Selective Ethanolysis of Fish Oil Catalyzed by Immobilized Lipases

Selective ethanolysis of fish oil was catalyzed by immobilized lipases and their derivatives in organic media. Lipases from Candida antarctica B (CALB), Thermomyces lanuginosa (TLL) and Rhizomucor miehei (RML) were studied. The three lipases were immobilized by anion exchange and hydrophobic adsorption. The discrimination between the ethyl ester of eicosapentaenoic acid (EE-EPA) and the ethyl ester of docosahexaenoic acid (EE-DHA) depends on the lipase, the immobilization support, the physico-chemical modifications of the immobilized lipase derivatives and on the solvents used. TLL and RML were much more selective than CALB. EE-EPA is released 20-fold faster than EE-DHA when ethanolysis was catalyzed, in cyclohexane, by TLL hydrophobically adsorbed on Sepabeads C18. The selectivity and stability of the different derivatives in these polar organic solvents were further improved after physico-chemical modification. The best results for activity-selectivity-stability were obtained in cyclohexane for TLL adsorbed on Sepabeads C18 and further modified via solid-phase physical modification with a polyethylenimine polymer. In this case, the initial selectivity was higher than 20, and a 80 % of EPA was released as ethyl ester after 3 h at 25 °C. At this conversion, mixtures of ethyl esters highly enriched in the ethyl ester of EPA with less than 5 % of the EE-DHA were obtained. TLL derivatives remained fully active after incubation for 24 h in anhydrous solvents.     

Hydrolysis of Fish Oil by Lipases Immobilized Inside Porous Supports

A new assay was designed to measure the release of omega-3 acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] from the hydrolysis of sardine oil by lipases immobilized inside porous supports. A biphasic system was used containing the fish oil dissolved in the organic phase and the immobilized lipase suspended in the aqueous phase. The assay was optimized by using a very active derivative of Rhizomucor miehei lipase (RML) adsorbed onto octyl-Sepharose. Standard reaction conditions were: (a) an organic phase composed by 30/70 (v:v) of oil in cyclohexane, (b) an aqueous phase containing 50 mM methyl-cyclodextrin in 10 mM Tris buffer at pH 7.0. The whole reaction system was incubated at 25 °C. Under these conditions, up to 2% of the oil is partitioned into the aqueous phase and most of the 95% of released acids were partitioned into the organic phase. The organic phase was analyzed by RP-HPLC (UV detection at 215 nm) and even very low concentrations (e.g., 0.05 mM) of released omega-3 fatty acid could be detected with a precision higher than 99%. Three different lipases adsorbed on octyl-Sepharose were compared: Candida antarctica lipase-fraction B (CALB), Thermomyces lanuginosa lipase (TLL) and RML. The three enzyme derivatives were very active. However, most active and selective towards polyunsaturated fatty acids (PUFA) versus oleic plus palmitic acids (a fourfold factor) was CALB. On the other hand, the most selective derivatives towards EPA versus DHA (a 4.5-fold factor) were TLL and RML derivatives.     

Enrichment of salmon oil with n‐3 PUFA by lipolysis,filtration and enzymatic re‐esterification

Selective enzymatic hydrolysis of salmon oil extracted without solvent from by‐products was carried out under mild conditions, using a stereospecific sn‐1, sn‐3 lipase Novozyme^®. A modification of the lipid class composition was obtained by controlling the degree of hydrolysis (40%, 24 h). The mixture of acylglycerols and free fatty acids was submitted to a filtration step to retain in the retentate most of the saturated fatty acids, with melting peaks ranging from ‐31.9 °C to +14.7 °C obtained by differential scanning calorimetry. This step allowed a significant increase of polyunsaturated fatty acids (PUFA) from 39.2 mol‐% in the crude oil to 43.3% in the permeate. The remaining free fatty acids in the permeate (20.2 wt‐%) was re‐esterified with an immobilized 1, 3‐specific lipase IM60. Acylglycerols synthesis reached 90% in optimized conditions. After 48 h of reaction, the distribution of monoacylglycerols, diacylglycerols and triacylglycerols was 22.1, 28.7, 43.4 (w/w), respectively. The re‐esterification step did not modify the PUFA content obtained after membrane filtration.     

Release of Omega-3 Fatty Acids by the Hydrolysis of Fish Oil Catalyzed by Lipases Immobilized on Hydrophobic Supports

The release of omega-3 fatty acids by the mild enzymatic hydrolysis of sardine oil was studied. The derivatives of different lipases physically adsorbed on hydrophobic porous supports Hydrophobic Lipase Derivatives (HLD) were tested. These immobilized lipases can only hydrolyze oil molecules partitioned into the aqueous phase of a biphasic reaction system. HLD biocatalysts were compared to other enzyme derivatives that were obtained by very mild covalent immobilization on CNBr-activated Sepharose Cyanogen bromide Lipase Derivatives (CNLD) that behave almost identically to soluble enzymes (CNLD). In general, HLD biocatalysts were found to be more active and more selective for the release of eicosapentaenoic acid (EPA) than CNLD. The most interesting biocatalyst was the HLD derivative of Yarrowia lipolytica lipase, which was found to be sevenfold more active and tenfold more selective than CNLD. On the other hand, the most active (but non-selective) derivative was the HLD of Pseudomonas fluorescens lipase (PFL). The activity of this derivative was 0.6 International Units under non-optimal reaction conditions. High-loaded PFL derivatives could be very interesting for the release of mixtures of EPA and docosahexaenoic acid. Hydrophobic supports promote the interfacial activation of lipases, similar to the interaction promoted by oil drops on soluble enzymes. The most effective overactivation obtained in this work ranged from 6- to 20-fold. The hydrolytic process was carried out under very mild conditions (pH 7.0 and 25 °C), and all lipase derivatives remained fully active for at least 15 days under these conditions.     

Alcoholysis of waste fats with 2‐ethyl‐1‐hexanol using Candida antarctica lipase A in large‐scale tests

Commercial native lipase A from Candida antarctica was used to produce alkyl esters through the alcoholysis of (waste) fats with 2‐ethyl‐1‐hexanol. The process was carried out in batch stirred tank reactors (from 100 mL up to 3000 L). The content of alkyl esters in reaction mixtures was determined by gradient HPLC using an evaporative light scattering detector and the reaction progress was controlled by determining the ratio of the palmitic acid ester peak area to the oleic acid ester peak area in HPLC chromatograms. The results show that alcoholysis is the favoured reaction in presence of excess water and water‐insoluble alcohols in comparison with hydrolysis (fatty acid content <5%). The optimum amount of water for the alcoholysis was found to be 80–100% of the amount of fat. In the presence of low quantities of water both alcoholysis and hydrolysis are slow. Conversion rate increases with increasing temperature to 65–70 °C. Based on these results a large‐scale test to produce 3000 L of alkyl ester (to be used as lubricant coolant) was carried out. The experiments have proved that alcoholysis is completed after about 7–10 h depending on temperature.     

Enrichment of DHA from Tuna Oil in a Packed Bed Reactor via Lipase-Catalyzed Esterification

A packed-bed reactor (length 6.5 cm; id 4.65 mm) has been used to enrich docosahexaenoic acid (DHA) via the lipase-catalyzed esterification of the fatty acid from tuna oil with ethanol. Lipozyme RM IM (from Rhizomucor miehei) was used for the esterification reaction because of its ability to discriminate between different fatty acids, and several reaction parameters, including the temperature, molar ratio of substrates, and water content were explored as a function of residence time. In this way, the optimum conditions for the enrichment process were determined to be a temperature of 20 °C, a molar ratio of 1:5 (i.e., fatty acid to ethanol), and a water content of 1.0 % (based on the total substrate weight). Under these conditions, a residence time of 90 min gave a DHA concentration of 70 wt% and a DHA recovery yield of 87 wt% in the residual fatty acid fraction.     

Experimental and Computation Studies on Candida antarctica Lipase B‐Catalyzed Enantioselective Alcoholysis of 4‐Bromomethyl‐β‐lactone Leading to Enantiopure 4‐Bromo‐3‐hydroxybutanoate

Both enantiomers of optically pure 4‐bromo‐3‐hydroxybutanoate, which is an important chiral building block in the syntheses of various biologically active compounds including statins, were synthesized from rac‐4‐bromomethyl‐β‐lactone through kinetic resolution. Candida antarctica lipase B (CAL‐B) enantioselectively catalyzes the ring opening of the β‐lactone with ethanol to yield ethyl (R)‐4‐bromo‐3‐hydroxybutanoate with high enantioselectivity (E>200). The unreacted (S)‐4‐bromomethyl‐β‐lactone was converted to ethyl (S)‐4‐bromo‐3‐hydroxybutanoate (>99% ee), which can be further transformed to ethyl (R)‐4‐cyano‐3‐hydroxybutanoate, through an acid‐catalyzed ring opening in ethanol. Molecular modeling revealed that the stereocenter of the fast‐reacting enantiomer, (R)‐bromomethyl‐β‐lactone, is ∼2 Å from the reacting carbonyl carbon. In addition, the slow‐reacting enantiomer, (S)‐4‐bromomethyl‐β‐lactone, encounters steric hindrance between the bromo substituent and the side chain of the Leu278 residue, while the fast‐reacting enantiomer does not have any steric clash.     

Synthesis of Oligoester α,ω‐diols by Alcoholysis of PET through the Reactive Extrusion Process

The alcoholysis of PET with diols in the presence of dibutyltinoxide was carried out in a twin‐screw extruder with residence times of 1 min and without solvent. The reaction led to scissions of PET chains and to the synthesis of oligoester α,ω‐diols with average number molecular weights of about 1000 g·mol^—1 characterised by conventional techniques such as NMR, SEC and MALDI‐TOF. The alcoholysis kinetics was studied with a rheological tool under selected conditions, and it was shown that this reaction is quite compatible with the residence times in an extruder. This study clearly shows that the oligoesters synthesised by reactive extrusion have characteristics similar to the oligoesters synthesised by batch processes over many hours. Furthermore, the melting temperature of these oligoesters can be controlled between room temperature and 220°C by using diols with different structures for the alcoholysis.     

Mono‐Estolide Synthesis from trans‐8‐Hydroxy‐Fatty Acids by Lipases in Solvent‐Free Media and Their Physical Properties

Pseudomonas aeruginosa 42A2 is known to produce two hydroxy‐fatty acids, 10(S)‐hydroxy‐8(E)‐octadecenoic and 7,10(S,S)‐dihydroxy‐8(E)‐octadecenoic acids, when cultivated in a mineral medium using oleic acid as a single carbon source. These compounds were purified, 91 and 96 % respectively, to produce two new families of estolides: trans‐8‐estolides and saturated estolides from the monohydroxylated monomer. trans‐8‐estolides were produced by three different lipases (Novozym 435, Lipozyme RM IM and Lipozyme TL IM) with reaction yields between 68.4 ± 2.1 and 94.7 ± 2.4 % in a solvent‐free medium at 80 °C in 168 h under vacuum. Novozym 435 was found to be the most efficient biocatalyst for both hydroxy‐fatty acids with reaction yields of 71.7 ± 2.3 and 94.7 ± 2.4 %, respectively. Moreover, saturated estolides were also produced from a saturated 10(S)‐hydroxy‐8(E)‐octadecenoic. These estolides were chemically and enzymatically synthesized with Novozym 435, under the previous described reaction conditions with yields of 60.7 ± 2.1 and 71.2 ± 2.3 % respectively. Finally, viscosity, glass transition temperature, decomposition temperatures and enthalpies were determined to characterize both types of estolides. Thermal applications for both types of polyesters were improved since glass transition temperatures were lowered and decomposition temperatures were increased, with respect to their corresponding substrates.     

Modulation of the Selectivity of Immobilized Lipases by Chemical and Physical Modifications: Release of Omega-3 Fatty Acids from Fish Oil

Three different lipases (from Candida antarctica fraction B (CALB), Thermomyces lanuginose (TLL), and Rhizomucor miehei (RML)) were immobilized by two different methods, immobilization on CNBr-activated Sepharose via a mild covalent immobilization or adsorption onto hydrophobic supports (Octyl-Sepharose). These immobilized preparations were chemically and physically modified on the protein surface (enzyme carboxylic groups with ethylenediamine, amino groups with succinic anhydride, or coating with polyethyleneimine).The activity and selectivity in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by enzymatic hydrolysis of sardine oil were evaluated. Activity and selectivity were dependent on the different lipases, the immobilization protocols, the modification methods, and the pH of the reaction media. The selectivity (EPA/DHA ratio) of RML immobilized on CNBr-activated Sepharose was increased after succinylation from 7.5 to 34 at pH 6.0. The selectivity of octyl-RML improved from 1.5 to 8.5 when pH was increased from 6 to 8. The selectivity and activity of octyl-TLL increased twofold after PEI coating at pH 6. The properties of CAL-B derivatives were slightly altered after modification.     

Synthesis of 2′‐Deoxynucleosides by Transglycosylation with New Immobilized and Stabilized Uridine Phosphorylase and Purine Nucleoside Phosphorylase

Multimeric uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) of Bacillus subtilis have been expressed from genes cloned in Escherichia coli, purified, characterized, immobilized and stabilized on solid support. A new immobilization strategy has been developed for UP onto Sepabeads coated with polyethyleneamine followed by cross‐linking with aldehyde‐dextran. PNP has been immobilized onto glyoxyl‐agarose. At pH 10 and 45 °C these derivatives catalyzed the transglycosylation of 2′‐deoxyuridine to 2′‐deoxyguanosine in high yield (92%). Under the same conditions the not immobilized enzymes were promptly inactivated.     

Synthesis and Characterization of 1‐Azido‐2‐Nitro‐2‐Azapropane and 1‐Nitrotetrazolato‐2‐Nitro‐2‐Azapropane

1‐Azido‐2‐nitro‐2‐azapropane ( 1 ) was synthesized in high yield from 1‐chloro‐2‐nitro‐2‐azapropane and sodium azide. 1‐Nitrotetrazolato‐2‐nitro‐2‐azapropane ( 2 ) was synthesized in high yield from 1‐chloro‐2‐nitro‐2‐azapropane and silver nitrotetrazolate. The highly energetic new compounds ( 1 and 2 ) were characterized using vibrational (IR and Raman) and multinuclear NMR spectroscopy (¹H, ¹³C, ¹⁴N), elemental analysis and low‐temperature single crystal X‐ray diffraction. 1‐Azido‐2‐nitro‐2‐azapropane ( 1 ) represents a covalently bound liquid energetic material which contains both a nitramine unit and an azide group in the molecule. 1‐Nitrotetrazolato‐2‐nitro‐2‐azapropane ( 2 ) is a covalently bound room‐temperature stable solid which contains a nitramine group and a nitrotetrazolate ring unit in the molecule. Compounds 1 and 2 are hydrolytically stable at ambient conditions. The impact sensitivity of compound 1 is very high (<1 J) whereas compound 2 is less sensitive (<6 J).     

Stabilization of Telomeric I‐Motif Structures by (2′S)‐2′‐Deoxy‐2′‐C‐Methylcytidine Residues

G‐quadruplexes and i‐motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH‐sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C‐rich oligonucleotides carrying conformationally restricted (2′S)‐2′‐deoxy‐2′‐C‐methyl‐cytidine units. The effect of this modified nucleoside on the stability of intramolecular i‐motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C‐core with C‐modified residues induced the formation of stable intercalated tetraplexes at near‐neutral pH. This study demonstrates the possibility of enhancing the stability of the i‐motif by chemical modification.     

Long Chain Branched Polypropene Prepared by Means of Propene Copolymerization with 1,7‐Octadiene Using MAO‐Activated rac‐Me2Si(2‐Me‐4‐Phenyl‐Ind)2ZrCl2

Small amounts of 1,7‐octadiene (OD) comonomer, ranging from 0.5–5.0 mol‐%, were added during propene polymerization, catalyzed with methylalumoxane (MAO) activated rac‐Me₂Si(2‐Me‐4‐phenyl‐Ind)₂ZrCl₂ (MPI), in order to incorporate long chain branches and small amounts of high molecular mass polypropene (PP), thus improving melt processability of isotactic metallocene‐polypropene. As a function of the OD content the PP melting temperatures varied from 120 to 160°C. The presence of long chain branches was reflected by increased zero shear viscosities combined with pronounced shear thinning behavior in the case of propene/OD copolymers with molecular mass distribution of M̄_w/M̄_n < 4. Rheological measurements clearly revealed crosslinking occurring at high OD content. OD addition impaired catalyst activities. However, in the presence of trace amounts of ethene, catalyst activities increased significantly even in the presence of high OD content.     

Dual,Site‐Specific Modification of Antibodies by Using Solid‐Phase Immobilized Microbial Transglutaminase

Microbial transglutaminase (MTG) was stably solid‐phase immobilized on glass microbeads by using a second‐generation dendronized polymer. Immobilized MTG enabled the efficient generation of site‐specifically conjugated proteins, including antibody fragments, as well as whole antibodies through distinct glutamines and, unprecedentedly, also through lysines with various bifunctional substrates with defined stoichiometries. With this method, we generated dual, site‐specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling—a strategy anticipated to design antibodies for imaging and simultaneous therapy. Furthermore, we provide evidence that immobilized MTG features higher siteselectivity than soluble MTG.     

Tail‐Tied Ligands: An Immobilized Analogue of (R)‐2‐Piperidino‐1,1,2‐triphenylethanol with Intact High Catalytic Activity and Enantioselectivity

A functional analogue of (R)‐2‐piperidino‐1,1,2‐triphenylethanol was synthesized and anchored to different polymeric supports by a position remote from the active region. This strategy, leading to what we call a tail‐tied ligand, allows for the achievement of the optimal transition state geometry in the catalytic process. The catalytic activity of the resulting heterogenized ligands was investigated by online FTIR analysis. The optimum polymer was assayed in the addition reaction of diethylzinc to a large family of aldehydes rendering essentially intact high catalytic activity and enantioselectivity compared to its homogeneous counterpart.