Difference between revisions of "Part:BBa K258006:Experience"

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In our experiment, we observed that TliA fused proteins were excreted
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to supernatant culture succesfully by detecting lipase activity  with tributyrin and spectrophotometric detection
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with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-
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fold more with ABC transporter PrtDEF of Erwinia chrysanthemi.
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Same results took place lipase activity at size of zone around supernatant on tributyrin mixed agar plate

Revision as of 20:31, 9 October 2009

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Applications of BBa_K258006

Lipase activity To identify a secretion phenotype on solid medium, E. coli was grown at 25°C for 48 h on LAT (LB medium, 1.5% Bacto Agar, 0.5% tributylin). The phenotype was evident by the development of a halo due to the secreted lipase [12]. In addition, lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate [12]. Ten millimolar pNPP dissolved in acetonitrile was mixed with ethanol and 50 mM Tris-HCl (pH 8.5) to a final ratio of acetonitrile:ethanol:Tris-HCl of 1:4:95 (v/v/v). The reaction was started by adding 50 μl of culture supernatant to 200 μl of reaction mixture at 42°C, and absorbance at 420 nm was monitored with Hitachi Spectrophotometry for 20 min. The activity was measured by the increase of optical density (OD).


procedure from:

Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD) Chan Woo Chung1, Jinsun You1, Kyeongyeon Kim1, Yuseok Moon2, Hoeon Kim3 and Jung Hoon Ahn* Published: 29 January 2009 Microbial Cell Factories 2009, 8:11 doi:10.1186/1475-2859-8-11 Received: 18 November 2008 Accepted: 29 January 2009 This article is available from: http://www.microbialcellfactories.com/content/8/1/11

User Reviews

UNIQ990842870d898422-partinfo-00000000-QINU UNIQ990842870d898422-partinfo-00000001-QINU In our experiment, we observed that TliA fused proteins were excreted

to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection

with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-

fold more with ABC transporter PrtDEF of Erwinia chrysanthemi. Same results took place lipase activity at size of zone around supernatant on tributyrin mixed agar plate