Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.
Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-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 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-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 alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees 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.
Liebl
W
W
Lehrstuhl für Mikrobiologie, Technische Universität München, Munich, Germany.
Stemplinger
I
I
Ruile
P
P
D000595:Amino Acid Sequence; D000688:Amylose / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D008074:Lipoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins; D013213:Starch; D000688:Amylose; D000516:alpha-Amylases; D002118:Calcium