Publication: Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
Issued Date
2008-10-06
Resource Type
ISSN
01410229
Other identifier(s)
2-s2.0-49749105587
Rights
Mahidol University
Rights Holder(s)
SCOPUS
Bibliographic Citation
Enzyme and Microbial Technology. Vol.43, No.4-5 (2008), 321-328
Suggested Citation
Shuichiro Murakami, Kenji Nagasaki, Haruka Nishimoto, Ryo Shigematu, Jun Umesaki, Shinji Takenaka, Jarunee Kaulpiboon, Manchumas Prousoontorn, Tipaporn Limpaseni, Piamsook Pongsawasdi, Kenji Aoki Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli. Enzyme and Microbial Technology. Vol.43, No.4-5 (2008), 321-328. doi:10.1016/j.enzmictec.2008.05.006 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/18843
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
Title
Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli
Other Contributor(s)
Abstract
A newly isolated strain, MS-2-5, identified as Bacillus halodurans, produced five alkaline and thermotolerant amylases. The five amylases, named amylases A-E, were separated from each other, and purified to homogeneity. The molecular masses of these amylases were different from each other, and estimated to be 90, 85, 70, 65, and 58 kDa. These amylases showed the maximal activities at 60-65 °C and pH 10.5-11. A predominant product by each enzyme reaction was maltotetraose. These amylases were classified as an α-amylase by anomeric form analysis of the reaction products. Internal amino acid sequence analyses of the purified enzymes suggested that these enzymes were produced from a single polypeptide by proteolytic degradation. The gene, named amyA, was cloned and expressed in the T7 promoter systems of Escherichia coli. To increase yield and productivity of recombinant enzyme, cultivation conditions were examined. The maximal amount of enzyme was produced when an E. coli transformant carrying amyA was cultivated at 25 °C in Luria-Bertani medium supplemented with 1.0% d-glucose, 1.0% d-sorbitol, 0.1% MgSO4·7H2O, and 2.0% yeast extract. The yield of the transformant increased 104-fold as, compared with that of the parent strain MS-2-5. © 2008 Elsevier Inc. All rights reserved.