Coupling of Metabolism and Bioconversion: Microbial Esterification of Citronellol with Acetyl Coenzyme A Produced via Metabolism of Glucose in an Interface Bioreactor

Microbial esterification of citronellol with acetyl coenzyme A (acetyl-CoA) produced via metabolism of glucose was performed with Hansenula saturnus IFO 0809 in an interface bioreactor by using a hydrophilic carrier (an agar plate or an agar-coated filter pad) and a hydrophobic organic solvent (decane). An increase in the glucose concentration on an agar plate led to a decrease in oxidation of citronellol and an increase in the amount of citronellyl acetate. Fed-batch addition of citronellol efficiently alleviated substrate toxicity and resulted in the accumulation of high levels of citronellyl acetate. Furthermore, triple coupling of acetyl-CoA formation, microbial reduction of citronellal to citronellol, and esterification of the resulting citronellol with acetyl-CoA was also an efficient way to prepare citronellyl acetate. By using coupling systems, citronellyl acetate could be efficiently produced without any acetyl donor.     

Optimization of lipase-catalyzed synthesis of citronellyl acetate in solvent-free medium

Summary Citronellol esterification catalyzed byCandida antarctica lipase is performed with high yields (74%) in a solvent-free medium. The use of dessicants gives a 10% yield enhancement and substrate excess consumption by adding calculated amounts of acetic acid lead to a 80% pure citronellyl acetate. Quantities up to 100 ml are treated with success.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Green synthesis of isoamyl acetate via silica immobilized novel thermophilic lipase from <Emphasis Type="Italic">Bacillus aerius</Emphasis>

Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the presence of a cross-linking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silica-bound lipase was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, substrate molar ratio 1: 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction. The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68% under the optimized reaction conditions. The product was identified as isoamyl acetate using gas-liquid chromatography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of an ester group at the wavenumber of 1720.5 cm^–1.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Effect of reaction parameters on synthesis of citronellyl methacrylate by lipase-catalyzed transesterification

The methacrylate ester of citronellol was synthesized using various lipases as catalyst. The effect of different reaction parameters such as amount of lipase, solvent, temperature, and acylating agent on the conversion of citronellol to citronellyl methacrylate was studied. Methyl methacrylate, vinyl methacrylate, and 2,3-butanedione mono-oxime methacrylate were used as acylating agents. Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Pseudomonas cepacia lipase (Amano-PS) were used as biocatalysts. Diisopropyl ether (DIPE) was found to be the most suitable solvent. The stereoselectivity of CRL in transesterification of (+/-)-citronellol was tested for the optimized reaction parameters.     

Studies of reaction parameters on synthesis of Citronellyl laurate ester via immobilized Candida rugosa lipase in organic media

Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

Gas phase ethyl acetate production in a batch bioreactor

Gas phase ethyl acetate production was studied using a porcine pancreatic lipase powder. It was observed that gaseous ethyl acetate was produced from gaseous ethanol and acetic acid. Accordingly, the effects of amount of lipase powder, gaseous ethanol and acetic acid concentrations, and reaction temperature on the performance of a batch bioreactor were investigated. Apparent Michaelis-Menten constant of ethanol was 0.163 [μM] and there was no inhibition by ethanol over the range investigated. As acetic acid concentration increased, ethyl acetate production increased to a maximum, then decreased, thus suggesting the inhibition effects by acetic acid. Over the reaction temperature of 25–55?°C, activation energy was calculated as 3.93 kcal/gmol and initial reaction rate was obtained as follows: r?=?75.7 exp(?1975.7/T) [μM/mg of lipase/hr]     

Lipase-catalyzed synthesis of geranyl acetate in n-hexane with membrane-mediated water removal.

The esterification of geraniol with acetic acid in n-hexane was investigated. A commercial lipase preparation from Candida antarctica was used as catalyst. The equilibrium conversion (no water removal) was found to be 94% for the reaction of 0.1 M alcohol and 0.1 M acid in n-hexane at 30 degrees C. This was shown by both hydrolysis and esterification reactions. The activation energy of reaction over the temperature range 10 degrees to 50 degrees C was found to be 16 kJ/mol. The standard heat of reaction was -28 kJ/mol. Membrane pervaporation using a cellulose acetate/ceramic composite membrane was then employed for selective removal of water from the reaction mixture. The membrane was highly effective at removing water while retaining all reaction components. Negligible transport of the solvent n-hexane was observed. Water removal by pervaporation increased the reaction rate by approximately 150% and increased steady-state conversion to 100%.     

Response surface methodological approach for Rhizomucor miehei lipase-mediated esterification of α-terpineol with propionic acid and acetic anhydride

Esterification of α-terpineol with acetic anhydride or propionic acid mediated by Rhizomucor miehei lipase was subjected to a response surface study in order to optimize conditions for maximum esterification. The variables were enzyme/substrate (acid) ratio, α-terpineol concentration and incubation period using lipase from R. miehei. Between acetic anhydride and propionic acid, the former showed better yields at lower enzyme/substrate ratios than the latter. Yields predicted by the models for α-terpinyl propionate and α-terpinyl acetate formation were found to be in agreement with the experimentally determined ones. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis of 4-nitrophenyl acetate using molecular sieve-immobilized lipase from <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

Extracellular lipase from Bacillus coagulans BTS-3 was immobilized on (3 Å × 1.5 mm) molecular sieve. The molecular sieve showed approximately 68.48% binding efficiency for lipase (specific activity 55 IU mg^?1). The immobilized enzyme achieved approx 90% conversion of acetic acid and 4-nitrophenol (100 mM each) into 4-nitrophenyl acetate in n-heptane at 65°C in 3 h. When alkane of C-chain length other than n-heptane was used as the organic solvent, the conversion of 4-nitrophenol and acetic acid was found to decrease. About 88.6% conversion of the reactants into ester was achieved when reactants were used at molar ratio of 1:1. The immobilized lipase brought about conversion of approximately 58% for esterification of 4-nitrophenol and acetic acid into 4-nitrophenyl acetate at a temperature of 65°C after reuse for 5 cycles.     

Anticonflict effects of rose oil and identification of its active constituents

The present study investigates the pharmacologically active constituents of rose oil, which possesses anti-conflict effects. Analysis using GC/MS revealed that rose oil contains 9 substances that were identified as myrcene, benzyl alcohol, 2-phenethyl alcohol, citronellol, geraniol, citronellyl acetate, eugenol, geranyl acetate and methyl eugenol. We examined the effects of each of these substances using the Geller and Vogel conflict tests in ICR mice. Myrcene, benzyl alcohol and citronellyl acetate did not produce any effects in either tests. Geranyl acetate and methyl eugenol produced no effect in the Geller conflict test. Geraniol and eugenol decreased the response rate during the safe period of the Geller conflict test, but did not affect the response rate during the alarm period. In contrast, 2-phenethyl alcohol and citronellol, like rose oil, produced an increasing effect on the response rate during the alarm period in the Geller conflict test. In addition, both chemicals increased the number of electric shocks mice received in the Vogel conflict test in a manner similar to that of rose oil. Given that 2-phenethyl alcohol and citronellol produced the same anti-conflict effects in both tests as rose oil, we concluded that they are the pharmacologically active constituents of anti-anxiety-like effect of rose oil.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

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.     

Chemical composition of volatiles from cortical oleoresin of Pseudotsuga menziesii

Pseudotsuga menziesii cortical oleoresin was found to contain 1·7% of oxygenated terpenoids and compounds of similar volatility composed of linalool, methylsalicylate, bornyl acetate, citronellol, geranyl acetate, methylthymol, citronellyl acetate, terpinen-4-ol, borneol, isopulegol, anethole, terpinen-4-ol acetate, camphor, geraniol, neryl acetate, and nerol. Sesquiterpenoid hydrocarbons were low (only 0·07%) and contained sibirene and longifolene as main constituents, with β-caryophyllene, γ-muurolene, γ-cadinene (identified by IR), and 20 additional compounds in small amounts. p-Cymen-8-ene was identified in monoterpene hydrocarbon fraction.     

A kinetic study of isoamyl acetate synthesis by immobilized lipase-catalyzed acetylation in n-hexane

The objective of this work was to propose a reaction mechanism and to develop a rate equation for the synthesis of isoamyl acetate by acylation of the corresponding alcohol with acetic anhydride using the lipase Novozym 435 in n-hexane. The reaction between isoamyl alcohol and acetic anhydride occurred at high rate in first place. Then, if excess alcohol was used, produced acetic acid further reacted with remaining alcohol, leading to yields higher than 100% (based on initial acetic anhydride content). This reaction was much slower and took place only when acetic anhydride had been totally consumed. Optimal pH for Novozym 435 was 7.7. Acetic acid strongly inactivated the enzyme but it was partially caused by the pH drop in the biocatalyst aqueous microenvironment. Acetic anhydride also showed an important inhibition effect. On the contrary, isoamyl alcohol and isoamyl acetate had no negative effect on the lipase. The analysis of the initial rate data showed that reaction followed a Ping-Pong Bi-Bi mechanism with inhibition by acetic anhydride. The kinetic constants were obtained by multiple regression analysis of experimental findings. Equation predictions and experimental reaction rate values matched very well at conditions where acetic acid concentration in the medium was low.     

Esterification In Organic Solvents: Selection Of Hydrolases And Effects Of Reaction Conditions

Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.     

Enzymatic synthesis of geraniol and citronellol esters by direct esterification in n-hexane

Summary Short chain fatty acid esters of geraniol and citronellol were synthesized by lipase-catalyzed direct esterification with yields ranging from 90 to 100% molar conversion. The effect of the order of addition of substrates, solvent and enzyme was investigated. Yields were highest when the solvent was added first followed by the substrates and then the lipase.