A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides

Abstract

A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.

Lipase regio‐ and stereoselectivities toward three enantiomeric pairs of didecanoyl‐deoxyamino‐O methyl glycerol: A kinetic study by the monomolecular film technique

“…These three enantiomeric pairs were previously used by Deveer et al 25 to characterize some kinetic parameters of the Pseudomonas glumae lipase. In this study we present the results of an extensive comparative study on the regioand stereoselectivities of 12 lipases of animal and microbial origin.…”

Staphylococcal lipases stereoselectively hydrolyse the sn-2 position of monomolecular films of diglyceride analogs. Application to sn-2 hydrolysis of triolein

“…Pure synthetic pseudodiglycerides were synthesised as previously described and their surface compression isotherms were published by Deveer et al [33]. Pure oleic acid, monoolein, diolein and triolein were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France).…”

“…Surface pressure-molecular area curves were drawn up with acylglycerol [41,42] and diglyceride analogs [33], using the methods described by the authors cited.…”

“…In previous studies, Deveer et al [33] and Douchet et al [34] used six pure chiral synthetic pseudodiglycerides ( Fig. 1) containing a single hydrolysable decanoyl ester and two lipase-resistant groups (one decanoylamino and one methyl ether from the didecanoyl-deoxyamino-O-methyl glycerol, DDG).…”

Kinetic Properties of a Novel Fusarium solani (phospho)lipase: A Monolayer Study

“…Lipases have been reported to have broad substrate specificity. Moreover, lipid monolayers technique represents an interesting approach to study protein-lipid interactions and to control physicochemical properties of the interface; thus, this methodology was extensively used to study lipase kinetics, 3,4 stereoselectivity, and regioselectivity of many lipases. Furthermore, some lipases can hydrolyze synthetic lipids and/or phospholipids.…”