Robert Verger

Lipases: Interfacial Enzymes with Attractive Applications

Unusually versatile substrate specificity is shown by lipases. Not only do they hydrolyze triacylglycerols-for example, in the stomach and intestine during digestion of dietary fat-and various synthetic esters and amides, but their high stability in organic solvents permits their use in transesterification reactions and ester synthesis as well. Reactions based on lipase catalysis usually proceed with high regio- and enantioselectivity. Thus, the Ca2+ antagonist diltiazem (1) was obtained with lipase from Serratia marcescens. Over 30 lipases have been cloned in the last few years. Since the tertiary structure of 12 lipases is known, there are presently significant efforts to improve this class of enzymes by protein engineering techniques, in view of their use in detergents and other fields of industrial application.

Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans

BACKGROUND The aim of this study was to quantitatively evaluate the relative contributions to in vivo lipolysis of gastric and pancreatic lipases. METHODS Gastric and pancreatic lipase secretions were measured, and their respective levels were determined in duodenal fluid during the digestion of a liquid test meal in healthy volunteers. Gastric lipase activity was clearly distinguished from that of pancreatic lipase by using both a specific enzymatic assay and an enzyme-linked immunosorbent assay. Lipolysis products were monitored throughout the digestion period. RESULTS On a weight basis, the ratio of pancreatic lipase to gastric lipase total secretory outputs was found to be around four after 3 hours of digestion. The level of gastric hydrolysis was calculated to be 10% +/- 1% of the acyl chains released from the meal triglycerides. Gastric lipase remained active in the duodenum where it might still hydrolyze 7.5% of the triglyceride acyl chains. CONCLUSIONS Globally during the whole digestion period, gastric lipase might hydrolyze 17.5% of the triglyceride acyl chains. In other words, gastric lipase might hydrolyze 1 acyl chain of 4, which need to be hydrolyzed for a complete intestinal absorption of monoglycerides and free fatty acids resulting from the degradation of two triglyceride molecules.

Purification from porcine pancreas of two molecular species with lipase activity

Abstract The two lipases existing in porcine pancreas and pancreatic juice have been fully purified by a method involving the six following steps: (1) almost complete delipidation of pancreas homogenates by solvent extraction; (2) (NH4)2SO4 fractionation; (3) removal of an acidic phosphatide by extraction and partition between n butanol and (NH4)2SO4; (4) chromatography on DEAE-cellulose (pH 9.0); (5) filtration through Sephadex G-100 and (6) separation of the two lipases by chromatography on CM-cellulose (pH 5.0). This method can be applied on a relatively large preparative scale. The two porcine lipases appear to be very closely related with regard to amino acid composition, molecular weight (45 000–50 000) and specific activity on long- or short-chain triglycerides.

Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions.

Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

A critical reevaluation of the phenomenon of interfacial activation.

Publisher Summary Lipases are carboxylic ester hydrolases and have been termed glycerolester-hydrolase in the international system of classification. They greatly differ with respect to both of their origins and their kinetic properties. They can catalyze in vitro the hydrolysis, or synthesis, of a wide range of different carboxylic esters; however, they all show a higher specific activity toward glyceridic substrates. Under physiological conditions, because natural triacylglycerols are water insoluble, lipases that are generally soluble in water, catalyze the hydrolysis of carboxylic ester bonds at lipid/water interfaces. Some lipases, such as gastric lipases, rapidly become denatured at an interface with a pure tributyrin or tributanoylglycerol emulsion. Consequently, it is impossible to assess experimentally what interfacial activation may have occurred. The three-dimensional structures of lipases suggested that the interfacial activation phenomenon might be due to the presence of an amphiphilic surface loop covering the active site of the enzyme in solution, just like a lid. When contact occurs with a lipid/water interface, this lid might undergo a conformational rearrangement as the result of which the active site becomes accessible.

Lipid-protein interactions in monolayers

Abstract We restricted ourselves to a few examples of the different methodological aspects of the investigation of lipid-protein interactions in monolayer assemblies. Experiments with monolayers have the unique advantage that the arrangement and packing of the molecules can be easily measured and controlled. The first section is devoted to proteins which do not degrade lipids. Soluble proteins are generally injected in the subphase below a preformed lipid monolayer and measurements are performed either at constant surface area or at constant surface pressure. These experiments can give information on the penetration capacity into the interface, its lipid specificity with a direct access to the area of the protein segment interacting with lipids. Reconstitution of functional enzymatic complexes can be achieved as well as the determination of the orientation of the protein at the interface. Most intrinsic membrane proteins are insoluble in water. In the absence of detergent they aggregate and display no affinity for lipid interfaces. These proteins can be spread from an organic solvent solution but with the risk of being denatured. In order to circumvent this difficulty a method for spreading an aqueous suspension of lipoproteins or natural membrane vesicles was developed. This spreading method allows the formation of lipoprotein films retaining biological activities and native membrane constituents. Formation of functional transmembrane complexes in planar bilayers from such lipoprotein films is the most fascinating application of this spreading technique. In the second section, we reviewed the use of pure or mixed lipid monolayers as substrates for lipolytic enzymes. Either long-chain lipids were used and their surface density was not controlled; or short-chain lipids were applied, again without control of surface density; or short-chain lipids were used at constant surface density. Recently a method was developed to study the hydrolysis of long chain lipids with control of surface density. The monolayer technique allows us to study accurately the influence of surface pressure and protein cofactors on the hydrolysis velocity and lag time in lipolysis. Two types of mixed lipid monolayers can be formed at the air/water interface: either by spreading, from a volatile organic solvent, a mixture of water insoluble lipids or by injecting a detergent solution into the water subphase covered with a preformed pure lipid monolayer. These techniques are ideally suited for the study of the mode of action of lipolytic enzymes on controlled mixed interfaces.

Structural basis for the substrate selectivity of pancreatic lipases and some related proteins

The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.

Human preduodenal lipase is entirely of gastric fundic origin

Lipase activity was measured in supernatant homogenates from various anatomic regions in the upper part of the human digestive tract of two organ donors. It is shown unambiguously that lipase activity occurs only in the fundic mucosa of the stomach, whereas no significant activity takes place in the antral, pharyngeal, or lingual areas, including the circumvallate papillae. In adults, the potential activity of human gastric lipase, as measured using tributyrin as substrate, amounts to 20% of its pancreatic counterpart. Lipase activity was also determined on human gastric biopsy samples taken during gastrofibroscopy tests on healthy adults. These results confirmed the finding that a lipolytic activity of gastric origin occurs uniformly and only in the fundic mucosa. Triacylglycerol hydrolysis is associated with a genuine gastric lipase activity that is clearly distinct from the classical esterase observed using p-nitrophenyl acetate as substrate. Lipase activity decreases significantly with age: it ranges on average from 4700 U/g of fresh mucosa in subjects aged up to 50 yr to 700 U/g of fresh mucosa in persons over 60 yr of age.