Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports

Biotechnol Prog. 2004 Mar-Apr;20(2):630-5. doi: 10.1021/bp0342957.

Abstract

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS-PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adsorption
  • Bacillus / classification
  • Bacillus / enzymology*
  • Bacillus / genetics
  • Enzyme Activation
  • Enzyme Stability
  • Enzymes, Immobilized / chemistry
  • Enzymes, Immobilized / isolation & purification
  • Hydrophobic and Hydrophilic Interactions
  • Lipase / chemistry*
  • Lipase / genetics
  • Lipase / isolation & purification*
  • Membranes, Artificial*
  • Octoxynol / chemistry*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Surface Properties
  • Ultrafiltration / instrumentation
  • Ultrafiltration / methods*

Substances

  • Enzymes, Immobilized
  • Membranes, Artificial
  • Recombinant Proteins
  • Octoxynol
  • Lipase