Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

Int J Mol Sci. 2019 Mar 30;20(7):1602. doi: 10.3390/ijms20071602.

Abstract

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

Keywords: cellulase; cellulose biodegradation; disulfide bonds; endoglucanase; protein engineering; rational design; thermostability.

MeSH terms

  • Cellulase / chemistry*
  • Cellulase / genetics
  • Cellulase / metabolism
  • Disulfides / chemistry*
  • Enzyme Stability
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Molecular Dynamics Simulation
  • Penicillium / enzymology*
  • Penicillium / genetics
  • Penicillium / metabolism
  • Protein Engineering / methods
  • Substrate Specificity
  • Thermotolerance*

Substances

  • Disulfides
  • Fungal Proteins
  • Cellulase