Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents.
Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.
D001203:Ascomycota / Q000201:enzymology; D001665:Binding Sites; D002021:Buffers; D002384:Catalysis; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005612:Freeze Drying; D006586:Hexanes / Q000737:chemistry; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D017186:Titrimetry / Q000379:methods; D014867:Water / Q000737:chemistry
D002021:Buffers; D004800:Enzymes, Immobilized; D006586:Hexanes; D012997:Solvents; D014867:Water; D008049:Lipase