Optimization of the expression of levansucrase L17 in recombinant E. coli
Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gen...
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2018-03-01
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Online Access: | http://dx.doi.org/10.1080/13102818.2018.1431056 |
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doaj-1f33fe24a1254b7090a180a1a71ef4822020-11-25T01:28:52ZengTaylor & Francis GroupBiotechnology & Biotechnological Equipment1310-28181314-35302018-03-0132247748610.1080/13102818.2018.14310561431056Optimization of the expression of levansucrase L17 in recombinant E. coliIlia Iliev0Tonka Vasileva1Veselin Bivolarski2Ayshe Salim3Sandrine Morel4Philippe Rabier5Valérie Gabriel6Plovdiv University ‘Paisii Hilendarski’Plovdiv University ‘Paisii Hilendarski’Plovdiv University ‘Paisii Hilendarski’Plovdiv University ‘Paisii Hilendarski’Toulouse UniversityUniversity Institute of Technology ‘Paul Sabatier’University Institute of Technology ‘Paul Sabatier’Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose.http://dx.doi.org/10.1080/13102818.2018.1431056Expression optimizationE. coliLeuconostoc mesenteroideslevansucraselow temperature |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Ilia Iliev Tonka Vasileva Veselin Bivolarski Ayshe Salim Sandrine Morel Philippe Rabier Valérie Gabriel |
spellingShingle |
Ilia Iliev Tonka Vasileva Veselin Bivolarski Ayshe Salim Sandrine Morel Philippe Rabier Valérie Gabriel Optimization of the expression of levansucrase L17 in recombinant E. coli Biotechnology & Biotechnological Equipment Expression optimization E. coli Leuconostoc mesenteroides levansucrase low temperature |
author_facet |
Ilia Iliev Tonka Vasileva Veselin Bivolarski Ayshe Salim Sandrine Morel Philippe Rabier Valérie Gabriel |
author_sort |
Ilia Iliev |
title |
Optimization of the expression of levansucrase L17 in recombinant E. coli |
title_short |
Optimization of the expression of levansucrase L17 in recombinant E. coli |
title_full |
Optimization of the expression of levansucrase L17 in recombinant E. coli |
title_fullStr |
Optimization of the expression of levansucrase L17 in recombinant E. coli |
title_full_unstemmed |
Optimization of the expression of levansucrase L17 in recombinant E. coli |
title_sort |
optimization of the expression of levansucrase l17 in recombinant e. coli |
publisher |
Taylor & Francis Group |
series |
Biotechnology & Biotechnological Equipment |
issn |
1310-2818 1314-3530 |
publishDate |
2018-03-01 |
description |
Levansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose. |
topic |
Expression optimization E. coli Leuconostoc mesenteroides levansucrase low temperature |
url |
http://dx.doi.org/10.1080/13102818.2018.1431056 |
work_keys_str_mv |
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1725099792449667072 |