Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

Chae Yoeng Lee; Kyung Ok Yu; Seung Wook Kim; Sung Ok Han

doi:10.1016/j.jbiosc.2009.10.014

Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

Chae Yoeng Lee, Kyung Ok Yu, Seung Wook Kim, Sung Ok Han

Research output: Contribution to journal › Article › peer-review

19 Citations (Scopus)

Abstract

The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher k_cat/K_m value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.

Original language	English
Pages (from-to)	331-336
Number of pages	6
Journal	Journal of Bioscience and Bioengineering
Volume	109
Issue number	4
DOIs	https://doi.org/10.1016/j.jbiosc.2009.10.014
Publication status	Published - 2010 Apr

Bibliographical note

Funding Information:
This work was supported in part by a Korea Research Foundation Grant funded by the Korean Government ( KRF-2008-331-D00172 ) and the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea ( No. 309016-5 ).

Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.

Keywords

Cellulase
Clostridium cellulovorans
Clostridium thermocellum
Thermostability
in vitro DNA recombination

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology

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10.1016/j.jbiosc.2009.10.014

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title = "Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE",

abstract = "The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher kcat/Km value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.",

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author = "Lee, {Chae Yoeng} and Yu, {Kyung Ok} and Kim, {Seung Wook} and Han, {Sung Ok}",

note = "Funding Information: This work was supported in part by a Korea Research Foundation Grant funded by the Korean Government ( KRF-2008-331-D00172 ) and the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea ( No. 309016-5 ). Copyright: Copyright 2010 Elsevier B.V., All rights reserved.",

year = "2010",

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doi = "10.1016/j.jbiosc.2009.10.014",

language = "English",

volume = "109",

pages = "331--336",

journal = "Journal of Bioscience and Bioengineering",

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AU - Yu, Kyung Ok

AU - Kim, Seung Wook

AU - Han, Sung Ok

N1 - Funding Information: This work was supported in part by a Korea Research Foundation Grant funded by the Korean Government ( KRF-2008-331-D00172 ) and the Technology Development Program for Agriculture and Forestry, Ministry for Agriculture, Forestry and Fisheries, Republic of Korea ( No. 309016-5 ). Copyright: Copyright 2010 Elsevier B.V., All rights reserved.

PY - 2010/4

Y1 - 2010/4

N2 - The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher kcat/Km value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.

AB - The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 °C and showed a higher kcat/Km value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.

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KW - Clostridium cellulovorans

KW - Clostridium thermocellum

KW - Thermostability

KW - in vitro DNA recombination

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Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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