Thermostability enhancement of chitosanase CsnA by fusion a family 5 carbohydrate-binding module

Biotechnol Lett. 2017 Dec;39(12):1895-1901. doi: 10.1007/s10529-017-2406-2. Epub 2017 Jul 26.

Abstract

Objective: To determine the effects of carbohydrate-binding modules (CBMs) on the thermostability and catalytic efficiency of chitosanase CsnA.

Results: Three CBMs (BgCBM5, PfCBM32-2 and AoCBM35) were engineered at the C-terminus of chitosanase CsnA to create hybrid enzymes CsnA-CBM5, CsnA-CBM32 and CsnA-CBM35. K m values of all the hybrid enzymes were lower than that of the wild type (WT) enzyme; however, only CsnA-CBM5 had an elevated specific activity and catalytic efficiency. The fusion of BgCBM5 enhanced the thermostability of the enzyme, which exhibited a 8.9 °C higher T50 and a 2.9 °C higher Tm than the WT. Secondary structural analysis indicated that appending BgCBM5 at the C-terminus considerably changed the secondary structure content.

Conclusions: The fusion of BgCBM5 improved the thermal stability of CsnA, and the obtained hybrid enzyme (CsnA-CBM5) is a useful candidate for industrial application.

Keywords: Carbohydrate-binding module; Chitosanase; Kinetic parameters; Thermostability.

MeSH terms

  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Enzyme Stability
  • Escherichia coli / genetics
  • Glycoside Hydrolases* / chemistry
  • Glycoside Hydrolases* / genetics
  • Glycoside Hydrolases* / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Protein Binding
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins* / chemistry
  • Recombinant Fusion Proteins* / genetics
  • Recombinant Fusion Proteins* / metabolism
  • Temperature

Substances

  • Bacterial Proteins
  • Recombinant Fusion Proteins
  • Glycoside Hydrolases
  • chitosanase