Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

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.     

Synthesis of N-Octyl Oleate with Lipase from Mucor miehei Immobilized onto Polyethylene Based Graft Copolymers

Lpase from Mucor miehei was immobilized onto partially hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE/HEMA) via spacer arms of 1,6-diaminohexane and glutaraldehyde-. The PE/HEMA-lipase system was used for the enzymatic esterification of n-octanol with oleic acid in the absence of organic solvents. The influence of lipase' concentration, in the attachment solution, on the ester production profile and initial reaction rate was studied. It was found that very small amounts of lipase gave preparations which reached good degrees of conversion. The effect of the initial oleic acid concentration on that pseudo-first order reaction, as well as the presence of water in the reactional medium and the influence of temperature were evaluated. It was found that initial oleic acid concentrations lesser than 1.2 M did not inhibit the immobilized lipase activity; the presence of small amount of water (10–30μ) solubilized in the reaction mixture (6.5 cm³) increased the lipase activity and a maximum of activity of the immobilized lipase preparation was found at 55d`C. The operational stability of the preparation was determined at 37d`C in a BSTR type reactor and a half-life time of three days for the immobilized lipase was obtained.     

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.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Stereoselective esterification of dl-menthol by polyurethane-entrapped lipase in organic solvent

Stereospecific esterification of dl-menthol was studied by the use of immobilized lipase in an adequate water-saturated organic solvent system. Lipase from Candida cylindracea immobilized by entrapment with urethane prepolymers and 5-phenylvaleric acid as the acyl donor were chosen based on the stereoselectivity and the yield of l-menthyl ester. Water-saturated cyclohexane or isooctane was found to be the most suitable solvent system. Entrapment significantly enhanced the operational stability of lipase.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Enzymatic propyl gallate synthesis in solvent-free system: Optimization by response surface methodology

The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterification of propanol with gallic acid was investigated and the antioxidant as well as the antimicrobial activities of the ester formed were evaluated. The response surface methodology, based on a three variables Box–Behnken design (reaction temperature, enzyme amount and 1-propanol/gallic acid molar ratio), was used to optimize the experimental conditions of propylgallate synthesis. The maximum conversion yield (90% ±3.5) was obtained by using 400 IU of immobilized lipase and a propanol/gallic acid at a molar ratio of 160 at 52 °C. The obtained ester was characterized by spectroscopic methods, NMR and FTIR. The antioxidant activity of propyl gallate was evaluated and compared to the synthetic classical antioxidants, BHA and ascorbic acid, taken as references. In addition, the antimicrobial activity of the propyl gallate was tested against S. xylosus, Escherchia coli and Staphylococcus aureus using disc diffusion and macrodilution methods. Our results show that the synthesized propyl gallate ester presents a higher antioxidant and antimicrobial power than the parent gallic acid as well as the synthetic classical antioxidants.     

Biocatalytic acylation of carbohydrates with fatty acids from palm fatty acid distillates

Palm fatty acid distillates (PFAD) are by-products of the palm oil refining process. Their use as the source of fatty acids, mainly palmitate, for the biocatalytic synthesis of carbohydrate fatty acid esters was investigated. Esters could be prepared in high yields from unmodified acyl donors and non-activated free fatty acids obtained from PFAD with an immobilized Candida antarctica lipase preparation. Acetone was found as a compatible non-toxic solvent, which gave the highest conversion yields in a heterogeneous reaction system without the complete solubilization of the sugars. Glucose, fructose, and other acyl acceptors could be employed for an ester synthesis with PFAD. The synthesis of glucose palmitate was optimized with regard to the water activity of the reaction mixture, the reaction temperature, and the enzyme concentration. The ester was obtained with 76% yield from glucose and PFAD after reaction for 74 h with 150 U ml⁻¹ immobilized lipase at 40°C in acetone.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68–93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50–55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4 days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).     

Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

In Situ Enzymatic Conversion of <Emphasis Type="Italic">Nannochloropsis oceanica</Emphasis> IMET1 Biomass into Fatty Acid Methyl Esters

Conventionally, production of methyl ester fuels from microalgae occurs through an energy-intensive two-step chemical extraction and transesterification process. To improve the energy efficiency, we performed in situ enzymatic conversion of whole algae biomass from an oleaginous heterokont microalga Nannochloropsis oceanica IMET1 with the immobilized lipase from Candida antarctica. The fatty acid methyl ester yield reached 107.7% for dry Nannochloropsis biomass at biomass to t-butanol to methanol weight ratio of 1:2:0.5 and a reaction time of 12 h at 25 °C, representing the first report of efficient whole algae biomass conversion into fatty acid methyl esters at room temperature. Different forms of algal biomass including wet Nannochloropsis biomass were tested. The maximum yield of wet biomass was 81.5%. Enzyme activity remained higher than 95% after 55 days of treatment (equal to 110 cycles of reaction) under the conditions optimized for dry algae biomass conversion. The low reaction temperature, high enzyme stability, and high yield from this study indicate in situ enzymatic conversion of dry algae biomass may potentially be used as an energy-efficient method for algal methyl ester fuel production while allowing co-product recovery.     

Efficient production of diltiazem chiral intermediate using immobilized lipase from <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The lipase from Serratia marcescence ECU1010 (Sml) was capable of enantioselectively catalyzing the synthesis of many chiral drug precursors. This paper investigated the immobilization of Sml on appropriate supporting materials and its performance in bioreactor. Chitosan, Celite 545, and DEAE-cellulose were found to be the ideal supports among 8 carriers tested with respect to enzyme load and activity recovery of lipase. When Sml was immobilized, significant improvements of stability against pH, thermal, and operational deactivation were observed with all the 3 better supports, and the best stability was observed when the lipase was immobilized on glutaraldehyde activated chitosan. As for the effect of organic solvent in the biphasic reaction system, the hydrolytic activity of the immobilized lipase on trans-3-(4′-methoxyphenyl)glycidic acid methyl ester ((±)-MPGM) observed in isopropyl ether was 6 and 3 times higher than those in toluene and methyl tert-butyl ether. The lipasecatalyzed production of (−)-MPGM by enzymatic resolution of (±)-MPGM with chitosan-Sml in isopropyl etherwater biphasic system was carried out in a 2 L stirred-tank reactor. The batch operation was more efficient operation mode for the enantioselective hydrolysis of (±)-MPGM, affording enantiopure (−)-MPGM in 44.3% overall yield, in contrast to 29.3% in a continuous reactor.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Improvement of catalytic activity of Candida rugosa lipase in the presence of calix[4]arene bearing iminodicarboxylic/phosphonic acid complexes modified iron oxide nanoparticles

In the present study, iron oxide magnetite nanoparticles, prepared through a co-precipitation method, were coated with phosphonic acid or iminodicarboxylic acid derivatives of calix[4]arene to modulate their surfaces with different acidic groups. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through sol–gel encapsulation. The catalytic activities and enantioselectivities of the two encapsulated lipases in the hydrolysis reaction of (R/S)-naproxen methyl ester and (R/S)-2-phenoxypropionic acid methyl ester were assessed. The results showed that the activity and enantioselectivity of the lipase were improved when the lipase was encapsulated in the presence of calixarene-based additives; the encapsulated lipase with the phosphonic acid derivative of calix[4]arene had an excellent rate of enantioselectivity against the (R/S)-naproxen methyl and (R/S)-2-phenoxypropionic acid methyl esters, with E = 350 and 246, respectively, compared to the free enzyme. The encapsulated lipases (Fe-Calix-N(COOH)) and (Fe-Calix–P) showed good loading ability and little loss of enzyme activity, and the stability of the catalyst was very good; they only lost 6–11% of the enzyme’s activity after five batches.     

Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst

The novel whole-cell biocatalyst Candida antarctica lipase B displaying-Pichia pastoris (Pp-CALB) is characterized by its low preparation cost and could be an alternative to the commercial immobilized Candida antarctica lipase B (CALB). This study addresses the feasibility of using Pp-CALB in large scale glucose fatty acid esters production. 1,2-O-Isopropylidene-α-d-glucofuranose (IpGlc) was used as the acyl acceptor to overcome the low solubility of glucose in an organic solvent and to avoid the addition of toxic co-solvents. IpGlc significantly improved the Pp-CALB catalyzing esterification efficiency when using long chain fatty acids as the acyl donor. Under the preferred operating conditions (50 °C, 40 g/L molecular sieve dosage and 200 rpm mixing intensity), 60.5% of IpGlc converted to 6-O-myristate-1, 2-O-isopropylidene-α-d-glucofuranose (C14-IpGlc) after a 96-h reaction in a 2-L stirred reactor. In a 5-L pilot scale test, Pp-CALB also showed a similar substrate conversion rate of 55.4% and excellent operational stability. After C14-IpGlc was collected, 70% trifluoroacetic acid was adopted to hydrolyze C14-IpGlc to myristate glucose ester (C14-Glc) with a high yield of 95.3%. In conclusion, Pp-CALB is a powerful biocatalyst available for industrial synthesis, and this study describes an applicable and economical process for the large scale production of myristate glucose ester.     

Synthesis of acetylenic sugars and uronic acids via two-carbon chain extension of d-ribose

Treatment of methyl β-d-ribofuranoside with acetone gave methyl 2,3-O-isopropylidene-β-d-ribofuranoside (1, 90%), whereas methyl α-d-ribofuranoside gave a mixture (30%) of 1 and methyl 2,3-O-isopropylidene-α-d-ribofuranoside (1a). On oxidation, 1 gave methyl 2,3-O-isopropylidene-β-d-ribo-pentodialdo-1,4-furanoside (2), whereas no similar product was obtained on oxidation of 1a. Ethynylmagnesium bromide reacted with 2 in dry tetrahydrofuran to give a 1:1 mixture (95%) of methyl 6,7-dideoxy-2,3-O-isopropylidene-β-d-allo- (3) and -α-l-talo-hept-6-ynofuranoside (4). Ozonolysis of 3 and 4 in dichloromethane gave the corresponding d-allo- and l-talo-uronic acids, characterized as their methyl esters (5 and 6) and 5-O-formyl methyl esters (5a and 6a). Ozonolysis in methanol gave a mixture of the free uronic acid and the methyl ester, and only a small proportion of the 5-O-formyl methyl ester. Malonic acid reacted with 2 to give methyl 5,6-dideoxy-2,3-O-isopropylidene-β-d-ribo-trans-hept-5-enofuranosiduronic acid (7).