Transesterification activity of a novel lipase from Acinetobacter venetianus RAG-1

Transesterification activity and the industrial potential of a novel lipase prepared from Acinetobacter ventiatus RAG-1 were evaluated. Purified lipase samples were dialyzed against pH 9.0 buffer in a single optimization step prior to lyophilization. The enzyme and organic phase were pre-equilibrated (separately) to the same thermodynamic water activities (a _w) ranging from a _w 0.33 to 0.97. Production of 1-octyl butyrate by lipase-catalyzed transesterification of vinyl butyrate with 1-octanol in hexane was monitored by gas chromatography. Production of 1-octyl butyrate and initial rate of reaction depended on water activity. Product synthesis and rate of transesterification increased sharply with increase from a _w 0.33 to 0.55. Highest product concentration (218 mM) and rate of reaction (18.7 μmol h⁻¹ · 10 μg protein) were measured at a _w 0.86. Transesterification activity in hexane represented 32% of comparable hydrolytic activity in aqueous buffer.     

Enzymatic transesterification of Jatropha oil

Background  

Transesterification of Jatropha oil was carried out in t-butanol solvent using immobilized lipase from Enterobacter aerogenes. The presence of t-butanol significantly reduced the negative effects caused by both methanol and glycerol. The effects of various reaction parameters on transesterification of Jatropha oil were studied.     

Kinetic modelling of the production of methyl oleate by Celite® supported lipase sol–gels

This study illustrates the benefits of Celite^® supported lipase sol–gels for the transesterification of triolein to produce methyl oleate. A ping–pong bi–bi kinetic model was developed and validated taking into account the inhibition effects of methanol and glycerol as well as the effect of temperature. Although initial reaction rate models are useful for predicting the kinetics in the absence of products, a kinetic model beyond the initial conditions that considers glycerol inhibition is important. The model developed was consistent with the experimental data (R² = 0.95) predicting an increase in methyl oleate production with increasing methanol concentration up to an optimal range of 1.3 M to 2.0 M depending on the temperature. In general, increasing the temperature increased the initial reaction rate for the immobilized lipase over the temperature range of 40–60 °C. Based on the kinetic constants, the maximum velocity of the reverse reaction is about 25% slower than that of the forward reaction and glycerol inhibition has a more significant effect on the reaction kinetics than methanol inhibition. The model developed would be useful for understanding the effects of methanol and glycerol inhibition as well as temperature on the production of methyl oleate using lipase-mediated enzymatic transesterification.     

Lipases are soluble and active in glycerol carbonate as a novel biosolvent

Glycerol carbonate was synthesized as biosolvent for the development of soluble enzymatic system. The effects of various reaction parameters on activity and stability of lipases were investigated using the transesterification of ethyl butyrate with n-butanol as a model reaction. Enzymatic activity in glycerol carbonate was compared with that in water and in conventional organic solvents with different ionizing and dissociating abilities. The pK_a value of trichloroacetic acid and transesterification activities of Candida antarctica lipase B and Candida rugosa lipase in glycerol carbonate are similar to those in water, indicating that ionizing and dissociating powers are capable of satisfactorily predicting the biocompatibility of organic solvents for soluble enzymatic systems.     

Two-step synthesis of fatty acid ethyl ester from soybean oil catalyzed by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase

Background  

Enzymatic biodiesel production by transesterification in solvent media has been investigated intensively, but glycerol, as a by-product, could block the immobilized enzyme and excess n-hexane, as a solution aid, would reduce the productivity of the enzyme. Esterification, a solvent-free and no-glycerol-release system for biodiesel production, has been developed, and two-step catalysis of soybean oil, hydrolysis followed by esterification, with Yarrowia lipolytica lipase is reported in this paper.     

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica–PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 °C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1:15 at 55 °C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production.     

Enzymatic synthesis of trieicosapentaenoylglycerol in a solvent-free medium

The enzymatic synthesis of trieicosapentaenoylglycerol from glycerol and eicosapentaenoic ethyl ester in a solvent-free medium is studied here. Novozym SP 435 (immobilized lipase from Candida antarctica) has appeared as a very efficient biocatalyst for this transesterification. A nitrogen bubbling has allowed a good mixing and also the shifting of the reaction toward synthesis by eliminating the ethanol formed. The effect of temperature and of the quantity of lipase has been studied. In the optimal conditions (T=80°C, 5% (w/w) of lipase, 1 mol glycerol for 3 mol ethyl ester), pure triglyceride has been obtained after 10 h.     

Lipase-catalyzed transesterification of soybean oil for biodiesel production in <Emphasis Type="Italic">tert</Emphasis>-amyl alcohol

Lipase-catalyzed transesterification of soybean oil and methanol for biodiesel production in tert-amyl alcohol was investigated. The effects of different organic medium, molar ratio of substrate, reaction temperature, agitation speed, lipase dosage and water content on the total conversion were systematically analyzed. Under the optimal conditions identified (6 mL tert-amyl alcohol, three molar ratio of methanol to oil, 2% Novozym 435 lipase based on the soybean oil weight, temperature 40°C, 2% water content based on soybean oil weight, 150 rpm and 15 h), the highest biodiesel conversion yield of 97% was obtained. With tert-amyl alcohol as the reaction medium, the negative effects caused by excessive molar ratio of methanol to oil and the by-product glycerol could be reduced. Furthermore, there was no evident loss in the lipase activity even after being repeatedly used for more than 150 runs.     

A novel,two consecutive enzyme synthesis of feruloylated monoacyl- and diacyl-glycerols in a solvent-free system

Feruloylated mono- and di-acylglycerols were synthesized in a two step reaction: ethyl ferulate was first transesterified with glycerol and then this was esterified with oleic acid. The yield of the combined feruloylated mono- and di-acylglycerols in the second reaction reached 96% when esterification of 0.3 g transesterification products with 2.22 g oleic acid was catalyzed with 0.25 g Candida antarctica lipase at 60°C under a vaccum of 10 mmHg for 1.33 h.     

Biosynthesis of glycerol carbonate from glycerol by lipase in dimethyl carbonate as the solvent

Glycerol carbonate was synthesized from renewable glycerol and dimethyl carbonate using lipase in solvent-free reaction system in which excess dimethyl carbonate played as the reaction medium. A variety of lipases have been tested for their abilities to catalyze transesterification reaction, and Candida antartica lipase B and Novozyme 435 exhibited higher catalytic activities. The silica-coated glycerol with a 1:1 ratio was supplied to prevent two-phase formation between hydrophobic dimethyl carbonate and hydrophilic glycerol. Glycerol carbonate was successfully synthesized with more than 90% conversion from dimethyl carbonate and glycerol with a molar ratio of 10 using Novozyme 435-catalyzed transesterification at 70 °C. The Novozyme 435 [5% (w/w) and 20% (w/w)] and silica gel were more than four times recycled with good stability in a repeated batch operation for the solvent-free synthesis of glycerol carbonate.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Effect of Solvents and Kinetic Parameters on Synthesis of Ethyl Propionate Catalysed by Poly (AAc-co-HPMA-cl-MBAm)-Matrix-Immobilized Lipase of Pseudomonas aeruginosa BTS-2.

Summary A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa BTS-2 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel network. The hydrogel showed approximately 95% binding efficiency for lipase (specific activity 1.96 U mg⁻¹). The immobilized enzyme achieved 65.1% conversion of ethanol and propionic acid (100 mM each) into ethyl propionate in n-nonane at 65 °C in 9 h. When alkane of C-chain length lower than n-nonane was used as the organic solvent, the conversion of ethanol and propionic acid into ethyl propionate decreased with a decrease in the log P value of alkanes. The immobilized lipase retained approximately 30% of its original catalytic activity after five cycles of reuse for esterification of ethanol and propionic acid into ethyl propionate at temperature 65 °C in 3 h. Addition of a molecular sieve (3 ?) to the reaction mixture enhanced the formation of ethyl propionate to 89.3%. Moreover, ethanol and propionic acid when taken a molar ratio of 3:1 further promoted the conversion rate to 94%. However, an increase in the molar ratio of propionic acid with respect to ethanol resulted in a decline of ethyl propionate synthesis.     

Lipase-catalyzed regioselective synthesis of steryl (6′-<Emphasis Type="Italic">O</Emphasis>-acyl)glucosides

The regioselective acylation of cholesteryl β-d-glucoside, at the C-6 of the glucose moiety, was achieved using microbial lipases in organic solvents. With palmitic acid as an acyl donor 81 or 63% conversions of cholesteryl glucoside to its 6′-O-palmitoyl derivative were obtained using Candida antarctica or Rhizomucor miehei enzymes, respectively. High yields (64–92%) were also obtained with fatty acids 6:0–22:0 and 16:1 (n-7). The synthesis of cholesteryl (6′-O-palmitoyl)glucoside was also achieved via transesterification, using mono-, di- and tri-palmitoylglycerols or methyl and ethyl palmitate as acyl sources. With R. miehei lipase transesterification between methyl palmitate (80 mM) and cholesteryl glucoside (1 mM) proceeded after 24 h with a nearly quantitative yield (97%).     

Efficient synthesis of 6-<Emphasis Type="Italic">O</Emphasis>-palmitoyl-1,2-<Emphasis Type="Italic">O</Emphasis>-isopropylidene-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucofuranose in an organic solvent system by lipase-catalyzed esterification

In order to synthesize a sugar ester at high concentration, 1,2-O-isopropylidene-α-d-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value (240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1,200 mmol palmitic acid/kg solvent were used in this solvent mixture.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

Development of an amphiphilic matrix for immobilization of <Emphasis Type="Italic">Candida antartica</Emphasis> lipase B for biodiesel production

Biodiesel has been greatly interested as an alternative fuel and is produced by a transesterification reaction of oil with alcohol. Recently, microbial lipases have been used for biodiesel production. Among the microbial lipase, immobilized Candida antartica lipase B (CALB) is the most widely used. However, CALB is unstable and shows low catalytic efficiency in the reaction media because the reaction media contains a high concentration of methanol and the lipase is also inhibited by the by-product glycerol. In this study, to overcome these limitations, we developed an amphiphilic matrix to immobilize CALB. The immobilized lipase in an amphiphilic matrix with 80% ethyltrimethoxysilane (ETMS) in tetramethoxysilane (TMOS) and pretreated with oil showed the highest specific activity and biodiesel conversion ratio; about 90% biodiesel conversion in 24 h at an initial molar ratio of 1: 1 (oil: methanol) with stepwise methanol feeding in order to adjust the net molar ratio to be 1: 3.     

Solvent-free synthesis of glyceryl ferulate using a commercial microbial lipase

A process was optimized for the enzymatic synthesis of glyceryl ferulate with a yield of up to 96% using a vacuum-rotary evaporation strategy under following conditions: 15 mmol glycerol, 1.5 mmol ethyl ferulate, 170 mg Candida antarctica lipase, at 60°C for 10 h and under a vacuum of 10 mm Hg. The immobilized lipase can be used 10 times.     

Enantioselective behavior of lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4°C and could be reused at least six times.     

Highly regioselective enzymatic synthesis of 5′-O-stearate of 1-β-D-arabinofuranosylcytosine in binary organic solvent mixtures

In this paper, highly regioselective enzymatic acylations of 1-β-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5′-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS–ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50°C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol·L⁻¹h⁻¹, 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13(¹³C) NMR and confirmed to be 5′-O-stearate of ara-C.