like the α-amylases of many other organisms, the activity of the T. maritima α-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima α-amylase (about 5.6 x 103 U/mg of protein at 80°C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.

Original languageEnglish
Pages (from-to)941-948
Number of pages8
JournalJournal of Bacteriology
Issue number3
StatePublished - 1997


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