Applied Microbiology and Biotechnology

, Volume 102, Issue 19, pp 8389–8402 | Cite as

Rational engineering of Cel5E from Clostridium thermocellum to improve its thermal stability and catalytic activity

  • Ibrahim Torktaz
  • Ali Asghar KarkhaneEmail author
  • Jafar HemmatEmail author
Biotechnologically relevant enzymes and proteins


The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of − 64.964 KJ/mol for wild-type Cel5E and − 176.148, − 200.921, and − 120.038 KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity.


Clostridium thermocellum celH Cel5E Rational mutagenesis Activity 


Funding information

The Iranian National Institute of Genetic Engineering and Biotechnology and Iran National Science Foundation (INSF; grant No. 96003289) supported this research.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This study does not contain any experiments involving human participants or animals performed by any of the authors.

Supplementary material

253_2018_9204_MOESM1_ESM.pdf (374 kb)
ESM 1 (PDF 374 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.National Institute of Genetic Engineering and Biotechnology (NIGEB)TehranIran
  2. 2.Biotechnology DepartmentIranian Research Organization for Science and Technology (IROST)TehranIran

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