Biodiesel Fuel Production by Enzymatic Transesterification of Oils: Recent Trends, Challenges and Future Perspectives

Liquid fuels have been used for many years as the most dominant and basic fuel for motor engines. However, declining fossil fuel resources as well as the tendency for developing new renewable biofuels have shifted the interest of the society towards finding novel alternative fuel sources. Biodiesel (monoalkyl esters of long-chain fatty acids) has a great potential as an alternative diesel fuel. From an environmental point of view it shows clear advantages over conventional fuel: it comes from renewable sources, and hence does not contribute to new carbon dioxide emission, it is biodegradable, its combustion products have reduced levels of particulates, sulphur oxides, carbon oxides, nitrogen oxides, and therefore, significantly reduces pollution (Al-Zuhair, 2007; Salis et al., 2005). One of the advantages of biodiesel in comparison to other biofuels is that biodiesel can be pumped, stored and handled using the same infrastructure employed for conventional diesel fuel (Robles-Medina et al., 2009). Also, major advantage of biodiesel as an alternative fuel is that its energy content is similar to conventional fuels, so it can be used either on its own or mixed with conventional diesel fuel, with no need of altering existing engines (Bozbas, 2005). European countries have recognized need for alternative fuels and issued the Directive on the Promotion of the use of biofuels and other renewable fuels for transport (2003/30/EC). The Directive stipulates that EU countries should replace 5.75% of fossil fuels with alternative, biofuels until 31. December 2010. This Directive has been amended by Directive 2009/28/EC which also promotes the usage of energy from renewable sources (aims at achieving a 20% share of energy from renewable sources in the EU’s final consumption of energy by 2020). In 2005, the estimated world production of biodiesel was 2.92 million tones of which 87% was obtained in EU. More importantly, between 2000 and 2005 world production increased threefold, indicating that share of biodiesel in global fuel production will significantly increase in future (Mousdale, 2008). ..

Višeslojne ugljenične nanocevi kao nosač lipaze za organsku sintezu: pregled najnovijih trendova

Lipase-catalyzed organic reactions have been widely practiced in the past three decades. Especially interesting are insoluble/immobilized forms due to providing a possibility of facile use and recyclability, thus reducing process costs, and making the procedure more environmentally friendly. Carbon-based supports have been extensively exploited for this purpose, because of neutral and biodegradable nature and thermal and chemical stability. Their high specific surface area, characteristic surface morphology and lower mass transfer resistances play a vital role in the performance of the attached enzyme. This review paper presents an overview of the main aspects of lipase immobilized on multi-walled carbon nanotubes (MWCNTs). Moreover, different immobilization strategies to achieve a biocatalyst with improved performances are discussed. Furthermore, as lipases are considered to have high commercial worth for synthesis of valuable organic molecules, the second part of the paper is dedicated to the overview of the most important industrial sectors in which these nanobiocatalysts have been used. In specific, applications in biodiesel production, flavour ester synthesis and racemization are summarized.Lipaze su poslednjih decenija široko rasprostanjeni katalizatoriu raznovrsnim organskim reakcijama. Posebno su interesantne u imobilisanom/nerastvornom obliku jer je na ovaj način olakšana njihova upotreba uz mogućnost recikliranja i ponovne upotrebe čime se smanjuju troškovi samog procesa i postupak je ekološki prihvatljiviji. Kao nosači za vezivanje nanomaterijali na bazi ugljenika, posebno ugljenične nanocevi, su našli primenu zbog svojih izuzetnih fizičkih, mehaničkih i hemijskih svojstava. Njihova velika specifična površina, karakteristična površinska morfologija i smanjen otpor prenosu mase igraju vitalnu ulogu u performansama vezanog enzima. Ovaj pregledni rad predstavlja prikaz glavnih aspekata lipaze imobilisane na višeslojne ugljenične nanocevi i različitih strategija imobilizacije za dobijanje biokatalizatora sa poboljšanim svojstvima. Takođe, kako su lipaze enzimi od velikog komercijalnog značaja za organsku sintezu i primenu u biotehnologiji, drugi deo rada posvećen je pregledu najvažnijih industrijskih sektora u kojima su ovi nanobiokatalizatori našli primenu. Shodno tome, dat je pregled proizvodnje biodizela, mirisnih estara i racemizacij

Proizvodnja lipaze iz Pseudozyma aphidis i utvrđivanje aktivnosti i stabilnosti lipaze u polarnim organskim rastvaračima

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was observed in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the value of 35.0 U cm(-3) in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude lipase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity towards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23 % residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents.Proizvodnja lipaze iz Pseudozyma aphidis utvrđena je u šest različitih medijuma. Najviša proizvodnja uočena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. Praćenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uočeno je da se najviša proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, što je četri puta veća proizvodnja od one do sada prijavljene u literaturi. Utvrđen je efekat različitih polarnih organskih rastvarača, mešljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. Hidrolitička aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaračima utvrđena je upotrebom istog spektrofotometrijskog testa. Pokazano je da lipaza ima aktivnost prema p-NPP-u samo u acetonu i acetonitrilu, dok je enzim stabilan jedino u acetonu i zadržava 23% aktivnosti nakon 24 časa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti korišćena kao biokatalizator za potencijalne primene u acetonu kao medijumu

Enzymatic synthesis of vitamin B6 precursor

3-Cyano-4-ethoxymethyl-6-methyl-2-pyridone is an important precursor in the synthesis of vitamin B6, obtained in the addition reaction between 2-cyanoacetamide and 1-ethoxy-2,4-pentanedione catalyzed by lipase from Candida rugosa (triacylglycerol ester hydrolases, EC 3.1.1.3). This work shows new experimental data and mathematical modeling of lipase catalyzed synthesis of 3-cyano-4-ethoxymethyl-6-methyl-2-pyridone, starting from 1-ethoxy-2,4-pentanedione and 2-cyanoacetamide. Kinetic measurements were done at 50 oC with enzyme concentration of 1.2 % w/v. Experimental results were fitted with two kinetic models: the ordered bi-ter and ping-pong bi-ter model, and the initial rates of the reaction were found to correlate best with a ping-pong bi-ter mechanism with inhibition by 2-cyanoacetamide. Obtained specificity constants indicated that lipase from C. rugosa had higher affinity towards 1-ethoxy-2,4-pentanedione and less bulky substrates. [Projekat Ministarstva nauke Republike Srbije, br. 172013, br. III 46010 and br. 172049

Design of a polyaniline based biosensor electrode for glucose: A comparative study of two immobilized systems

The present study compares the results of two different methods employed for preparation of polyaniline based glucose biosensor with respect to enzyme loading, biosensing efficiency and potential stability. Kinetic analysis of the potentiometric data for two enzyme immobilized electrode systems show that the GOx/PANI electrode is suitable for assaying samples with low analyte concentrations, whereas the GOx/m-ABA/PANI electrode system exhibits a better potential stability. It may therefore be possible to achieve high level of biosensing efficiency by chemical modeling and synthesis combined with careful selection of the immobilization method

Effect of different reaction parameters on lipase-catalyzed esterification of naringin and esculin

Fatty acid flavonoid esters are antioxidants with high potential for food, cosmetic and pharmaceutic industry. However, their application is still not wide-spread due to low efficiency of traditional chemical esterifications, as well as the necessity for comprehensive optimization of parameters for enzymatic synthesis to increase the economic viability of process. Hereby, esterification of two important flavonoid glycosides, naringin and esculin, with oleic acid as acyl donor using Novozym® 435 was optimized in order to broaden their commercial application. The effect of solvents, flavonoid concentrations, as well as substrate molar ratio on molar conversion and product yield, were investigated. The highest molar conversions of both flavonoids were reached in acetonitrile. Also, optimal flavonoid concentrations for synthesis of naringin and esculin esters were 30 mM and 70 mM, respectively achieving concentrations of 21.2 mg/ml naringin oleate and 27.5 mg/ml esculin oleate after 72h. Further investigation has shown that substrate molar ratio significantly influences esterification of both flavonoids and it was concluded that fivefold excess of oleic acid was optimal for both reactions. Maximum conversions of 70 and 86% were achieved with esculin and naringin, respectively, under optimized reaction conditions, making obtained results promising for further investigations, including product purification and process scale-up

enzimska lipofilizacija vitamina C linolnom kiselinom - određivanje antioksidativnih i difuzionih svojstava L-askorbil-linolata

Lipophilic derivatives of vitamin C are additives with antioxidant properties, attractive for application in food, cosmetics and pharmaceutics. They could be synthesized in lipase-catalyzed processes by using various acyl donors. Hereby, we present application of linoleic acid, which is polyunsaturated fatty acid essential in human nutrition, for esterification of vitamin C catalyzed by immobilized enzyme preparation Novozym® 435 in acetone. Highest specific ester yield, 9.7 mmol/g of immobilized lipase, was accomplished with 0.15 M of vitamin C, 0.6 M of linoleic acid, 3 g/l of enzyme and 0.07% (v/v) of water, at 60°C. NMR analyses of purified product proved that synthesized molecule was identical to 6-O-ascorbyl linoleate. Capacity of ester for scavenging 2,2-diphenyl-1-picrylhydrazyl radicals was two times higher comparing to parent molecule, vitamin C. Its diffusion coefficient, determined using Franz cell and cellulose acetate membrane, was 40% higher than palmitate and 62% higher than oleate. Obtained results showed that L-ascorbyl linoleate could be successfully synthesized in biocatalyzed processes. Furthermore, it was demonstrated that it possess high potential for application in different lipophilic products due to its liposolubility, high antioxidant efficiency and good diffusion properties.Lipofilni derivati vitamina C su aditivi sa antioksidativnim dejstvom pogodni za primenu u prehrambenim, kozmetičkim i farmaceutskim proizvodima. Mogu biti sintetisani u procesima katalizovanim lipazama korišćenjem različitih acil-donora. U ovom radu, opisana je primena linolne kiseline, polinezasićene masne kiseline esencijalne u ljudskoj ishrani, u esterifikaciji vitamina C katalizovanoj imobilisanim enzimskim preparatom Novozym® 435 u acetonu. Najviši specifični prinos estra od 9,7 mmol/g imobilisane lipaze, ostvaren je sa 0,15 M vitamina C, 0,6 M linolne kiseline, 3 g/l enzima i 0,07 zapr. % vode, na 60°C. NMR analize prečišćenog proizvoda dokazale su da je sintetisani molekul identičan 6-O-askorbil-linolatu. Kapacitet estra za vezivanje 2,2- difenil-1-pikrilhidrazil radikala bio je dva puta viši u odnosu na sam vitamin C. Njegov koeficijent difuzije, određen korišćenjem Franz-ove ćelije i celuloza-acetatne membrane, bio je za 40% viši u odnosu na palmitat i za 62% u odnosu na oleat. Ostvareni rezultati pokazali su da L-askorbil-linolat može uspešno biti sintetisan u biokatalizovanom procesu. Pored toga, dokazano je da ovaj estar poseduje značajan potencijal za primenu u različitim lipofilnim proizvodima zbog svoje liposolubilnosti, snažnog antioksidativnog dejstva i pogodnih difuzionih karakteristika

Food products modification by direct enzymatic synthesis of fructo-oligosaccharides with purpose of reduction of sucrose content and enrichment of the products with prebiotics

Predmetni pronalazak se odnosi na "in situ" enzimski postupak sinteze frukto-oligosaharida direktnom transfruktozilacijom saharoze prisutne u prehrambenim proizvodima korišćenjem enzima fruktozil-transferaze. Na taj način sharoza prisutna u proizvodu se konvertuje u prebiotike, frukto-oligosaharide, pri čemu se dobija proizvod poboljšanih funkcionalnih svojstava, sa smanjenim sadržajem saharoze, tj. manjim glikemijskim indeksom. Takođe, predmetni postupak se odnosi i na proizvode sa prebiotskom aktivnošću proizvedene prema metodama ovog pronalask

Stabilizacija lipaza iz Candida rugosa jednostavnom i efikasnom imobilizacijom na hidroksiapatitu

Razvili smo brz i efikasan metod imobilizacije industrijski veoma vrednih Candida rugosa lipaza (CRL) na ekonomičan, biokompatibilan nosač - hidroksiapatit, sa visokim prinosom imobilizacije (blizu 100 %) i prinosom aktivnosti od 50 %. Imobilizovane lipaze su pokazale značajno višu stabilnost nego slobodni enzim, nakon termalnog tretmana na 60 oC i u prisustvu različitih 95 % polarnih organskih rastvarača, pre svega kratkolančanih alifatičnih alkohola, značajnih polaznih sirovina u sintezi brojnih estara i drugih značajnih proizvoda. Predstavljeni rezultati ukazuju na veliki upotrebni potencijal dobijenog preparata u različitim industrijskim procesima, koji iziskuju rad u nekonvencionalnim reakcionim uslovima.We have developed a simple and highly effective method for immobilising industrially very appreciated and valuable Candida rugosa lipases from commercial preparation on ecologically suitable, biodegradable and economical hydroxyapatite support. Our immobilisation protocol resulted in excellent immobilisation yield of nearly 100 % and activity yield of 50 %, which is significantly higher in comparison to other immobilisation protocols for different enzymes on the same support. Immobilised lipase formulation has proven to have superior stability, compared to free enzyme, at both high temperature (60 o C) and in the presence of different polar organic solvents, especially short-chain alcohols: methanol, ethanol and iso-propanol. Therefore, presented experimental data strongly support the great future potential of the prepared Candida rugosa immobilisat