Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.     

Substrate Specificity and Mode of Action of the Lipase Produced by Pseudomonas fragi22.39 B

The lipase purified from Pseudomonas fragi 22.39 B hydrolyzed not only triglycerides but also synthetic esters such as Tween, Span and methyl oleate. Of the saturated monoacid triglycerides tested, tributyrin was hydrolyzed most quickly. The lipase did not produce 1,3-diolein as a hydrolysis product from triolein. The addition of the Ca²⁺ ion to the reaction mixture promoted the hydrolysis rate for triglycerides and monoesters with longer-chain fatty acids (C₁₄, C₁₆, C₁₈). The enzyme could hydrolyze various kinds of natural fats and oils, and the extent their hydrolysis reached above 90%.     

Enzymatic transesterification with the lipase from Pseudomonas fragi 22.39 B in a non-aqueous reaction system

The lipase from Pseudomonas fragi 22.39 B catalyzed the transesterification in ester and alcohol mixtures without any other solvent. Activated esters, such as vinyl and phenyl esters, were excellent acyl donors for the reaction, and the activity was enhanced by increasing the carbon number of the fatty acid fraction of the esters. Primary alcohols were esterified faster than secondary ones in this reaction system, while tertiary alcohols such as alpha-terpineol did not react at all. The lipase exhibited stereoselectivity in the esterification of alcohols such as 2-octanol.     

Lipase from Pseudomonas fragi. I. Purification of the Enzyme

The experimental conditions required to isolate a lipase from Pseudomonas fragi were determined. The organism was grown in a buffered tryptone medium for 4 to 5 days at 20 C. The lipase in the culture supernatant fluid was isolated by fractionation with ammonium sulfate at 60% saturation, followed by acetone precipitation at 30-60% concentration. Further purification was made by using Sephadex G-200 gel-filtration and diethylaminoethyl cellulose chromatography. Electrophoretic analysis of the purified lipolytic fraction showed apparent homogeneity by both cellulose polyacetate and disc electrophoresis. The specific activity of the purified enzyme was about 100 times that of the starting culture filtrate, and the yield was about 1.8% of the original activity.     

Lipase from Pseudomonas fragi. II. Properties of the Enzyme

The optimal pH value of a lipase from Pseudomonas fragi was between 7.5 and 8.9, and a high reaction rate was observed at 54 C. Heating the enzyme solution at 63 C for 30 min inactivated only 27.6% of its activity; however, total inactivation was observed at 66 C after 1 hr and at 71 C after 10 min. The lipase was inhibited strongly by Fe⁺⁺⁺ and Fe⁺⁺ ions, and to a lesser extent by Co⁺⁺, Cu⁺⁺, Zn⁺⁺. No inhibition was observed with Ca⁺⁺ or NaF. Ethylenediaminetetraacetate was effective in removing the toxicity of Fe⁺⁺⁺. The activity of the enzyme was inhibited markedly by p-chloromercurobenzoate, but the effects of N-ethylmaleimide and iodoacetate were moderate. The enzyme was able to hydrolyze natural fats, synthetic triglycerides, and alcohol esters. The order of the rate of hydrolysis of some triglycerides under experimental conditions was, from the fastest to the lowest, trilaurin, tricaprin, tricaprylin, tripalmitin, tributyrin, tricaproin, and tristearin. The enzyme was capable of hydrolyzing methyl butyrate, but the rate of hydrolysis was about one-fifth that for triolein and one-thirteenth that for coconut oil. The enzyme lost its activity rapidly when held frozen, at 20 C, and at the extremes in pH. Glutathione, cysteine, and mercaptoethanol did not preserve the activity of the enzyme.     

Ester Production by Pseudomonas fragi IV. Demonstration of Esterase Activity

Assay of the esterase activity of sonically treated cell-free extracts, whole cell suspensions, and supernatant fluid of Pseudomonas fragi cultures with a differential respirometer revealed that the esterases were intracellular. Polyacrylamide-gel electrophoresis demonstrated six bands of esterase activity, which revealed substrate specificity differences. Band 1 exhibited slow mobility, bands 2, 3, and 4 moderate mobility, and bands 5 and 6 rapid mobility. Six bands were active with alpha-naphthyl acetate, four bands with alpha-naphthyl propionate, and 5 bands with alphanaphthyl butyrate. These esterases appeared to be more active with aromatic esters than with aliphatic esters.     

Ester Production by Pseudomonas fragi. II. Factors Influencing Ester Levels in Milk Cultures

Cultures of Pseudomonas fragi were grown at 21 C in sterile homogenized milk and reconstituted skim milk media supplemented with ethyl alcohol. Quantitative determinations of ethyl butyrate and ethyl hexanoate by gas-liquid chromatography showed definite increases in the concentrations of the two esters produced in these media in comparison to media not supplemented with ethyl alcohol. Supplementation with butyric acid in addition to ethyl alcohol generally elevated the ethyl butyrate concentration and usually depressed the cell count slightly. Aeration of any of the media during growth tended to reduce the cell population slightly. A relationship between increase in cell number and increase in concentration of esters during the growth of the culture was observed. Media containing high concentrations of ethyl alcohol plus milk fat or low-molecular-weight fatty acids were conducive to the production of a fruity aroma by P. fragi.     

Enzymatic Synthesis of Various Aromatic Polyesters in Anhydrous Organic Solvents

Lipases and proteases from various sources were tested in aromatic polyester synthesis in organic solvents. A commercial protease from Bacillus licheniformis efficiently catalyzed the transesterifica-tion of a diester of terephthalic acid and 1,4-butanediol in anhydrous tetrahydrofuran (THF). This protease was used as a catalyst in the synthesis of aromatic polyesters in THF. Oligomers with average molecular weights from 400 to 1000 daltons were obtained using various diols and aromatic diesters.     

Kinetics of Enantioselective Esterification of Naproxen by Lipase in Organic Solvents

The kinetics of stereoselective esterification of racemic Naproxen with trimethylsilyl methanol by Candida cylindracea lipase in organic solvents has been investigated. A Ping-Pong Bi Bi mechanism with competitive inhibition by this alcohol for each enantiomer -has been identified. The rate equations were further analyzed in the time-course reaction after considering the effect of enzyme deactivation in the organic mixtures, but not in isooctane. Effects of the hydrophobicity of solvent on the solubility of the racemate, the kinetic parameters and their combinations are also discussed.     

Synthesis of Ester Oligomer by Aspergillus niger Lipase

Lipase from Aspergillus niger NRRL 337 catalyzed the synthesis of esters from various dicarboxylic acids and diols. Among the esters synthesized, those from 1,13-tridecanedioic acid and 1,3-propanediol were separated by gel permeation chromatography. The constitution of the purified ester was determined by using IR and MS. The dominant components of synthesized esters were pentamer and heptamer, and both end groups of the pentamer and heptamer were hydroxyl.

The possible reaction sequence in synthesis of ester oligomer by Aspergillus niger lipase is described.     

Thermal Resistance of Pseudomonas fragi in Milk Containing Various Amounts of Fat

Similar populations of Pseudomonas fragi were grown at 25 C for 20 hr or at 7 C for 7 days in milk containing 0, 10, and 20% fat; they were then heated at 48, 50, and 52 C in milk containing 0, 10, and 20% fat. After inoculation, the heating medium contained 2.1 x 10(6) to 6.9 x 10(6) organisms per milliliter. The P. fragi cells grown in skim milk had greater thermal resistance (D(52) = 3.0 to 3.1) than those grown in milk containing fat (D(52) = 1.9 to 2.5). The organisms grown at 7 C for 7 days in milk containing 10% fat were more resistant (D(52) = 3.0) than those grown in the same medium at 25 C for 20 hr (D(52) = 2.0). The presence of 0 to 20% milk fat in the heating medium had no apparent effect on the thermal resistance.     

Chymotrypsins Modified by Neutral or Charged Amphiphiles as Catalysts for Ester Synthesis in Polar Organic Solvents

-Chymotrypsin has been modified by a series of neutral liposaccharidic or charged lipocarboxylic amphiphile reagents. In the esterification of N-acetyl tyrosine in three polar solvents, the new biocatalysts have been compared to chymotrypsins modified by reductive alkylation with glyoxylic acid, melibiose or octanal. This comparison indicates that the rate accelerations observed with the neutral or anionic amphiphile-coated enzymes are mainly due to the hydrophobization of the protein surface in the neigbourhood of the external lysine residues. This interpretation is strengthened by the favorable effect of supports more hydrophobic than celite on the reaction.     

Lipase from Pseudomonas fragi CRDA 323: Partial purification,characterization and interesterification of butter fat

Several strains of Pseudomonas were screened for lipase production using the rhodamine agar diffusion test. On the basis of the diameter of the halo produced, P. fragi CRDA 323 and P. putida ATCC 795 were considered to be good and weak lipase producers respectively. P. fragi, cultured in a 2-1 fermenter, produced a maximal amount of lipase after 3–4 days of incubation at 27°C. The lipase extract of P. fragi was obtained by acidification of culture supernatant at pH 4.0 and partially purified with ammonium sulphate precipitation. The majority of lipase activity (42%) was located in fraction IV, precipitated at 20%–40% of saturation, with a 19-fold enzyme purification. The K _m and V _max values for the partially purified enzymatic extract (fraction IV) were 0.70 mg/ml and 0.97 × 10^–3 U/min respectively. Fraction IV, which showed and optimum activity at pH 8.5, was used for the interesterification of butter fat in a microemulsion free co-surfactant system containing Span 60 (sorbitol monostearate) and Tween 60 (polyoxyethylene sorbitan monostearate) in the ratio 48:52 (v/v). The results showed that the interesterification of butter fat resulted in a considerable decrease in long-chain saturated fatty acids (C12:0, C14:0 and C16:0) with a concomitant increase in C18:0 and C18:1 at the sn-2 position of selected triacylglycerols. In addition, the results demonstrated an increase in the fatty acids (C12:0, C14:0 and 16:0) among the 1 and 3 positions of the triacylglycerol molecules of modified buffer fat accompanied by a decrease in C18:0 and C18:1.     

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Hydrophobic Substitution of Surface Residues Affects Lipase Stability in Organic Solvents

A novel lipase has been recently isolated from a local Pseudomonas sp. (GQ243724). In the present study, we have tried to increase the organic solvent stability of this lipase using site-directed mutagenesis. Eight variants N219L, N219I, N219P, N219A, N219R, N219D, S251L, and S251K were designed to change the surface hydrophobicity of this enzyme with respect to the wild-type. Among these variants, the stability of N219L and N219I significantly increased in the presence of all tested organic solvents, whereas two mutants (N219R and N219D) significantly exhibited decreased stabilities in all the organic solvent studied, suggesting that improvement of hydrophobic patches on the enzyme surface enhances the stability in organic media. Furthermore, replacing Ser²⁵¹ with hydrophobic residues on the enzyme surface dramatically diminished its stability in the tested condition. In spite of the distance of the mutated sites from the active site, the values of k _cat and K _m were affected. Finally, structural analysis of the wild-type and mutated variants was carried out in the presence and absence of some organic solvents using circular dichroism and fluorescence spectroscopy.     

非水溶剂中Rhizopus arrhizus脂肪酶催化合成三种酯的最佳条件(英文)

以少根根霉 (Rhizopusarrhizus)脂肪酶为催化剂 ,有机溶剂为反应介质 ,合成了 3种短链脂肪酸酯 .研究了反应温度、溶剂、底物浓度、底物摩尔比、吸水剂用量等因素对酯化反应的影响 .确定了3种酯的最佳合成条件 :(1)己酸乙酯 :反应温度为 4 0℃ ,环己烷为溶剂 ,0 2 5mol L底物浓度 ,酸醇摩尔比为 1∶1 2 ;(2 )乙酸异丙酯 :5 0℃ ,环己烷为溶剂 ,0 15mol L底物浓度 ,摩尔比为 1∶1;(3)乙酸异戊酯 :5 0℃ ,异辛烷为溶剂 ,0 2 0mol L底物浓度 ,摩尔比为 1∶1.三种酯合成时均需 0 12 5g ml的0 5nm分子筛为吸水剂 ,在 8h后 ,合成酯转化率达到 97%～ 99% .     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

神经网络优化非水相脂肪酶催化合成抗坏血酸油酸酯的条件研究

抗坏血酸油酸酯具有强抗氧化作用．为了获得脂肪酶催化合成抗坏血酸油酸酯的最适条件,主要研究了反应温度、脂肪酶量、油酸量对抗坏血酸油酸酯合成效果的影响．采用中心组合设计和动量梯度下降神经网络对反应条件网络进行训练仿真,并利用训练好的网络对催化酯化工艺条件进行预测．研究结果表明：经过训练的网络可以很好的模拟反应条件,得到了脂肪酶催化反应的最佳工艺参数．当抗坏血酸0．8g时,反应温度56℃,油酸量0．95g,固定化脂肪酶量0．74g,添加分子筛条件下,抗坏血酸油酸酯的转化率为46.5％．该方法为抗坏血酸酯化催化效果的预测提供了一条可行的途径．     

Characteristics of Lipase Catalysis During Ester Synthesis in Reversed Micellar Systems

The ability of lipase from Candida cylindracea to catalyze ester synthesis from a long chain fatty acid (palmitic acid) and alcohols of varying chain length, is examined. The enzyme is located in the minimal-water environment of reversed micelles. Lipase activity is a strong function of the mode of encapsulation. Direct solid lipase addition to reversed micelles leads to encapsulation in an inactive state unless the enzyme is contacted with the acyl substrate. The alcohol inhibits activity, with low molecular weight alcohols tending to denature the enzyme. Implications to reversed micelle based biocatalyst preparation are briefly discussed.