Production of lipolyzed butteroil by a calf pregastric esterase immobilized in a hollow fiber reactor: III. Effect of glycerol

Lipolysis of butter oil in a hollow fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C, a pH of 6.0, and glycerol concentrations of 0, 150, and 500 g/L in the buffer solution. The concentrations of 10 fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography. The rate of loss of enzyme activity and the relative selectivities of this esterase depended on the glycerol concentration. By contrast, the overall rate of release of fatty acids was not affected by the glycerol concentration. Loss of enzyme activity was modeled using first-order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The model was successful in describing the rates of release of all 10 fatty acid species for a range of space times from 0 to 25 h. The parameters of the model were tested for dependence on glycerol concentration.     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil

A lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat-sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35°C. For the reaction conditions used in this research, a pseudo-zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis–Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell-shaped (or Γ-type) distribution. Data taken at five different pH values were fit assuming a Dixon–Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first-order kinetics with a half-life of 19.9 days at 35°C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped-substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.     

Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409

Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5 mM. The enzymes were stable at 50 degrees C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70 degrees C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64 kDa by SDS-PAGE. The 23 amino acid sequence from the NH(2)-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.     

Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach

The genus Pectinatus has been often reported in beer spoilage with off-flavours. The bacteria are strictly anaerobic, Gram-negative rods. Propionate and acetate are the main fermentation products from glucose in the two species belonging to the genus, P. cerevisiiphilus and P. frisingensis. Amino acids routinely present at a high level in beer were not growth substrates for both species, and a significant accumulation of succinate was observed with lactate as growth substrate. Both Pectinatus ssp. showed almost identical fermentation balances on glucose. Growth kinetics of both glucose-grown species were unchanged under a N₂, H₂ or 20% CO₂-containing atmosphere. Combinations of culture medium pH values from pH 3·9 to pH 7·2, of glucose levels between 5 and 55 mmol l^-1, and of lactate concentrations varied from 4 to 40 mmol l^-1 demonstrated that biomass and volatile fatty acids production were proportional to glucose concentration for both Pectinatus species. A significant increase of volatile fatty acid production was measured for both species at the lowest pH values with a lactate or a glucose concentration increase. The maximum biomass production was observed at pH 6·2 for P. cerevisiiphilus , and between pH 4·5 and pH 4·9 for P. frisingensis. Glucose and lactate or pH value were dependent with regard to propionate and acetate production in P. frisingensis. On the other hand, the variations of these three parameters were independent with regard to biomass production for both strains, and to volatile fatty acids production for P. cerevisiiphilus. Addition of ethanol to glucose-grown cultures completely inhibited growth at 1·3 mol l^-1 ethanol for P. cerevisiiphilus , and at 1·8 mol l^-1 for P. frisingensis.     

一株嗜盐菌新种的分离与鉴定

从舟山册子岛船舶压载水泥样中分离到一株细菌S3-22,其与已知细菌的16S rDNA序列相似性低于97%,G+C mol%为54.9 mol%,主要脂肪酸iso-C17:1ω9c(24.99%),细胞醌型为甲基萘醌MK-5。革兰氏染色阴性,最适生长条件为30～37℃、pH7、3%NaCl。嗜盐,氧化酶、接触酶、淀粉酶、酯酶呈阳性,可还原硝酸盐。依据其16S rDNA序列相似性、系统发育学分析及细胞与分子水平的鉴定表明,该菌是Kordiimonas属的一个新种,菌株S3-22的16S rDNA序列登陆号为FJ847942。     

Fatty acid specificity of immobilized kid goat pre-gastric esterase: Effects of pH

Summary Kid goat pre-gastric esterase immobilized in a hollow fiber reactor was used to hydrolyse butteroil at buffer pH values of 5.0, 6.0 and 7.0. Overall hydrolysis proceeded fastest at pH 6.0, but changes in volatile fatty acid ratios with pH suggest that the same enzyme:substrate system can produce different flavor profiles, e. g., at pH 6.0 the relative rate of production of undesirable soaplike flavors is minimized.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part III. Multiresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the continuous hydrolysis of the glycerides of butter oil at 40 degrees C and pH 7.0. The effluent concentrations of 10 different free fatty acid products were measured by highperformancee liquid chromatography (HPLC). Multiresponse nonlinear regression methods were used to fit the data to a multisubstrate rate expression derived from a Ping Pong Bi Bi mechanism in which the rate-controlling step is deacylation of the lipase. Thermal deactivation of the enzyme was also included in the mathematical model of reactor performance. A postulated normal distribution of v(max) with respect to the chain length of the fatty acid (with an additive correction for the degree of unsaturation) was tested for statistical significance. The model is useful for predicting the free fatty acid profile of the lipolyzed butteroil product over a wide range of flow rates.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

The uptake of oleic acid by rat small intestine: a comparison of methodologies

The interaction between long-chain and medium-chain lipids during intestinal absorption was examined using several model systems. A decrease in steady-state triolein (LCT) output in thoracic duct lymph after addition of trioctanoin (MCT) to the duodenal infusion confirmed previous studies in unanesthetized rats which demonstrated inhibition of steady-state LCT uptake from the small intestinal lumen by MCT. In slices of everted rat jejunum octanoic acid reduced incorporation into triglyceride and initial uptake of (14)C-labeled oleic acid from micellar solutions. Inhibition of uptake did not occur at 0 degrees C, when triglyceride synthesis was blocked. Incubation of slices at low pH (5.8) or in the presence of dimethyl sulfoxide also reduced uptake of oleic acid and its incorporation into triglyceride. However, when everted sacs of jejunum were similarly incubated, octanoate, dimethyl sulfoxide, or low pH caused no inhibition of oleic acid uptake or esterification. The results indicate that the significance of kinetic data describing intestinal fatty acid absorption which were obtained from experiments conducted in vitro is highly questionable, and that suitable models for in vivo uptake kinetics have yet to be developed. However, analysis of the in vitro kinetic data suggests that the intestinal mucosal membrane does not function as a simple lipid interface with respect to fatty acid absorption.     

Evidence of evolutionary optimization of fatty acid length and unsaturation

The lipid composition of cell membranes exerts a crucial influence on cell physiology. Indeed, one double bond triggers membrane fluidity, essential for cell functionality, but additional double bonds increase the susceptibility to peroxidation, which produces reactive compounds that impair the viability of cells. It has therefore been suggested, but never tested in an extensive comparative context, that the composition of membrane fatty acids has been optimized during evolution. A similar prediction has been made for fatty acid chain length, on which susceptibility to peroxidation also depends. Here I tested for stabilizing selection on fatty acid composition by evaluating the fitting of the single stationary peak (SSP) model of evolution to a large data set from 107 species of birds, against alternative evolutionary models. I found that across‐species variation in average chain length and in the proportion of monounsaturated fatty acids (MUFAs), but not in the proportion of polyunsaturated (PUFAs) nor saturated (SFAs) fatty acids, was better explained by SSP models than by other models. Results show optimum values of fatty acid chain length and proportion of MUFAs of 18 C atoms and 25.5% mol, respectively, the strength of stabilizing selection being particularly high in chain length. This is the first evidence of evolutionary optimization in fatty acid composition, suggesting that certain values may have been selected because of their adaptive capacity to minimize susceptibility to lipid peroxidation.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

Modeling dynamic experiments on the anaerobic degradation of molasses wastewater

The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Esterase activities of cell-free extracts from 'wild' strains of leuconostocs and heterofermentative lactobacilli isolated from traditional Greek cheese

Cell-free extracts from strains of leuconostocs and heterofermentative lactobacilli were tested for esterase activities using synthetic substrates. Results showed that esterases from both lactic acid bacteria groups preferentially degraded short-chain fatty acids. Moreover, even though some similarities between strains of the same species were recorded, the observed differences were not strong enough to distinguish between species and therefore commend this technique as a tool for taxonomy of either leuconostocs or heterofermentative lactobacilli.     

Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: a role of plasma esterases

Hydrolysis of acetylsalicylic acid (ASA, aspirin), an antiplatelet drug commonly used in the prevention of stroke and myocardial infarction, seems to play a crucial role in its pharmacological action. Thirty-eight healthy volunteers and 38 type 2 diabetic patients were enrolled to test the hypothesis that the enhanced plasma degradation and lowered bioavailability of ASA in diabetic patients is associated with the attenuation of platelet response. Aspirin esterase activities were tested at pH 7.4 and 5.5. A significantly higher overall aspirin esterase activity was noted at pH 7.4 in the diabetic patients (P<0.003), corresponding to faster ASA hydrolysis (P<0.006). This increased activity was attributable to butyrylcholinesterase and probably to albumin, because it was effectively inhibited by eserine and 4-bis-nitrophenyl phosphate (P<0.01). No significant differences between control and diabetic subjects were found at pH 5.5 in either enzymatic activities or ASA hydrolysis rates. The enhanced plasma ASA degradation in diabetic subjects was significantly associated with the refractoriness of blood platelets to ASA (P<0.05) and modulated by plasma cholesterol (P<0.01). No direct effects of plasma pH or albumin were observed. In conclusion, higher aspirin esterase activity contributes to the lowered response of diabetic platelets to ASA-mediated antiplatelet therapy.     

Hydrolysis of Butteroil by Immobilized Lipase Using a Hollow-Fiber Reactor: Part VI. Multiresponse Analyses of Temperature and pH Effects

A lipase from Aspergillus niger, immobilized by physical adsorption on hydrophobic hollow fibers made of microporous polypropylene, was used to effect the hydrolysis of the glycerides of melted butterfat at 40, 50, 55, and 60°C (pH 7.0), and at pH 3.0, 4.0, 5.0, 7.0, 8.0, and 9.0 (40°C). McIlvane buffer and melted butterfat were pumped cocurrently through the hollow fiber reactor. The concentrations of ten different free fatty acids in the effluent oil stream were measured by HPLC. Multiresponse nonlinear regression methods were employed to fit the data to multisubstrate rate expressions derived from a Ping Pong Bi Bi mechanism in which the rate controlling step is deacylation of the enzyme. Thermal deactivation of the immobilized lipase was also included in the mathematical model of reactor performance. A postulated normal distribution of v_max with respect to the number of carbon atoms of the fatty acid residue (with an additive correction for the number of double bonds) was found to provide the best statistical fit of the data. The models developed can be used to independently predict the effects of either the pH or the temperature, as well as the reactor space time and the time elapsed after immobilization, on the free fatty acid profile of the lipolyzed butteroil product.     

Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.     

Ionization and phase behavior of fatty acids in water: application of the Gibbs phase rule

The phase behavior of several medium-chain (10- and 12-carbon) and long-chain (18-carbon) fatty acids in water was examined as a function of the ionization state of the carboxyl group. Equilibrium titration curves were generated above and below fatty acid and acid-soap chain melting temperatures and critical micelle concentrations, and the phases formed were characterized by X-ray diffraction, 13C NMR spectroscopy, and phase-contrast and polarized light microscopy. The resulting titration curves were divided into five regions: (i) at pH values less than 7, a two-phase region containing oil or fatty acid crystals and an aqueous phase; (ii) at pH approximately 7, a three-phase region containing oil, lamellar, and aqueous (or fatty acid crystals, 1:1 acid-soap crystals, and aqueous) phases; (iii) between pH 7 and 9, a two-phase region containing a lamellar fatty acid/soap (or crystalline 1:1 acid-soap) phase in an aqueous phase; (iv) at pH approximately 9, a three-phase region containing lamellar fatty acid-soap (or crystalline 1:1 acid-soap), micellar, and aqueous phases; and (v) at pH values greater than 9, a two-phase region containing micellar and aqueous phases. Interpretation of the results using the Gibbs phase rule indicated that, for oleic acid/potassium oleate, the composition of the lamellar fatty acid/soap phase varied from approximately 1:1 to 1:3 un-ionized to ionized fatty acid species. In addition, constant pH regions observed in titration curves were a result of thermodynamic invariance (zero degrees of freedom) rather than buffering capacity. The results provide insights into the physical states of fatty acids in biological systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a feruloyl esterase from Fusarium oxysporum catalyzing esterification of phenolic acids in ternary water-organic solvent mixtures

An extracellular feruloyl esterase (FAE-II) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by SP-Sepharose, t-butyl-HIC and Sephacryl S-200 column chromatography. The protein corresponded to molecular mass and pI values of 27 kDa and 9.9, respectively. The enzyme was optimally active at pH 7 and 45 degrees C. The purified esterase was fully stable at pH 7.0-9.0 and temperature up to 45 degrees C after 1 h incubation. Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 but inactive to the C-2 positions of arabinofuranose such as 4-nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside. In the presence of Sporotrichum thermophile xylanase, there was a significant release of ferulic acid from destarched wheat bran by FAE-II, indicating a synergistic interaction between FAE-II and S. thermophile xylanase. FAE-II by itself could release only little ferulic acid from destarched wheat bran. The potential of FAE-II for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsion formed in ternary mixture consisting of n-hexane, 1-propanol and water.