Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40 |
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Authors: | Masuo Naohisa Ito Kotaro Yoshimune Kazuaki Hoshino Mitsuyo Matsushima Kenichiro Koyama Yasuji Moriguchi Mitsuaki |
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Affiliation: | Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu 700, Oita 870-1192, Japan. |
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Abstract: | We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote. |
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Keywords: | Glutaminase Micrococcus luteus K-3 Aspergillus oryzae RIB40 Salt tolerance |
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