Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68026
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dc.contributor.authorLin, L.L.en_US
dc.contributor.authorChou, P.R.en_US
dc.contributor.authorHua, Y.W.en_US
dc.contributor.authorHsu, W.H.en_US
dc.date2006zh_TW
dc.date.accessioned2014-06-11T05:56:10Z-
dc.date.available2014-06-11T05:56:10Z-
dc.identifier.issn0175-7598zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/68026-
dc.description.abstractA truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg2+, K+, and Na+ can activate BLrGGT, whereas that of Pb2+ dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 mu M when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds.en_US
dc.language.isoen_USzh_TW
dc.relationApplied Microbiology and Biotechnologyen_US
dc.relation.ispartofseriesApplied Microbiology and Biotechnology, Volume 73, Issue 1, Page(s) 103-112.en_US
dc.relation.urihttp://dx.doi.org/10.1007/s00253-006-0440-4en_US
dc.subjectescherichia-coli k-12en_US
dc.subjectn-terminal nucleophileen_US
dc.subjecttransferaseen_US
dc.subjecttranspeptidaseen_US
dc.subjectamino-acidsen_US
dc.subjectintramolecular proteolysisen_US
dc.subjectautoproteolyticen_US
dc.subjectactivationen_US
dc.subjectprotein-productionen_US
dc.subjectcrystal-structuresen_US
dc.subjectmolecular-cloningen_US
dc.subjectsignal peptidesen_US
dc.titleOverexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformisen_US
dc.typeJournal Articlezh_TW
dc.identifier.doi10.1007/s00253-006-0440-4zh_TW
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