Inhibition of lipases by proteins. A kinetic study with dicaprin monolayers

We report further investigations on protein inhibition of pancreatic and microbial lipases carried out with the monolayer technique. When beta-lactoglobulin A, melittin, serum albumin, myoglobin, and a protein inhibiting lipase from soybean were preincubated with a dicaprin film at a surface pressure of 35 dynes/cm, no activity was detected with horse pancreatic or Rhizopus delemar lipases. By contrast, Rhizopus arrhizus and Geotrichum candidum lipase activities were not impaired under the same conditions. Experiments using mixed lipid-protein film transfer clearly show that the inhibition of pancreatic lipase is due to the protein associated with lipid and not caused by direct protein-enzyme interaction in the aqueous phase. Three parameters were used to determine the surface properties of the various proteins at the dicaprin/water interface; namely, the initial rate of surface pressure increase, (delta pi/delta t)t = 0, the maximal surface pressure increase, delta pi max, and the critical surface pressure, pi c. A positive correlation was observed between values of (delta pi/delta t)t = 0 of proteins and their respective capacity to inhibit pancreatic and R. delemar lipases. By contrast, there was no apparent correlation with the two other parameters, delta pi max or pi c.     

Display of lipase on the cell surface of Escherichia coli using OprF as an anchor and its application to enantioselective resolution in organic solvent

We have developed a new cell surface display system using a major outer membrane protein of Pseudomonas aeruginosa OprF as an anchoring motif. Pseudomonas fluorescens SIK W1 lipase gene was fused to the truncated oprF gene by C-terminal deletion fusion strategy. The truncated OprF-lipase fusion protein was successfully displayed on the surface of Escherichia coli. Localization of the truncated OprF-lipase fusion protein was confirmed by western blot analysis, immunofluorescence microscopy, and whole-cell lipase activity. To examine the enzymatic characteristics of the cell surface displayed lipase, the whole-cell enzyme activity and stability were determined under various conditions. Cell surface displayed lipase showed the highest activity at 37 degrees C and pH 8.0. It retained over 80% of initial activity after incubation for a week in both aqueous solution and organic solvent. When the E. coli cells displaying lipases were used for enantioselective resolution of racemic 1-phenylethanol in hexane, (R)-phenyl ethyl acetate was successfully obtained with the enantiomeric excess of greater than 96% in 36 h of reaction. These results suggest that E. coli cells displaying lipases using OprF as an anchoring motif can be employed for various biotechnological applications both in aqueous and nonaqueous phases.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Water activity dependence of performance of surface-displayed lipase in yeast cells: a unique water requirement for enzymatic synthetic reaction in organic media

Water activity (a(w)) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the a(w) dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing a(w) of the salt solution, to give the best performance at a(w) of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique a(w) profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher a(w) resulted in a higher reaction rate. Moreover, increasing a(w) was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique a(w) dependence probably because of the variation in cell surface characteristics.     

Inhibition of pancreatic and microbial lipases by proteins

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.     

Effects of gum arabic on lipase interfacial binding and activity.

We investigated the surface behavior of gum Arabic (GA) as well as its effects on the lipolytic activity of human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HLL), using emulsions of triacylglycerols (TAG) with various chain lengths. The effects of GA on the interfacial binding of HPL were also investigated. In the presence of 4 mM sodium taurodeoxycholate (NaTDC), GA (3% w/v, final concentration) had no effect on the HPL activity measured in the presence of colipase, whatever the type of TAG used. However, in the absence of bile salts or at low bile salt concentrations, GA inhibited the HPL activity when trioctanoin (TC8) and purified soybean oil (PSO) were used as substrates. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. Both crude and dialyzed GA solutions were found to be highly tensioactive at the air-water as well as the oil-water interface using the drop technique. In conclusion, GA, or a putative contaminant present in GA, was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL.     

Long-chain fatty acyl-CoA esters induce lipase activation in the absence of a water-lipid interface

In most lipases a mobile element or lid domain covers the catalytic site of the enzyme and the lid opening event, which usually proceed at a lipid-water interface, is required to form the catalytically competent lipase. We report here a noticeable increase in activity of two fungal lipases assayed in aqueous solution in absence of any interface when adding submicellar concentrations of amphipathic physiological molecules like long-chain acyl-CoAs. The catalytic activity was dramatically dependent on the acyl chain length of the amphiphile and could be related with a lid-opening process. Our data support that lipase activation can be triggered in the absence of a well-defined interface, and stresses the notion that other non-aggregated amphipathic constituents of the local microenvironment can act as putative regulators of lipase activity.     

A study of the surface-active properties of the Mg2+-activated ATPase from cytoplasmic membranes of Streptococcus faecalis.

The surface activity and enzymic properties of the factor F1, the catalytic moiety of Streptococcus faecalis H+-ATPase, has been studied at the air-water and phospholipid-water interfaces. F1 does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F1 molecule at the air-water interface have been estimated. In the presence of Mg2+, base area is S = 1.8 . 10(4) A2, height h = 27 A. Bearing in mind the size of a globular subunit, it follows from the measurements that the major F1 subunits should all lie in the same plane. The ATPase activity of F1 at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution. Adsorption of F1 at the interface shifts the isoelectric point of tiscussed relative to the proton-active transport mechanism.     

Orientation and conformation of a lipase at an interface studied by molecular dynamics simulations

Electron density profiles calculated from molecular dynamics trajectories are used to deduce the orientation and conformation of Thermomyces lanuginosa lipase and a mutant adsorbed at an air-water interface. It is demonstrated that the profiles display distinct fine structures, which uniquely characterize enzyme orientation and conformation. The density profiles are, on the nanosecond timescale, determined by the average enzyme conformation. We outline a computational scheme that from a single molecular dynamics trajectory allows for extraction of electron density profiles referring to different orientations of the lipase relative to an implicit interface. Profiles calculated for the inactive and active conformations of the lipase are compared with experimental electron density profiles measured by x-ray reflectivity for the lipase adsorbed at an air-water interface. The experimental profiles contain less fine structural information than the calculated profiles because the resolution of the experiment is limited by the intrinsic surface roughness of water. Least squares fits of the calculated profiles to the experimental profiles provide areas per adsorbed enzyme and suggest that Thermomyces lanuginosa lipase adsorbs to the air-water interface in a semiopen conformation with the lid oriented away from the interface.     

Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: Description of a lipase-activating pretreatment

The influence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak of fluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.     

脱脂棉固定化脂肪酶的非水活性与稳定性

脂肪酶具有非水催化作用,但其非水催化活性和稳定性需进一步提高,这是非水酶学的瓶颈问题之一。理想的策略是模拟脂肪酶的界面活化机制,以大分子代替水,优化、稳定化和有效分散酶蛋白,阻止其在有机相中变性。因此,选用多羟基、比表面积大、惰性、且与酶蛋白能兼容的大分子--脱脂棉纤维,作为固定化载体,以1∶0.9的质量比,通过物理吸附,将假单胞菌脂肪酶(Pseudomonas cepacia lipase)固定在脱脂棉纤维上。在催化己醇与乙酸乙烯酯的转酯反应中,反应1 h,脱脂棉固定化脂肪酶转化底物的能力是酶粉的3.7倍。在每次6 h共6次的循环催化中,固定化酶和酶粉转化底物的能力分别平均每次降低约0.3%和2.4%。表明脱脂棉固定化脂肪酶的非水活性,尤其是稳定性明显提高。这为通过固定化有效提高脂肪酶的非水催化作用,以满足工业应用的需要,提供了一种有效的途径和重要参考。     

Production of diacylglycerols from glycerol monooleate and ethyl oleate through free and immobilized lipase-catalyzed consecutive reactions

The ability of free and immobilized lipase on the production of diacylglycerols (DAG) by transesterification of glycerol monooleate (GMO) and ethyl oleate was investigated. Among three free lipases such as lipase G (Penicillium cyclopium), lipase AK (Pseudomonas fluorescens) and lipase PS (Pseudomonas cepacia), lipase PS exhibited the highest DAG productivity, and the DAG content gradually increased up to 24 hours reaction and then remained steady. The comparative result for DAG productivity between free lipase PS and immobilized lipases (lipase PS-D and Lipozyme RM IM) during nine times of 24 hours reaction indicated that total DAG production was higher in immobilized lipase PS-D (183.5mM) and Lipozyme RM IM (309.5mM) than free lipase PS (122.0mM) at the first reaction, and that the DAG production rate was reduced by consecutive reactions, in which more sn-1,3-DAG was synthesized than sn-1,2-DAG. During the consecutive reactions, the activity of lipase PS was relatively steady by showing similar DAG content, whereas DAG production of lipase PS-D and Lipozyme RM IM was gradually decreased to 69.9 and 167.1mM at 9th reaction, respectively, resulting in 62% and 46% reduced production when compared with 1st reaction. Interestingly, from 7th reaction lipase PS produced more DAG than immobilized lipase PS-D, and exhibited a stable activity for DAG production. Therefore, the present study suggested that DAG productivity between GMO and ethyl oleate was higher in immobilized lipases than free lipases, but the activity was reduced with repeated uses.     

Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases

Hydrophobins are fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. This attribute can be used to introduce hydrophobic foci on the surface of hydrophilic supports where hydrophobins are attached by covalent binding. In this paper, we report the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) to construct a support for noncovalent immobilization and activation of lipases from Candida antarctica, Humicola lanuginosa, and Pseudomonas flourescens. Lipase immobilization on agarose-bound hydrophobins proceeded at very low ionic strength and resulted in increased lipase activity and stability. The enzyme could be desorbed from the support using moderate concentrations of Triton X-100, and its enantioselectivity was similar to that of lipases interfacially immobilized on conventional hydrophobic supports. These results suggest that lipase adsorption on hydrophobins follows an "interfacial activation" mechanism; immobilization on hydrophobins offers new possibilities for lipase study and modulation and reveals a new application for fungal hydrophobins.     

Oil-bodies as substrates for lipolytic enzymes

Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown. One of the questions that arises is whether the protein/phospholipid coat of oil-bodies prevents the access of lipase to the oil core. We have investigated the susceptibility of almond oil-bodies to in vitro lipolysis by various purified lipases with a broad range of biochemical properties. We have found that all the enzymes assayed were capable of releasing on their own free fatty acids from the TAG of oil-bodies. Depending on the lipase, the specific activity measured on oil-bodies using the pH-stat technique was found to range from 18 to 38% of the specific activity measured on almond oil emulsified by gum arabic. Some of these lipases are known to have a dual lipase/phospholipase activity. However, no correlation was found to exist between the ability of a lipase to readily and efficiently hydrolyse the TAG content of oil-bodies and the presence of a phospholipase activity. Kinetic studies indicate that oil-bodies behave as a substrate as other proteolipid organelles such as milk fat globules. Finally we have shown that a purified water-soluble plant lipase on its own can easily hydrolyse oil-bodies in vitro. Our results suggest that the lipolysis of oil-bodies in seedlings might occur without any pre-hydrolysis of the protein coat.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

Structure and evolution of the lipase superfamily.

The lipase superfamily includes three vertebrate and three invertebrate (dipteran) proteins that show significant amino acid sequence similarity to one another. The vertebrate proteins are lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL). The dipteran proteins are Drosophila yolk proteins 1, 2, and 3. We review the relationships among these proteins that have been established according to gene structural relatedness and introduce our findings on the phylogenetic relationships, distance relationships, and evolutionary history of the lipase gene superfamily. Drosophila yolk proteins contain a 104 amino acid residue segment that is conserved with respect to the lipases. We have used the yolk proteins as an outgroup to root a phylogeny of the lipase family. Our phylogenetic reconstruction suggests that ancestral PL diverged earlier than HL and LPL, which share a more recent root. Human and bovine LPL are shown to be more closely related to murine LPL than to guinea pig LPL. A comparison of the distance (a measure of the number of substitutions between sequences) between mammalian and avian LPL reveals that guinea pig LPL has the largest distance from the other mammals. Human, rodent, and rabbit HL show marked divergence from one another, although they have similar relative rates of amino acid substitution when compared to human LPL as an outgroup. Human and porcine PL are not as divergent as human and rat HL, suggesting that PL is more conserved than HL. However, canine PL demonstrates an unusually rapid rate of substitution with respect to the other pancreatic lipases. The lipases share several structurally conserved features. One highly conserved sequence (Gly-Xaa-Ser-Xaa-Gly) contains the active site serine. This feature, which agrees with that found in serine esterases and proteases, is found within the entire spectrum of lipases, including the evolutionarily unrelated prokaryotic lipases. We review the location and possible activity of putative lipid binding domains. We have constructed a conservation index (CI) to display conserved structural features within the lipase gene family, a CI of 1.0 signifying perfect conservation. We have found a correlation between a high CI and the position of conserved functional structures. The putative lipid-binding domains of LPL and HL, the disulfide-bridging cysteine residues, catalytic residues, and N-linked glycosylation sites of LPL, HL, and PL all lie within regions having a CI of 0.8 or higher. A number of amino acid substitutions have been identified in familial hyperchylomicronemia which result in loss of LPL function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. (c) 1993 John Wiley & Sons, Inc.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.     

The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.