Asymmetric short-chain phosphatidylcholines: defining chain binding constraints in phospholipases

Several short-chain asymmetric lecithins with a total of 14 carbons in the acyl chains (ranging from 1-lauroyl-2-acetylphosphatidylcholine to 1-hexanoyl-2-octanoylphosphatidylcholine) have been synthesized and characterized. The specific activities of phospholipase A2 from cobra venom, phospholipase A2 from porcine pancreas, and phospholipase C from Bacillus cereus toward these lecithins as micelles have been determined. The results of these kinetic studies allow the definition of hydrophobic binding requirements in the active sites of these water-soluble phospholipases. For phospholipase C, with the exception of monomyristoylphosphatidylcholine, each of the asymmetric short-chain lecithins exhibits high activity, comparable to the 14-carbon symmetric short-chain species, diheptanoylphosphatidylcholine. Therefore, for phospholipase C, in addition to the acyl linkages, only a certain degree of hydrophobicity in the fatty acyl chains is requisite for substrate binding and appreciable hydrolysis; there is no chain specificity. The activity of phospholipase A2 from cobra venom toward the same asymmetric lecithins is quite different. As the sn-2 chain lengthens, activity is increased to a maximum for diheptanoyl-PC. Further increase in the number of carbons in the sn-2 chain has no effect on hydrolysis rates. For this enzyme, seven carbons in the sn-2 chain are necessary for optimal activity. In contrast, porcine pancreatic phospholipase A2 activity shows very little dependence on sn-2 chain length.     

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate

Lymphoid cell lines established by Epstein-Barr virus transformation of blood B lymphocytes from a patient with Wolman's disease exhibited the acid lipase deficiency characteristic for this disease. Comparison of hydrolysis by normal and Wolman's cells of 4-methylumbelliferyl-acyl esters with variable chain length demonstrates that: (1) the best substrates for acid lipase were characterized by an acyl chain length of 12-18 carbon atoms; (2) the acid residual activity in Wolman's cells showed a slightly different substrate specificity and this is probably due to an acid carboxylesterase different from the lysosomal acid lipase, and (3) the 'nonspecific' carboxylesterases (at pH 6.0 and 8.0) not inhibited by taurocholate showed a characteristic substrate specificity for short-chain fatty acids. In the used assay conditions (optimal for acid lipase), methylumbelliferyl-palmitate, -elaidate and -lignocerate are the most accurate synthetic substrates for the diagnostic of Wolman's disease.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis–Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure–function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis–Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme–substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.     

Lysophospholipase A2 activity in guinea-pig heart microsomal fractions displaying high activities with 2-acylglycerophosphocholines with linoleic and arachidonic acids.

Lysophospholipases A1 which catalyse the hydrolysis of acyl groups from 1-acylglycerophosphocholine (GPC) have been characterized in a number of mammalian tissues and do not exhibit any acyl specificity. In the present study lysophospholipase activity in guinea-pig heart microsomes (microsomal fractions) that hydrolyses 2-acyl-GPC was detected and characterized. The enzyme showed a high degree of acyl specificity. The relative rates of hydrolysis of individual 2-acyl-GPCs with different fatty acids was as follows: C18:2/C20:1/C18:1/C16:0, 14:6:1:1. When substrates were presented in pairs, the hydrolysis of each substrate by the enzyme was inhibited, but to very different extents. Of each pair of lysolipids examined (2-arachidonoyl- and 2-palmitoyl-GPC; 2-arachidonoyl- and 2-linoleoyl-GPC), the one with the expected higher rate of hydrolysis was more severely inhibited and the degree of inhibition was dependent on the concentration of the other lysolipid. The characteristics of the lysophospholipase A2 suggest the enzyme could work in concert with phospholipase A1 to release arachidonic and linoeic acids for further metabolism. The properties of lysophospholipase A2 and A1 suggest that they are different enzymes.     

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.     

Zero-order interfacial enzymatic degradation of phospholipid tubules.

The first study of enzymatic hydrolysis of phospholipid tubules is reported. Phosphatidylcholines with acyl chains containing diacetylene groups are known to form tubular microstructures in which the lipids are tightly packed and crystalline. These tubules can be used to probe the role of microstructural form in the mechanics of interfacial enzymatic degradation by such enzymes as phospholipase A2 (PLA2). Hydrolysis by PLA2 may occur most rapidly in regions having the greatest number of bilayer packing defects, such as those that must be found at tubule ends. A microstructure that degrades primarily from its ends should exhibit zero-order kinetics, because the area of the degrading tubule and remains constant as the length of the microstructure decreases. Free fatty acid concentration was measured to follow the generation of PLA2 hydrolysis products in suspensions of diacetylenic phospholipid tubules. The kinetics of tubule hydrolysis were essentially zero-order until conversion was complete, as predicted. However, microscopy of partially hydrolyzed tubules revealed the formation of multiple discrete anionic product domains along the length of degrading tubules as well as in insoluble reaction product microstructures. Furthermore, the rate of tubule hydrolysis was only moderately enhanced by increasing the number of tubule ends, which is consistent with the conclusion that tubule ends are not the only sites of hydrolysis. A model that reconciles the overall kinetics with the morphological evidence is proposed.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

A facile purification procedure of phospholipase D from cabbage and its characterization.

Phospholipase D (PLD), an enzyme predestined for the preparation of new phospholipids, was isolated from cabbage and purified in a highly efficient way by using a combination of hydrophobic chromatography and a specific calcium effect. In the presence of calcium ions (50mM), PLD is bound from the crude enzyme solution to Octyl-Sepharose and subsequently selectively eluted by removing the calcium ions. The obtained enzyme is electrophoretically pure (95%), its molecular mass and isoelectric point were determined to be 87,000 Da and 4.7, respectively. The purified enzyme was kinetically characterized by use of mixed phosphatidylcholine-SDS micelles as well as the short-chain lecithins 1,2-dihexanoyl- and 1,2-diheptanoyl-sn-glycero-3-phosphocholine as substrates. A hyperbolic upsilon/[S]-characteristic was obtained for the mixed micellar system, whereas the upsilon/[S] curves of the short-chain lecithins reflect the dependence of velocity on the physical state of the substrate. A small velocity increase was observed up to a critical substrate concentration near the critical micelle concentration, from where the velocity increases hyperbolically.     

Fatty acyl chain specificity of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II and its (56-79) synthetic fragment

Mixed acyl chain phosphatidylcholine molecules in Triton N-101 micelles were employed as substrates for lipoprotein lipase to test which substrate acyl chain has the greatest effect on activation of the enzyme by apolipoprotein C-II. The phospholipase A1 activity of lipoprotein lipase was measured by pH-stat. The activation factor (lipoprotein lipase activity plus apolipoprotein C-II/activity minus apolipoprotein C-II) increased monotonically with apolipoprotein C-II concentration up to 1 microM apolipoprotein C-II at an enzyme concentration of 0.01 microM. The maximal activation factor for phosphatidylcholine substrate molecules with sn-2 acyl chain lengths of 14 averages 14.8. By contrast, for sn-2 acyl chain lengths of 16 the activation factor was 29.2. Varying the sn-1 acyl chain length had no significant effect on the activation factor. The chain-length dependence of the activation factor is similar with the apolipoprotein C-II peptide fragment comprising residues 56-79, which does not include the lipid-binding region of apolipoprotein C-II. These data are consistent with a model for activation of lipoprotein lipase in which residues 56-79 bind to lipoprotein lipase and alter the interaction of the sn-2 acyl chain of the phosphatidylcholine (PC) substrate or the lysoPC product within the activated state complex.     

Chain length dependence of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) on the bovine milk lipoprotein lipase (LpL)-catalyzed hydrolysis of a homologous series of saturated phosphatidylcholines was examined with respect to the fatty acyl chain length of the substrates. Dilauryl-, dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine solubilized by Triton X-100 and sonicated vesicles of dimyristoylphosphatidylcholine were used as substrates. The maximal rate of the LpL-catalyzed hydrolysis of each of these lipids was determined in the absence and presence of apoC-II. The activation factor (the ratio of enzyme activity with apoC-II to that without the activator protein) increased with increasing mol ratios of apoC-II to LpL and was maximal at a ratio of approximately 50. At all apoC-II/LpL mole ratios tested, the activation factor increased as a function of fatty acyl chain length. A quantitative relationship between fatty acyl chain length and the extent of maximal activation of LpL by apoC-II was observed: the logarithm of the activation factor is a linear function of the number of carbon atoms of a single fatty acyl chain of the substrates.     

Protein depalmitoylases

Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.     

Kinetic studies on the reactivation of d-β-hydroxybutyrate dehydrogenase with mixtures of short-chain lecithins

d-β-hydroxybutyrate dehydrogenase, purified as soluble, lipid-free apoenzyme (inactive) from rat liver mitochondria can be reactivated by the short-chain dihexanoyl, diheptanoyl, and dioctanoyl lecithins at the monomeric state, upon formation of a reversible enzyme-lecithin complex. Previous studies with these lecithins suggested that reactivation of the apoenzyme requires the simultaneous occupation of two identical, noninteracting lecithin binding sites via a rapid equilibrium random mechanism. The short-chain lecithins exhibited similar reactivating capacities, differing only in their affinities towards the enzyme. In order to further test that model, the reactivation of the apoenzyme was studied when two or three short-chain lecithins were simultaneously present in the reaction medium. The initial velocities were measured either as a function of the concentration of one lecithin while the other(s) were kept constant, or as a function of the total phospholipid concentration with mixtures of different lecithins at a constant molar ratio. The pertinent equations were derived on the principles of multiple equilibria with identical, noninteracting sites able to be occupied by any of the different lecithins present in the reaction medium, with the doubly occupied enzyme as the only active species. In agreement with the above-proposed model, the results obtained indicates that the molar fraction of the doubly occupied (active) enzyme species can be calculated from equilibrium considerations and that the maximal attainable with the different short-chain lecithins are similar.     

Synthesis of dermorphin-(1-4) derivatives catalyzed by proteases in organic solvents.

In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing D-amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) with potent analgesic activity. The N-terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin-(1-4) derivatives, Boc-Tyr-D-Ala-Phe-Gly-N(2)H(2)Ph and Boc-Tyr-D-Ala-Phe-Gly-NH(2), which contained a d-amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin-(1-4) derivatives could be catalyzed by subtilisin with Boc-Tyr-D-Ala-OCH(2)CF(3) as an acyl donor substrate in AcOEt. The synthesis of dermorphin-(1-2) derivative Boc-Tyr-D-Ala-N(2)H(2)Ph was catalyzed by alpha-chymotrypsin in different organic solvents and D-Ala-N(2)H(2)Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

Short-chain lecithin/long-chain phospholipid unilamellar vesicles: asymmetry, dynamics, and enzymatic hydrolysis of the short-chain component

Asymmetric unilamellar vesicles are produced when short-chain phospholipids (fatty acyl chain lengths of 6-8 carbons) are mixed with long-chain phospholipids (fatty acyl chain lengths of 14 carbons or longer) in ratios of 1:4 short-chain/long-chain component. Short-chain lecithins are preferentially distributed on the outer monolayer, while a short-chain phosphatidylethanolamine derivative appears to localize on the inner monolayer of these spontaneously forming vesicles. Lanthanide NMR shift experiments clearly show a difference in head-group/ion interactions between the short-chain and long-chain species. Two-dimensional 1H NMR studies reveal efficient spin diffusion networks for the short-chain species embedded in the long-chain bilayer matrix. The short-chain lecithin is considerably more mobile than the long-chain component but has hindered motion compared to short-chain lecithin micelles. This differentiation in physical characteristics of the two phospholipid components is critical to understanding the activity of phospholipases toward these binary systems.     

Influence of mechanical agitation on cutinases and protease activity towards polyamide substrates

Two polyamide 6,6 substrates with different constructions, namely a model substrate and a fabric, were hydrolyzed using native cutinase and L182A cutinase mutant (from Fusarium solani pisi) and a protease (subtilisin from Bacillus sp.). The catalytic efficiency of these enzymes, measured in terms of hydrolysis products release, was measured for both substrates and the protease released five times more amines to the bath treatment. The L182A cutinase mutant showed higher activity when compared with the native enzyme.

All enzymes have shown activity additive effects with higher levels of mechanical agitation for polyamide fabrics. The results achieved are of paramount importance on the design of a process of enzymatic functionalization of polyamide.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Recombinant Candida rugosa lipase 2 from Pichia pastoris: immobilization and use as biocatalyst in a stereoselective reaction

The characterization of the recombinant Candida rugosa Lip2 (r-Lip2) isoenzyme obtained from fed-batch cultures of Pichia pastoris under PAOX promoter was carried out, determining the optimal pH and temperature as well as their catalytic performance in both hydrolysis and synthesis reactions comparing with purified native Lip2 (n-Lip2) previously determined. The substrate specificity of r-Lip2 in hydrolysis reactions was determined with a series of triacylglycerols and p-nitrophenyl esters of variable acyl chain length. r-Lip2 showed the maximum specificity for both substrates towards medium-chain esters (C-8), similar behavior was observed with n-Lip2. However, significant differences were observed towards unsaturated substrates (triolein) or short-chain esters. A statistical design applied to study the effect of pH and temperature on lipase stability shown that r-Lip2, like n-Lip2, was more sensitive to pH than temperature changes. Nevertheless, the overall stability of soluble r-Lip2 was lower than soluble n-Lip2. The stability of r-lip2 was significantly improved by immobilization onto EP100, an excellent support for lipases with yields around 95% for offered lipolytic activity lower than 600 AU/mL. Finally, immobilized r-Lip2 was tested in the resolution of ibuprofen in isooctane by means of enantioselective esterification using 1-butanol as esterifying agent. r-Lip2 showed a better performance in terms of enantiomeric excess (74%) and enatiomeric factor (96%) than n-Lip2 (56 and 80%, respectively) for the same conversion (40%). Thus, r-Lip2 should be considered a good and pure biocatalyst, easy to produce and with a remaining activity of ca. 90% after one reaction cycle when immobilized on EP100.