Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: a quantitative model

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(H(N epsilon) - O(alc)) between the histidine side chain and the alcohol oxygen increases, d(H(N epsilon) - O(alc)) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(H(N epsilon) - O(alc)) is >2.2 A for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(H(N epsilon) - O(alc)) is less than 2.0 A, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(H(N epsilon) - O(alc)) is around 2.1 A. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.     

Catalytic behavior of Pseudomonas cepacia lipase in w/o microemulsions

The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping-pong bi-bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be K(m octanol) = 310 mM, K(m lauric acid) = 78 mM, and V(max) = 250 mumol min(-1) mg(-1). The same system was used for the synthesis of mono- and diglycerides from glycerol and lauric acid, which was successful at very low w(o) values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C(12)E(4)). The optimum activity was found at about w(o) = 8. The apparent values of V(max app) and K(m app) for octanol were calculated and found to be 100 mumol min(-1) mg(-1) and 76 mM, respectively. (c) 1995 John Wiley & Sons, Inc.     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

Comparative fatty acid selectivity of lipases in esterification reactions with glycerol and diol analogues in organic media

Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C. For P. cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA. For R. miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA. For C. antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA. The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used. These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions.     

Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle

Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.     

Improving lipase enantioselectivity in organic solvents by forming substrate salts with chiral agents

We recently demonstrated (J Am Chem Soc 121:3334-3340, 1999) that enzymatic enantioselectivity in organic solvents can be markedly enhanced by temporarily enlarging the substrate via salt formation. In the present study, this approach was expanded by finding that, in addition to its size, the stereochemistry of the counterion can greatly affect the enantioselectivity enhancement. For example, the enantioselectivity [E = (k(cat)/K(M))(S)/(k(cat)/K(M))(R)] of crystalline Pseudomonas cepacia lipase in the propanolysis of phenylalanine methyl ester (PheOMe) in anhydrous acetonitrile was found to be 5.8 +/- 0.6; the E value doubled when PheOMe's salt with S mandelic acid was used as a substrate instead of the free ester, and rose sevenfold with R mandelic acid as a Bronsted-Lowry acid. Similar effects were observed with other bulky, but not petite, counterions. The greatest enantioselectivity enhancement was afforded by 10-camphorsulfonic acid: the E value increased to 18 +/- 2 for a salt with its R enantiomer and jumped to 53 +/- 4 for the S. These effects, also observed in other organic solvents, were explained by means of structure-based molecular modeling of the lipase-bound transition states of the substrate enantiomers and their diastereomeric salts.     

Regulation of exercise carbohydrate metabolism by estrogen and progesterone in women

To assess the roles of endogenous estrogen (E2) and progesterone (P4) in regulating exercise carbohydrate use, we used pharmacological suppression and replacement to create three distinct hormonal environments: baseline (B), with E2 and P4 low; estrogen only (E), with E2 high and P4 low; and estrogen/progesterone (E + P), with E2 and P4 high. Blood glucose uptake (R(d)), total carbohydrate oxidation (CHO(ox)), and estimated muscle glycogen utilization (EMGU) were assessed during 60 min of submaximal exercise by use of stable isotope dilution and indirect calorimetry in eight eumenorrheic women. Compared with B (1.26 +/- 0.04 g/min) and E + P (1.27 +/- 0.04 g/min), CHO(ox) was lower with E (1.05 +/- 0.02 g/min). Glucose R(d) tended to be lower with E and E + P relative to B. EMGU was 25% lower with E than with B or E + P. Plasma free fatty acids (FFA) were inversely related to EMGU (r(2) = 0.49). The data suggest that estrogen lowers CHO(ox) by reducing EMGU and glucose R(d). Progesterone increases EMGU but not glucose R(d). The opposing actions of E(2) and P(4) on EMGU may be mediated by their impact on FFA availability or vice versa.     

Fluorescent organophosphonates as inhibitors of microbial lipases

Short- and long-chain 1-O-alkyl-2-acylaminodeoxyglycero- and alkoxy-alkylphosphonic acid p-nitrophenyl esters were synthesized as inhibitors for analytical and mechanistic studies on lipolytic enzymes. The respective compounds contain perylene or nitrobenzoxadiazole as reporter fluorophores covalently bound to the omega-ends of the respective 2-acylamino- and alkoxy- residues. Their inhibitory effects on the activities of three selected lipases showing different substrate preferences were determined, including the lipases from Rhizopus oryzae, Pseudomonas species, and Pseudomonas cepacia. R. oryzae lipase reacted much better with the single-chain inhibitors than the two-chain deoxyglycerolipids. In contrast, P. cepacia lipase was inactivated by perylene-containing two-chain phosphonate (XXII) to a larger extent as compared to the other inhibitors whereas Pseudomonas species lipase interacted efficiently and without any preferences with all inhibitors used in this study. In summary, the different lipases show a very characteristic reactivity pattern not only with respect to triacylglycerol substrates but also to their structurally related inhibitors. Thus, the novel phosphonates might be useful tools not only for analysis and discrimination of known lipolytic enzymes but also for discovery of yet unknown lipases/esterases in biological samples.     

Statistical medium optimization and production of a hyperthermostable lipase from Burkholderia cepacia in a bioreactor

AIM: Statistical medium optimization for maximum production of a hyperthermostable lipase from Burkholderia cepacia and its validation in a bioreactor. METHODS AND RESULTS: Burkholderia cepacia was grown in shake flasks containing 1% glucose, 0.1% KH2PO4, 0.5% NH4Cl, 0.24% (NH4)2HPO4, 0.01% MgSO4.7H2O and 1% emulsified palm oil, at 45 degrees C and pH 7.0, agitated at 250 rev min(-1) with 6-h-old inoculum (2% v/v) for 20 h. A fourfold enhancement in lipase production (50 U ml(-1)) and an approximately three fold increase in specific activity (160 U mg(-1)) by B. cepacia was obtained in a 14 litre bioreactor within 15 h after statistical optimization following shake flask culture. The statistical model was obtained using face centred central composite design (FCCCD) with five variables: glucose, palm oil, incubation time, inoculum density and agitation. The model suggested no interactive effect of the five factors, although incubation period, inoculum and carbon concentration were the important variables. CONCLUSIONS: The maximum lipase production was 50 U ml(-1), with specific activity 160 U mg(-1) protein, in a 14 litre bioreactor after 15 h in a medium obtained after statistical optimization in shake flasks. Further, the model predicted reduction in time for lipase production with reduction in total carbon supply. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistical optimization allows quick optimization of a large number of variables. It also provides a deep insight into the regulatory role of various parameters involved in enzyme production.     

Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication.

The effect of concentrated cell-free extracellular material from stationary-phase cultures of Burkholderia cepacia 10661 and Pseudomonas aeruginosa PAO1 on virulence factor production in B. cepacia was assessed. While increasing concentrations of the B. cepacia exoproduct caused a slight increase in siderophore, lipase, and protease production in the producing organism, a significant in productivity was observed for all three virulence factors with the addition of the PAO1 exoproduct. Moreover, the addition of the exoproduct from a strain of P. aeruginosa producing reduced amounts of autoinducer caused only a slightly greater response than that of the control. Both B. cepacia 10661 and P. aeruginosa PAO1, along with two matched clinical isolates of both organisms obtained from a cystic fibrotic patient, were shown to produce variable amounts of three different types of autoinducer. The potential for interspecies signalling in microbial pathogenicity is discussed.     

Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.     

Novel route for the resolution of both enantiomers of dropropizine by using oxime esters and supported lipases of Pseudomonas cepacia

Resolution of (R)- and (S)-dropropizine which is an antitussive and central sedative therapeutic agent in high optical and chemical yields was achieved by lipases of Pseudomonas cepacia supported on ceramic particles (lipase PS-C) and on diatomite (lipase PS-D) with oxime esters in organic solvents. The influence of several factors (lipase source, structural variations in oxime esters, the amount of lipase and its recyclability) on the enantioselectivity have been investigated. Different properties were used to describe the solvents, namely the hydrophobicity (quantified by log P) and the dielectic constant (epsilon). This enzymatic acylation using oxime esters was significant as only (S)-dropropizine and (R)-dropropizine monoacetate was obtained. (R)-Dropropizine monoacetate was chemically hydrolyzed to obtain (R)-dropropizine. The highest enantioselectivity was observed when O-acetyl benzophenone oxime was used. This enzymatic resolution provides a versatile method for getting the pure enantiomers of dropropizine by effectively optimizing the various reaction parameters.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

Genotypic associations of the hepatic secretion of VLDL apolipoprotein B-100 in obesity

We examined the effect of genetic polymorphisms of proteins regulating intrahepatic processing of apolipoprotein B-100 (apoB) and the supply of neutral lipids to the liver on the hepatic secretion of very low density lipoprotein (VLDL) apoB in obesity. Hepatic secretion of very low density apolipoprotein B-100 (VLDL apoB) was measured using an infusion of [1-(13)C]leucine in 29 obese men. Isotopic enrichment and turnover of VLDL apoB was determined using gas chromatography-mass spectrometry and multi-compartmental modelling, respectively. Visceral fat was measured by magnetic resonance imaging. Genotypes for the apoB signal peptide (SP27/SP24 alleles), microsomal triglyceride transfer protein promoter (MTP, -493 G/T alleles), apoE (E2, E3, E4 alleles), hepatic lipase promoter (-514 C/T alleles), and cholesteryl ester transfer protein (CETP, Taq1B B1/B2 alleles) were determined using polymerase chain reaction. Statistically significant associations were found between hepatic secretion of apoB and allelic combinations of i) apoB SP with apoE (P = 0.02), hepatic lipase (P = 0.02), and CETP (P = 0. 006) genes, ii) MTP promoter with CETP genes (P = 0.03); the association with apoBSP/MTP promoter allelic combinations just failed to reach significance (P = 0.06), however. The CETP/apoBSP allelic combination was the most significant predictor of apoB secretion, and this was independent of visceral fat, plasma lathosterol and insulin levels, and dietary fat. SP24 carriers who were homozygous for CETP B1 had 60% lower apoB secretion than B2 heterozygotes who were non-carriers of SP24 (10.5 +/- 1.74 mg/kg fat free mass/day, n = 7 vs. 26.1 +/- 3.16, n = 22). The data suggest that variation in both the apoB and CETP genes may be a major genetic determinant of the hepatic secretion of apoB in men with visceral obesity.     

Efficient chemoenzymatic synthesis of (S)- and (R)-5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide: key intermediate for Src-SH2 inhibitor

A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity.     

Enhancing the activity and regioselectivity of lipases for 3'-benzoylation of floxuridine and its analogs by using ionic liquid-containing systems

Ionic liquid-containing systems offered new opportunities to enzymatic acylation of nucleosides. In Pseudomonas cepacia lipase-mediated benzoylation of floxuridine (FUdR), the enzyme performances, including enzyme activity and 3'-regioselectivity, were significantly enhanced by using [C(4)MIm]PF(6)-containing systems. It was observed that the enzyme performances not only depended on the anion of IL, but also on the cation, and that a proper combination between the cation and anion was critical to allow the enzyme to exhibit excellent performances. The optimal IL content in IL-containing systems is 5% (v/v). To gain a deeper insight into enzyme recognition, we extended benzoylation of FUdR catalyzed by P. cepacia lipase to its analogs. FUdR and its analogs could be biotransformed to the desired product by P. cepacia lipase in excellent conversion (>99%) and good to excellent 3'-regioselectivity (81-99%). Furthermore, the 3'-regioselectivity of P. cepacia lipase was enhanced with the increase of hydrophobicity of 5-substituent of the substrates, due to better hydrophobic interactions with Leu287 present in the active site.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Synthesis, protonation behavior, conformational analysis, and regioselective enzymatic acylation of the novel diamino analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)

(E)-3',5'-Diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through 1H NMR spectroscopy. In addition, a convenient synthesis of N-3' and N-5' acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5'-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3'-amino group, thus affording derivatives 9.     

从植物根际定向批量筛选广谱有机溶剂耐受性脂肪酶产生菌

洋葱伯克霍尔德菌脂肪酶是一类具有重要工业应用价值的优良脂肪酶之一。根据已公布的洋葱伯克霍尔德菌基因组信息, 在传统的洋葱伯克霍尔德菌选择性培养基中添加适量的氨苄青霉素和卡那霉素, 从植物根际的土壤中筛选洋葱伯克霍尔德菌。对获得的单菌落再用含罗丹明B指示剂的产脂肪酶定性检测平板检测, 从4个根际土壤中筛选到35株产脂肪酶的洋葱伯克霍尔德菌, 阳性率达到65%。其中15株对体积浓度为10%的苯、己烷和正庚烷同时具有耐受性。用recA基因分子鉴定上述15株菌种, 全部属于洋葱伯克霍尔德菌菌群。