Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate

Abstract

pase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate. Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids, Elsevier, 2018, 1863 with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid 37 monomolecular films spread at the air-water interface, and UV absorption using a novel 38 MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter 39plates. The kinetic properties of TTL were compared to those of the homologous lipase from 40Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium 41 solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on 42 micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV 43 absorption assay with coated MGDG was highly sensitive and allowed measuring significant 44 activities with about ten ng of enzymes, against hundred ng to ten µg with the pHstat. TTL 45 showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with 46 monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL 47 based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions 48 in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was 49 found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed 50 micelles and galactolipid monomolecular films. These later experiments suggest that the 51 second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated 52 substrate. 53 -3 -

Causal Enzymology and Physiological Aspects May Be Accountable to Membrane Integrity in Response to Salt Stress in Arabidopsis thaliana Lines

“…They have very important functions such as receiving signals, involved in transport of chemicals inwards and outwards. At the same case and in addition to cellulose and hemicellulose, the membrane contains lipids, phospholipids, and galactolipids [11, 27]. In particular, those last compounds were judged playing very important roles on the membrane integrity and also functionality against various biotic and abiotic stresses [11, 31].…”

“…Aliquots of frozen fresh shoot material (0.5 g) were ground to a fine powder with liquid nitrogen and homogenized in a cold solution containing 100 mM Tris-HCl buffer (pH 8), 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM KCl, 20 mM MgCl 2 , 0.5 mM PMSF (Phenyl-Methyl-Sulfonyl-Fluoride), and 2 % (w/v) PVP. The homogenate was centrifuged at 14.000 × g for 30 min at 4°C and the supernatant was used for determination of the enzyme activities [27]. Protein concentration was determined according to Bradford method [28].…”

“…As indicated by [27], lipase activities were measured potentiometrically at 40°C and pH 8 meaning automatically titrating the free fatty acids released from purified egg L- α -phosphatidylcholine (Sigma) as substrate, as previously described [11, 19, 27]. Lipase activities were measured with mechanically stirred triglyceride emulsions [29].…”

The digestion of galactolipids and its ubiquitous function in Nature for the uptake of the essential α-linolenic acid

“…were found to display optimum activities at NaTDC to galactolipid molar ratios of 1.3, 0.5 and 0.25 on mixed micelles of NaTDC/medium chain-MGDG, NaTDC/long chain-DGDG and NaTDC/medium chain-DGDG, respectively 67 . It is worth mentioning that these conditions were also found to be optimum for other enzymes like human and bovine pancreatic CEH 88 , Thermomyces lanuginosus lipase 79 , Fusarium solani cutinase 79 , Fusarium solani (phospho)lipase 80 and Talaromyces thermophilus lipase 81 ( Table 4) which suggests that all these enzymes preferentially recognize a specific organization and presentation of galactolipids in mixed micelles. Mixed micelles made of NaTDC and medium chain galactolipids were characterized by dynamic light scattering at 37 °C and pH 8, and their hydrodynamic diameters (z-average) were found to be 9.4 ± 0.3 nm for NaTDC/di-octanoyl-MGDG mixed micelles at a molar ratio of 1.33 and 24.5 ± 0.2 nm for NaTDC /di-octanoyl-DGDG mixed micelles at a molar ratio of 0.25 81 .…”

“…For instance, the lipase from the fungus Rhizopus arrhizus (or Rhizopus Orizae) has been commonly used since 1973 to study the FA composition and distribution in galactolipids because it has specificity for the hydrolysis of ester bond at the sn-1 position of galactolipids 63 . Several microbial lipases with galactolipase activity have been identified [79][80][81] Figure 7B), as well as of Fusarium solani cutinase ( Figure 7C), but absent from the classical human pancreatic lipase (HPL) 85 . Molecular docking of a 18:3 n-3/18:3 n-3 DGDG molecule in the active site of HPLRP2, GPLRP2 and cutinase reveals a similar topology for the binding sites of the digalactosyl polar head (sn-3 position) and the acyl chain found at the sn-1 position, that closely interact with apolar residues of the β9-loop in PLRPs.…”