B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus

J Biotechnol. 2018 Jun 10;275:17-23. doi: 10.1016/j.jbiotec.2018.03.013. Epub 2018 Mar 29.

Abstract

The α-L-Rhamnosidase is an important enzyme with applications in the food and pharmaceutical industries because it can release terminal L-rhamnose residues from various natural products. In this study, the B-factor-saturation mutagenesis strategy was used to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus. The D594Q and G827K/D594Q mutant enzymes were obtained by screening a series of mutants; they prolonged the half-life of the enzyme at 70 °C by 2.1-fold and 2.3-fold, respectively. Analysis of the 3D structure showed that in the thermostable variants the number of hydrogen bonds and salt bridges was increased, explaining the enhanced thermostability. Kinetic studies showed that the KM values for the D594Q and G827K/D594Q mutant enzymes decreased by 4.0% and 3.8%, respectively. Additionally, the kcat/KM values for the D594Q and G827K/D594Q mutant enzymes increased by 15.5% and 9.2%, respectively. Moreover, the D594Q and G827K/D594Q mutant enzymes exhibited markedly improved isoquercitrin yield at 70 °C that increased by 13.5% and 11.0%, respectively. Therefore, the D594Q and G827K/D594Q mutant enzymes are more suitable for the industrial processes of isoquercitrin preparation.

Keywords: 3D structure model; B-factor-saturation mutagenesis; Isoquercitrin yield; Thermostability; α-L-rhamnosidase.

MeSH terms

  • Aspergillus / enzymology*
  • Aspergillus / genetics
  • Crystallography, X-Ray
  • Enzyme Stability
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Glycoside Hydrolases / chemistry*
  • Glycoside Hydrolases / genetics*
  • Half-Life
  • Hot Temperature
  • Hydrogen Bonding
  • Models, Molecular
  • Mutation*
  • Protein Structure, Tertiary
  • Quercetin / analogs & derivatives
  • Quercetin / metabolism

Substances

  • Fungal Proteins
  • isoquercitrin
  • Quercetin
  • Glycoside Hydrolases
  • alpha-L-rhamnosidase