Substrate Report for: Butylacetate

It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis of butyl acetate using MCI-CALB under ultrasonic energy, comparing the results against those obtained using the commercial preparation, Novozym 435. The optimal conditions were determined using response surface methodology (RSM) evaluating the following parameters: reaction temperature, substrate molar ratio, amount of biocatalyst, and added water. The optimal conditions for butyl acetate synthesis catalyzed by MCI-CALB were: temperature, 48.8 degrees C; substrate molar ratio, 3.46:1 alcohol:acid; amount of biocatalyst, 7.5%; and added water 0.28%, both as substrate mass. Under these conditions, 90% of conversion was reached in 1.5 h. In terms of operational stability, MCI-CALB was reused in seven cycles while keeping 70% of its initial activity under ultrasonic energy. The support pore size and resistance are key points for the enzyme activity and stability under mechanical stirring. The use of ultrasound improved both activity and stability because of better homogeneity and reduced mechanical stress to the immobilized system.

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50 degrees C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50 degrees C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50 degrees C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.

The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, P seudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in E scherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase.

It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis of butyl acetate using MCI-CALB under ultrasonic energy, comparing the results against those obtained using the commercial preparation, Novozym 435. The optimal conditions were determined using response surface methodology (RSM) evaluating the following parameters: reaction temperature, substrate molar ratio, amount of biocatalyst, and added water. The optimal conditions for butyl acetate synthesis catalyzed by MCI-CALB were: temperature, 48.8 degrees C; substrate molar ratio, 3.46:1 alcohol:acid; amount of biocatalyst, 7.5%; and added water 0.28%, both as substrate mass. Under these conditions, 90% of conversion was reached in 1.5 h. In terms of operational stability, MCI-CALB was reused in seven cycles while keeping 70% of its initial activity under ultrasonic energy. The support pore size and resistance are key points for the enzyme activity and stability under mechanical stirring. The use of ultrasound improved both activity and stability because of better homogeneity and reduced mechanical stress to the immobilized system.

The influence of low-frequency ultrasound (40 kHz) in the esterification reaction between acetic acid and butanol for flavor ester synthesis catalyzed by the commercial immobilized lipase B from Candida antarctica (Novozym 435) was evaluated. A central composite design and the response surface methodology were used to analyze the effects of the reaction parameters (temperature, substrate molar ratio, enzyme content and added water) and their response (yields of conversion in 2.5 h of reaction). The reaction was carried out using n-hexane as solvent. The optimal conditions for ultrasound-assisted butyl acetate synthesis were found to be: temperature of 46 degrees C; substrate molar ratio of 3.6:1 butanol:acetic acid; enzyme content of 7%; added water of 0.25%, conditions that are slightly different from those found using mechanical mixing. Over 94% of conversion was obtained in 2.5h under these conditions. The optimal acid concentration for the reaction was determined to be 2.0 M, compared to 0.3 M without ultrasound treatment. Enzyme productivity was significantly improved to around 7.5-fold for each batch when comparing ultrasound and standard mechanical agitation. The biocatalyst could be directly reused for 14 reactions cycles keeping around 70% of its original activity, while activity was virtually zeroed in the third cycle using the standard mixing system. Thus, compared to the traditional mechanical agitation, ultrasound technology not only improves the process productivity, but also enhances enzyme recycling and stability in the presence of acetic acid, being a powerful tool to improve biocatalyst performance in this type of reaction.

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50 degrees C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50 degrees C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50 degrees C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.

The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, P seudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in E scherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase.

A new lipase preparation from Rhizopus oryzae was used to catalyze the esterification reaction between acetic acid and butanol to produce butyl acetate ester (pineapple flavor). This flavor compound can be used in food, cosmetic and pharmaceutical industries. Only 3% of butyl acetate was obtained when free lipase was used in the synthesis containing only the substrates. In contrast, the conversion yield reached 25% when immobilized lipase was used under the same conditions. The synthesis of butyl acetate catalyzed by immobilized lipase in nonconventional media was optimized. A maximum conversion yield of 60% in a solvent-free system was obtained under the following conditions: amount of immobilized lipase, 500 IU; amount of initially added water, 45%; acetic acid/butanol molar ratio, 1:1; and in incubation temperature, 37 degrees C. Immobilized lipase could be repeatedly used for three cycles without a decrease in synthesis activity. The production of butyl acetate esters by immobilized R. oryzae lipase was also studied in the presence of organic solvents. Compared with a solvent-free system, the synthesis activity was improved in the presence of heptane and hexane with conversion yields of 80% and 76%, respectively. However, solvent-free systems tend to purify more easily the products without any toxicity and inflammability problems.

Five different ionic liquids (ILs) based on quaternary ammonium cations, with functional side chains ((3-hydroxypropyl)-trimethyl-, (3-cyanopropyl)-trimethyl-, butyl-trimethyl-, (5-cyanopentyl)-trimethyl- and hexyl-trimethyl-) associated with the same anion (bis(trifluoromethane)sulfonyl amide)), were synthesized, and their suitability for Candida antarctica lipase B (CALB)-catalyzed ester synthesis in IL/supercritical carbon dioxide (scCO(2)) biphasic systems was assayed. Catalytic efficiency of the system has been analyzed as a function of both enzyme properties and mass-transfer phenomena criteria. First, the suitability of these ILs as enzymic reaction media was tested for the kinetic resolution of rac-phenylethanol. All ILs were found to be suitable media for enzyme catalysis, the best catalytic parameter (5.3 U/mg specific activity, 94.9% selectivity) being obtained for the (5-cyanopentyl)-trimethylammonium. Second, enzyme stability in all of the ILs was studied at 50 degrees C over a period of 50 days, and data were analyzed by a two-step kinetic deactivation model. All of the ILs were shown to act as stabilizing agents with respect to hexane, producing an increase in the free energy of deactivation (to 25 kJ/mol protein) and an improvement in the half-life time of the enzyme (2000-fold), which agrees with the observed increased hydrophobicity of the cation alkyl side chain (measured by Hansen's solubility parameter, delta). By using two different CALB-IL systems with different hydrophobicity in the cation, continuous processes to synthesize six different short chain alkyl esters (butyl acetate, butyl propionate, butyl butyrate, hexyl propionate, hexyl butyrate, and octyl propionate) in scCO(2) at 10 MPa and 50 degrees C were carried out. Both rate-limiting parameters (synthetic activity and scCO(2)-ILs mass-transfer phenomena) were related with the delta-parameter of the ILs-alkyl chain and reagents.

The feasibility of continuous ester synthesis in a membrane bioreactor (MBR) by a recombinant cutinase from Fusarium solani pisi was investigated, using the optimal conditions previously attained by medium engineering. The objective was to analyze the MBR behavior as a differential or an integral reactor. The main component of the reactor was an anisotropic ceramic membrane with 15,000 NMWCO. The operating variables included the influence of substrates ratio and flow rate on the conversion degree and on the productivity. The highest conversion degree was obtained using 1M of hexanol and 0.1M of butyl acetate as acyl donor. The use of these substrate concentrations led to a conversion degree of 79.3% and a specific productivity of 41 g hexyl acetate/(d x g cutinase), when the permeate flow rate was 0.025 mL/min. The increase of flow rate to 0.4 mL/min decreased the conversion to 35.6%, although the productivity was enhanced to 294 g product/day x g enzyme. The MBR characterization involved the calculations of mass balance, recirculation rate, conversion per pass, number of cycles, and hydraulic residence time. The operational stability was also evaluated in a longterm experiment over 900 hours and the enzyme half-life was estimated to be approximately 2 years.

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.

Esters are a widespread class of organic compounds found both in industry and the environment. Because esters are often volatile and, therefore, readily inhaled, the capacity of respiratory tract tissues as well as liver S-9 homogenates from rats, rabbits, and Syrian hamsters to hydrolyze a variety of esters was investigated. A new technique to determine hydrolysis rates by measuring carboxylic acid residues using ion chromatography was proven effective. The results indicated that esters, including potentially carcinogenic beta-lactones, are readily hydrolyzed by respiratory tract enzymes. Species and tissue differences were apparent. The nasal ethmoturbinates had especially high levels of esterase activity with tissue weight-normalized activities from rabbits and hamsters for most substrates exceeding all other tissues tested, including liver. Phenyl acetate was the most rapidly hydrolyzed by ethmoturbinate tissue of the esters tested. Among straight chain aliphatic alcohol acetates, hydrolysis rates increased with carbon number up to pentyl alcohol and then decreased. Branched 4-carbon alcohol acetates were less rapidly hydrolyzed than n-butyl acetate. Correlation of hydrophobicity constants with hydrolysis rates indicated that, for the straight chain aliphatic acetates, a bilinear model best fit the data.

1-Bromopinacolone, BrPin, acts initially as a reversible competitive inhibitor for acetylcholinesterase, KI = 0.18 mM in hydrolysis of acetylcholine. Unlike bromoacetone, with time it acts as an irreversible covalent inhibitor. BrPin has a hydrolytic half-life of 30 h at the pH of incubation, 7.8. The enzyme-BrPin complex is 50% inactivated in 2 h. First order kinetics are observed; the rate constant is proportional to the concentration of complex. Retardation by cationic inhibitors of the inactivation is consistent with inactivation occurring as a result of binding of BrPin to the active site. Efficiency of irreversible inhibition by BrPin is essentially the same for hydrolysis of cationic and uncharged substrates, acetylcholine, 3,3-dimethylbutyl acetate, phenyl acetate, n-butyl acetate, and indophenyl acetate. In contrast, a cationic alkylating agent, N,N-dimethyl-2-phenylaziridinium ion, DPA, acts noncompetitively; it inactivates completely toward cationic, and partially toward uncharged substrates, and does so slightly more rapidly than BrPin, but less than would be commensurate with its greater intrinsic reactivity. Enzyme first treated with DPA is inactivated by BrPin toward hydrolysis of 3,3-dimethylbutyl acetate. It is proposed that BrPin, and not DPA, binds and reacts in, and may be a useful labeling agent for, the active site.

The Michaelis-Menten parameters kcat, Ks(app) and the second-order rate constants kII = k2/Ks of acetylcholinesterase-catalyzed hydrolysis of 25 acetic esters with non-ionic leaving groups have been determined at 25 degree C and pH 7.5 in 0.15 M KCL. A linear relationship between the substrate noncovalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group inductive effect, hydrophobicity, and steric effect, have been established. The acetyl-enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible. The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws-like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n-butyl acetate.