Purification and characterization of two alkaline, thermotolerant alpha-amylases from Bacillus halodurans 38C-2-1 and expression of the cloned gene in Escherichia coli |
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Authors: | Murakami Shuichiro Nishimoto Haruka Toyama Yosuke Shimamoto Etsuko Takenaka Shinji Kaulpiboon Jarunee Prousoontorn Manchumas Limpaseni Tipaporn Pongsawasdi Piamsook Aoki Kenji |
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Affiliation: | Laboratory of Applied Microbiology, Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Japan. |
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Abstract: | 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. |
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