Using response surface methodology in combination with Plackett-Burman design for optimization of culture media and extracellular expression of Trichoderma reesei synthetic endoglucanase II in Escherichia coli

Mol Biol Rep. 2018 Oct;45(5):1197-1208. doi: 10.1007/s11033-018-4272-y. Epub 2018 Jul 21.


Cellulases like endoglucanase II (EGII) from Trichoderma reesei are the industrial enzymes responsible for breakdown of cellulosic materials. Due to its importance for production of eco-friendly commercial products such as alternative biofuels, industrial EGII production and optimization of its production conditions merit consideration. The gene responsible for EGII expression was designed and sub-cloned in to pET26b expression vector and transformed into BL21 (DE3) pLysS cells. Protein expression and purification was followed by a RSM design (20 experiments) to optimize the IPTG Concentration, post induction period and cell density (OD600). Thereafter, another RSM design (20 experiments) was performed to find and optimize the most important permeabilizing factors to achieve higher extracellular EGII expression. The EGII expression levels were assessed by Ghose method. The EGII gene was sub-cloned and protein expression and purification were successfully performed. The RSM experiments indicated that 0.331 mM for IPTG Concentration, 10.89 H for post induction period and 3.41 for cell density (OD600) were the optimum culture. Glycine (0.99%), Triton X-100 (0.73%) and CaCl2 (0.232) have been assigned as the most effective membrane permeabalizing factors. Optimization of culture medium components has led to a 3.06 fold increase in extracellular expression of EGII. RSM is an amenable method to optimize the expression of commercially significant enzymes. Our results indicated that optimization of IPTG concentration, post induction period and cell density along with glycine, Triton X-100 and Ca2+ concentration could lead to more cost effective industrial production of EGII.

Keywords: Culture condition; Endoglucanase II; Optimization; Response surface methodology.

MeSH terms

  • Bacteriological Techniques
  • Calcium / chemistry
  • Cellulase / genetics*
  • Cellulase / metabolism*
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Culture Media / chemistry*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Glycine / chemistry
  • Isopropyl Thiogalactoside / chemistry
  • Octoxynol / chemistry
  • Protein Engineering
  • Trichoderma / enzymology*
  • Trichoderma / genetics


  • Culture Media
  • Fungal Proteins
  • Isopropyl Thiogalactoside
  • Octoxynol
  • endoglucanase 2
  • Cellulase
  • Calcium
  • Glycine