Enzymatic Assay Method for Evaluating the Lipase Activity in Complex Extracts from Transgenic Corn Seed

Abstract

A colorimetric method was established to determine the activity of recombinant lipase in extracts from transgenic corn seed. The system was an oil-in-water emulsion that was stabilized by a surfactant to accommodate the organic phase substrate and aqueous phase enzyme. The lipase activity was measured by monitoring the release of nitrophenol at 346 nm from the substrate, 4-nitrophenyl butyrate. Emulsions prepared with various surfactant types and concentrations were tested. For each surfactant, the measured activity was greatest when the surfactant concentration was close to the critical micelle concentration, consistent with the changing trend of oil droplet size as a function of surfactant concentration. The optimal system, with 0.01% (w/w) Tween 80, demonstrated good reproducibility, high sensitivity, robustness, and a linear response to lipase concentration. A colorimetric method was established to determine the activity of recombinant lipase in extracts from transgenic corn seed. The system was an oil-in-water emulsion that was stabilized by a surfactant to accommodate the organic phase substrate and aqueous phase enzyme. The lipase activity was measured by monitoring the release of nitrophenol at 346 nm from the substrate, 4-nitrophenyl butyrate.Emulsions prepared with various surfactant types and concentrations were tested. For each surfactant, the measured activity was greatest when the surfactant concentration was close to the critical micelle concentration, consistent with the changing trend of oil droplet size as a function of surfactant concentration. The optimal system, with 0.01% (w/w) Tween 80, demonstrated good reproducibility, high sensitivity, robustness, and a linear response to lipase concentration.

Effects of ensiling processes and antioxidants on fatty acid concentrations and compositions in corn silages

“…A high LOX activity would be plausible in high pH silages, whereas these enzymes would be inhibited by low pH [15,36]. In addition, lipid or FA oxidation requires oxygen, whereas ensiling is a process that progresses from aerobic conditions to anaerobic conditions.…”

Partial Characterization of a Lipase from Gypsy Moth (Lymantria dispar L.) Larval Midgut

“…Additionally, lipases hydrolyze nitrophenyl esters at the oil-water interface. The released nitrophenol is water soluble and can be monitored spectrophotometrically (ZHONG & GLATZ 2006). Lipase and esterase/lipase operate on different substrates, but share structural similarity, i.e.…”

“…Lipases are widely distributed in nature and their physical and biochemical properties, and activities, have been investigated in many species of animals (GROENER & KNAUER 1981;LAI et al 1998;DEERLI & ALI AKPINAR 2002;OKU et al 2006), plants (LAMIKANRA & WATSON 2004;ZHONG & GLATZ 2006), bacteria (SIMONS et al 1996;HONG & CHANG 1998;DOSANJH & KAUR 2002;TEO et al 2003;GUPTA et al 2004), yeast (CRABBE et al 1996 and fungi (GARCIA-LEPE et al 1997;PAN-DEY et al 1999;PERA et al 2006). By using nitrophenyl esters as substrates, lipases have been investigated in bacteria (HUMBLE et al 1977;ARAGON et al 2000), insects (COSTA & DA CRUZ-LANDIM 2005), and other arthropods (GIB- SON & BARKER 1979;L[PEZ-L[PEZ et al 2003).…”

Selenium in Germinated Chickpea (Cicer arietinum L.) Increases the Stability of Its Oil Fraction

“…Lipase activity was measured by the colorimetric method described by [39]. One unit of lipase activity corresponded to the production of 1 µmol of nitrophenol in 1 min.…”