Cloning and characterization of a thermostable intracellular alpha-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8.
The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed alpha-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da). The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases. The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C. Like the alpha-amylases of many other organisms, the thermostability of T. maritima MSB8 alpha-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca(2+) (10 mM).
Lim
Woo Jin
WJ
Division of Applied Life Science, Gyeongsang National University, 660-701, Chinju, Republic of Korea.
Park
Sang Ryeol
SR
An
Chang Long
CL
Lee
Jong Yeoul
JY
Hong
Su Young
SY
Shin
Eun Chule
EC
Kim
Eun Ju
EJ
Kim
Jong Ok
JO
Kim
Hoon
H
Yun
Han Dae
HD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D000516:alpha-Amylases