Selectivity in enzyme catalysed reaction: Preferential hydrolysis of saturated methyl ester over the corresponding unsaturated ester by pig liver esterase

Summary Saturated methyl ester is preferentially hydrolysed in presence of corresponding ,-unsaturated ester by Pig Liver Esterasse. This selectivity is known for aliphatic, aromatic and heterocyclic esters.Chemo, regio and stereoselective transformations are an important goal towards synthetic efficiency (Bartman and Trost, 1984). Among the various methods that are employed to achieve such reactions, enzymatic transformations are in the forefront of current research activities (Jones, 1986) because of several advantages over the chemical methods. These include extremely high degree of selectivity, easier isolation of products, specially with immobilized enzymes and their reusability. In the past decade this class of biocatalysts has provided a plathora of valuable selective transformations (Boland et.al, 1991). Pig Liver Esterase (PLE) is one such enzyme which has been thoroughly studied and used for asymmetric (Zhu and Tedford, 1990) and regioselective (Adachi et.al, 1986) hydrolysis of esters. Several chiral building blocks for the synthesis of complex natural products (Imori et.al., 1983) have been synthesized through PLE. In this communication, we report the preferential hydrolysis of a saturated ester over its unsaturated counterpart. The preference is total and works well for aliphatic, aromatic as well as heterocyclic systems.     

Activity and dynamics of an enzyme, pig liver esterase, in near-anhydrous conditions

Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 (±2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast (≤nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.     

Lipase-enhanced activity in flavour ester reactions by trapping enzyme conformers in the presence of interfaces

In order to improve the lipase-catalyzed synthesis of flavour esters, we have used the reported strategy of interfacial activation-based molecular (bio)imprinting [Mingarro et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3308], later called trapping in the presence of amphiphile interfaces (TPI) [Mingarro et al. 1996. Biochemistry 35: 9935]. Five lipases of fungal and mammalian origin typically used for esterification process have been explored to improve production by TPI treatment. A marked enhancement of enzymatic activity has been observed in all TPI-treated lipases assayed and the activation factor obtained was up to 90-fold. The dependence on chain length of acyl donors in the esterification of geranyl alcohol has been investigated, showing clear differences between activated and nonactivated lipase. The results indicate that this rational approach leads to conversion yields that are remarkably higher, not only than its counterpart pH-optimized control lipase, but also the "protected" lipase by conventional methods (lyoprotectans or salts). We propose this strategy as a promising tool to be used in more industrial biotransformations. Copyright 1998 John Wiley & Sons, Inc.     

Inhibition of the reactions catalysed by a type I topoisomerase and catenating enzyme of Trypanosoma cruzi by DNA-intercalating drugs. Preferential inhibition of the catenating reaction

A catenating enzyme and a type I topoisomerase were purified from Trypanosoma cruzi. We investigated the inhibitory effect of DNA-intercalating drugs on topoisomerisations catalysed by these enzymes. Inhibition of catenation was detected by electrophoretic analysis in neutral agarose gels. However, the inhibition of relaxation was not readily detectable in these gels since supercoiled DNA, which was relaxed in the presence of an intercalating drug, returned to a supercoiled state when the drug was removed. Thus electrophoretic analyses were made in gels containing chloroquine so that unreacted DNA could be distinguished from DNA relaxed by the enzyme. The results show that the catenation was more sensitive to DNA-intercalating drugs than the relaxation.     

A study of the reaction catalysed by the liver branching enzyme

The mechanism of action of liver branching enzyme has been studied by using as substrate two polysaccharides in which the non-reducing ends had been labelled by incubation with phosphorylase and a trace amount of [¹⁴C]glucose 1-phosphate. After these polysaccharides had been treated with branching enzyme, their structure was analysed by periodate oxidation, by degradation with phosphorylase and amylo-(1→6)-glucosidase and by degradation with pullulanase. All the results indicate that the branching enzyme catalyses the transfer from (1→4)- to (1→6)-linkage of a chain of glucose units, the minimum length of which is six glucose units. A maltodextrin containing 16 glucose units was not acted on by the branching enzyme.     

猪肝酯酶催化苯乙二醇环碳酸酯水解最佳反应条件的研究

猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度（15g/L）、pH值（8.0）、温度（25~30℃）及有机溶剂种类和浓度（二氧六环,65%v/v）,为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。     

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.     

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.     

Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study

We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

The effect of dimethyl sulfoxide on the enantioselectivity in the pig liver esterase catalyzed hydrolysis of dialkylated propanedioic acid dimethyl esters

The effect of dimethyl sulfoxide (DMSO) on the enantioselectivity in the pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of dialkylated propanedioic acid diesters was studied. Increasing concentrations of DMSO increased the ratio of the (R)-enantiomers of the produced monoesters. This effect was not due to the change in pH_app caused by the solvent. The reaction rate was lowered to one-tenth of the rate without DMSO when the DMSO concentration was increased to 50%.     

Lipase catalysed hydrolysis of ricebran oil by free and immobilised enzyme system in batch stirred reactor

A change of the reaction rate was observed for the lipasecatalysed hydrolysis of ricebran oil in a batch stirred tank reactor using immobilized lipase enzyme as compared to free enzyme. The reactor rate was observed to be controlled mainly by factors like temperature, pH, initial enzyme concentration, initial substrate concentration and initial products concentration.     

Apparent multiple catalytic sites involved in the ester hydrolysis of juvenile hormones by the hemolymph and by an affinity-purified esterase from Manduca sexta johannson (Lepidoptera: Sphingidae)

The esterases which metabolize juvenile hormone (JH) in some insects may be important in regulating the hormone titer. The JH ester-hydrolyzing activity (JHE) in the larval hemolymph of the tobacco hornworm (Manduca sexta) was found to be attributed to two forms of esterase with almost equivalent activity based on selectivity and kinetics of inhibition by two 3-substituted thio-1,1,1-trifluoropropan-2-ones and a phosphoramidothioate. Neither of the two forms were inhibited by diisopropyl phosphorofluoridate or iodoacetamide. Steady-state kinetics of JH II hydrolysis supported the inhibition studies and showed that the two forms were widely different in their affinity for JH II. The activity of the hemolymph was found to be bound selectively to an affinity column synthesized by the reaction of epoxy-activated Sepharose with 3-(4′-mercaptobutylthio)-1,1,1-trifluoropropan-2-one. This column offered a quantitative, one-step purification of JH esterase with a purification factor of ~800 and specific activity of ~573 nmol JH III hydrolyzed min⁻¹ mg protein⁻¹. The purified protein showed only a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of ~65,000. However, the purified enzyme apparently revealed the same two kinetic forms as the native enzyme, which indicates that two sites of the same protein are likely to be involved in JH hydrolysis.