Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli.
A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant alpha-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named alpha-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50-60 degrees C and pH 10-11, and 42 and 38% relative activities at 30 degrees C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding alpha-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl beta-D(-)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.
Murakami
Shuichiro
S
Laboratory of Applied Microbiology, Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Japan.
Nishimoto
Haruka
H
Toyama
Yosuke
Y
Shimamoto
Etsuko
E
Takenaka
Shinji
S
Kaulpiboon
Jarunee
J
Prousoontorn
Manchumas
M
Limpaseni
Tipaporn
T
Pongsawasdi
Piamsook
P
Aoki
Kenji
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021141:Nucleic Acid Amplification Techniques; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D002614:Chelating Agents; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D019216:Metals, Heavy; D004492:Edetic Acid; D000516:alpha-Amylases