Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports.
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.
Palomo
J M
JM
Departamento de biocatálisis, Instituto de catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Segura
R L
RL
Fernández-Lorente
G
G
Pernas
M
M
Rua
M L
ML
Guisán
J M
JM
Fernández-Lafuente
R
R
D000327:Adsorption; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000302:isolation & purification; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008567:Membranes, Artificial; D017830:Octoxynol / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013499:Surface Properties; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods*
D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D017830:Octoxynol; D008049:Lipase