Wheat Germ Lipase Research Articles

Role of isotropic lipid phase in the fusion of photosystem II membranes

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300–500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Novel activity of human BChE: Lipid hydrolysis

Acetylcholinesterase and butyrylcholinesterase (BChE) typically hydrolyze the neurotransmitter acetylcholine. The multifunctional enzyme BChE is associated with lipid metabolism through an undefined mechanism. Based on lipid-related studies and by comparing the structural similarities between lipases and BChE we postulated that the association of BChE with lipid metabolism could occur through hydrolytic activity. Utilizing purified BChE enzymes from different sources and several lipases as controls, the ability of BChE to hydrolyze 4-methylumbelliferyl (4-mu) palmitate is investigated. Using lectin affinity, inhibition kinetics, and molecular modeling, we demonstrated that purified BChE hydrolyzed 4-mu palmitate at pH 8 as effectively as wheat germ lipase. The affinity Km value of the enzymes for 4-mu palmitate as substrate is found as 10.4 μM, 34.2 μM, 129.8 μM, and 186 μM for wheat germ lipase, purified BChE, pancreatic lipase, and commercial BChE, respectively. Analysis of the inhibitory effect of 4-mu palmitate on BChE using butyrylthiocholine as substrate revealed competitive inhibition with Ki and IC50 values of 448 μM and 987.2 μM, respectively. The binding affinity and interactions of 4-mu palmitate with BChE and pancreatic lipase were predicted by molecular docking. These results suggest that BChE possesses lipolytic activity. The possibility that BChE hydrolyzes not only 4-mu palmitate but also other types of lipids will lead to a new approach to those disease states associated with increased BChE activity/expression.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs—indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase—suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that—in line with the Dynamic Exchange Model—the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane—Particles. I. 31P-NMR Spectroscopy

Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases—with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.

Study of the interfacial activity of wheat germ lipase

Wheat germ lipase (LA), which is abundant in wheat germ, could result in the deterioration of wheat germ. To explore the method of stabilising wheat germ and understand the regulatory mechanism of ...

Concurrent Asymmetric Reactions Combining Photocatalysis and Enzyme Catalysis: Direct Enantioselective Synthesis of 2,2-Disubstituted Indol-3-ones from 2-Arylindoles.

The combination of photoredox and enzymatic catalysis for the direct asymmetric one-pot synthesis of 2,2-disubstituted indol-3-ones from 2-arylindoles through concurrent oxidization and alkylation reactions is described. 2-Arylindoles can be photocatalytically oxidized to 2-arylindol-3-one with subsequent enantioselective alkylation with ketones catalyzed by wheat germ lipase (WGL). The chiral quaternary carbon center at C2 of the indoles was directly constructed. This mode of concurrent photobiocatalysis provides a mild and powerful strategy for one-pot enantioselective synthesis of complex compounds. The experiments proved that other lipases containing structurally analogous catalytic triad in the active site also can catalyze the reaction in the same way. This reaction is the first example of combining the non-natural catalytic activity of hydrolases with visible-light catalysis for enantioselective organic synthesis and it does not require any cofactors.

Evaluation of the possible non‐thermal effect of microwave radiation on the inactivation of wheat germ lipase

AbstractTo determine whether non‐thermal effect exist during the process of enzyme inactivation by microwave radiation, the conformation and activity of wheat germ lipase (WGL) treated by microwaves and conventional heating were investigated, respectively. The results indicated that, when the WGL was maintained at 20 °C, its Tyr, Trp residues, the secondary and tertiary structures did not changed, even though the microwave was sustained for 1,800 s with power of higher than 80 W. While, when the temperatures were up to 45 and 60 °C, the activity of WGL decreased 60% and 100%, respectively, and the tertiary structure of WGL was totally changed by microwave at 60 °C. Moreover, the inactivation efficient of microwave radiation was about 10% more than that of conventional heating based on the thermal effects. In conclusion, microwave inactive WGL based on thermal effects rather than non‐thermal effect, and was more effective than conventional heating.Practical ApplicationsMicrowave has been widely used in food thawing, drying, sterilization, enzyme inactivation and et al. In cereal industry, microwave is usually used to inactivate wheat germ lipase (WGL), which plays an important role in the storage of wheat products. However, because the effects of microwave was strongly interferes by the possible non‐thermal effect and cannot be separated easily, the potential inactivation mechanism of microwave on enzyme was not clear enough to guide the inactivation process of lipase by microwave.In this study, the effects of microwave and conventional heating on the stability of WGL were investigated to detect the possibility of non‐thermal effects during the inactivation process of lipase by microwave. The results make sure that, the microwave inactivated WGL based on thermal effect, rather than non‐thermal effect. Thus, we can focus on the factors that influence the heating efficiency to improve the effect of inactivation of WGL or other lipase by microwave.

Biocatalytic asymmetric Mannich reaction of ketimines using wheat germ lipase

Wheat germ lipase (WGL) was used for the first time as a biocatalyst in asymmetric Mannich reaction of ketimines.

Conditional Ultrasound Sensitivity of Poly[(N-isopropylacrylamide)-co-(vinyl imidazole)] Microgels for Controlled Lipase Release.

Triggering the release of cargo from a polymer network by ultrasonication as an external, noninvasive stimulus can be an interesting concept for on-demand release. Here, it is shown that, in pH- and thermosensitive microgels, the ultrasound sensitivity of the polymer network depends on the external conditions. Crosslinked poly[(N-isopropylacrylamide)-co-(vinyl imidazole)] microgels showed a volume phase transition temperature (VPTT) of 25-50 °C, which increases with decreasing pH. Above the VPTT the polymer chains are collapsed, while below VPTT they are extended. Only in the case of maximum observed swelling, where the polymer chains are expanded, the microgels are mechanically fragmented through ultrasonication. In contrast, when the polymer chains are partially collapsed it is not possible to manipulate the microgels by ultrasound. Additionally, the ultrasound-induced on-demand release of wheat germ lipase from the microgels could be demonstrated successfully. The principle of conditional ultrasound sensitivity is likely to be general and can be used for selection of matrix-cargo combinations.

A modified titrimetric method for measuring the activity of wheat germ lipase

Wheat germ lipase is a cereal lipase which plays an important role in the growth and storage of wheat products. The commonly used titrimetry is not reliable and accurate enough to determine lipase activity due to its difficulties in identifying the end point when used to measure the alkaline lipase of low activity. In this paper, acidic titration method was utilised, with triacetin as substrate to measure the activity of wheat germ lipase by an automatic potentiometric titrator. The optimum catalysis time is 4 h, the pH value of end point is 6.0, and the initial pH of substrate is 7.7. Under optimum test conditions, the activity of wheat germ lipase was found to be 50.398±0.848 U/mg, within a linear range of 1.0-10.0 mg, and with relative standard deviation equal to 1.682%. This method has proved to be precise and reproducible for measuring alkaline lipase of low activity in aqueous solution.

Harmful Effect of Detergents on Lipase

In order to study effects of detergents at molecular level, we have done activity measurements of wheat germ lipase in increasing concentration of some commercial detergents. Conformational changes in protein structure using circular dichroism and fluorescence spectroscopy were studied in increasing concentration of sodium dodecyl sulfate. Our study proves that detergents may lead to loss of enzymatic activity and structure of plant enzymes. Since detergents are common source of pollution in water bodies and the water from these resources can be used in fields, our study may prove helpful in creating awareness about harmful action of detergents.

A modified colorimetric method for determining the activity of wheat germ lipase in low-aqueous media

Copper soap colorimetry is a common way to determine the free fatty acids of grain. However, when determining the lipase activity of grain by copper soap colorimetry its accuracy is frequently affected by the degree of mixedness between lipase and substrate. Grain lipase extraction and purification steps are quite cumbersome as well. To overcome these shortcomings, a modified colorimetric method was established for the determining of wheat germ lipase activity in a low-aqueous media. First, we partially defatted wheat germ, then adjusted it to a certain water activity, added some olive oil, extracted the free fatty acids after incubating for a period of time, then determined the absorbance of reaction system by copper soap colorimetry. Finally we obtained the activity of wheat germ lipase by calculation. The optimum conditions were: coordinated the water activity of wheat germ to 0.84, the addition of 0.75 ml olive oil per gram wheat germ and incubation for 4 h at 55 °C. It was calculated that the lipase ...

The Effect of Humidity and Compactional Pressure on the Wheat Germ Lipase Activity

The use of proteins and peptides as human therapeutics has been increased in recent years. Lipase is a relatively homogeneous proteinaceous enzyme indicated in maldigestion. The purpose of the present study was to evaluate the effect of humidity along with compactional pressure on the enzymatic activity of wheat germ lipase. Samples of lipase powder were kept at different relative humidity (RH) conditions (24, 40, 63 and 75 %) before compaction under various pressures (74-372 Mega pascal or Mpa). The relative enzymatic activity of the compacts was then determined by titration method using Triacetin as substrate. Density measurements were also conducted in order to describe the possible mechanism of the enzymatic activity. The results indicated densities of the compacts prepared under various compactional pressures increase as humidity rises. Based on the results, activity loss of the compacts following relative humidity increase can be related to steric hindrance caused by higher pressures. However, there was no significant difference between densities at different compactional pressures at a given humidity condition.

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.

Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

AbstractThe progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter‐range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV‐detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L‐enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L‐esters of the two amino acids was also studied and it was found that the production of L‐threonine had priority over L‐serine. Chirality, 2010. © 2009 Wiley‐Liss, Inc.

Wheat germ lipase catalyzed kinetic resolution of secondary alcohols in non-aqueous media

The lipase from wheat germ was used for the kinetic resolution of secondary alcohols. It has the opposite enantioselectivity against the Kazlauskas rule and acts as an anti-Kazlauskas catalyst. The effect of initial water activity, organic solvent, acyl donor and temperature were investigated. Wheat germ lipase had a high activity and enantioselectivity only in n-hexane with a high initial water activity (alpha(w) = 0.97), especially with 1-phenylethanol (C 32%, E > 200). Its performance changed little with the chain length of acyl donor and temperature.

Identification of catalytically active groups of wheat (Triticum aestivum) germ lipase

The active site of wheat germ lipase was studied by the Dixon method and chemical modification. The profile of curve logV = f(pH), pK and ionization heat values, lipase photoinactivation, and lipase inactivation with diethylpyrocarbonate and dicyclohexylcarbodiimide led us to assume that the active site of the enzyme comprises the carboxylic group of aspartic or glutamic acid and the imidazole group of histidine. Apparently, the OH-group of serine plays a key role in catalysis: as a result of incubation for 1 h in the presence of phenylmethylsulfonyl fluoride, the enzyme activity decreased by more than 70%. It is shown that ethylenediamine tetraacetate is a noncompetitive inhibitor of lipase. Wheat germs are very healthful because they are rich in vitamins, essential amino acids, and proteins. For this reason, wheat germs are widely used in food, medical, and feed mill industries [1-3]. However, their use is limited by instability during storage, which is largely determined by the effect of hydrolytic and redox enzymes. Representative enzymes of this group are lipase (glycerol ester hydrolase, EC 3.1.1.3), which hydrolyzes triglycerides of higher fatty acids, and lipoxygenase (EC 1.13.11.13), which oxidizes polyunsaturated higher fatty acids.

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.

Chemoenzymatic synthesis of enantiomerically enriched α-hydroxyamides

A study on a chemoenzymatic synthesis of model α-hydroxyamide was performed. Special attention was paid to the optimization of the enzymatic process, both on the selection of enzyme and cosolvent. An intriguing influence of cosolvent on the enantioselectivity of Wheat Germ Lipase and Amano PS Lipase catalyzed hydrolysis was observed, as the results obtained proved that enzyme's enantioselectivity is directly correlated with cosolvent's hydrophobicity. In the best example ( Wheat Germ lipase, Et 2O used as a cosolvent), the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7%ee.

Multicomponent diversity and enzymatic enantioselectivity as a route towards both enantiomers of α-amino acids—a model study

A model study on a new, enantioconvergent method for the synthesis of chiral, nonracemic α-amino acids is presented. α-Acetoxyamides obtained in a Passerini multicomponent reaction are selectively hydrolyzed by Wheat Germ lipase. Studies on conversion of the thus obtained, enantiomerically enriched α-hydroxyamides into α-aminoamides are presented. Products of these reactions are then hydrolyzed to give α-amino acids.