Immobilized lipases as practical catalysts

Attractive features of lipase systems include versatility, substrate selectivity, regioselectivity, enantioselectivity and catalysis at ambient temperatures and pressures. To fully exploit the technical and economical advantages of lipases, it is recommended to use them in an immobilized form to reduce the cost and the poor stability of the free lipase. This paper summarizes various methods of lipases immobilization including covalent attachment to or adsorption on solid supports, encapsulation and entrapment within the membrane and in polymeric matrices. The effects of immobilization conditions on lipase properties and stability of biocatalysts are considered. Applications of immobilized lipases in the feasible reaction system as well as probable future trends in lipase catalyzed process are discussed.Lipaze su enzimi od velikog potencijalnog praktičnog značaja zbog osobine da selektivno katalizuju veliki broj reakcija pod blagim uslovima i bez primene kofaktora. Savremeni pristup primeni ovih enzima u industrijskim uslovima predstavqa primena imobilisanih enzima, čime se povećava njihova stabilnost i omogućuje primena u kontinualnim sistemima. U radu je dat pregled različitih metoda i nosača za imobilizaciju lipaza uključujući kovalentnu imobilizaciju adsorpciju, mikroinkapsulaciju i zarobljavanje enzima unutar polimerne matrice i u pore membrane, pri čemu je ukazano na prednosti i nedostatke svakog od imobilisanih sistema. Diskutovani su uticaji uslova imobilizacije i prirode nosača na osobine i stabilnost biokatalizatora. Takođe su razmatrane mogućnosti primene imobilisanih lipaza u reakcijama od praktičnog značaja kao i razvoj novih imobilisanih sistema

Utilization of baker's yeast (Saccharomyces cerevisiae) for the production of yeast extract: effects of different enzymatic treatments on solid, protein and carbohydrate recovery

Yeast extract (YE) was produced from commercial pressed baker's yeast (active and inactivated) using two enzymes: papain and lyticase. The effects of enzyme concentration and hydrolysis time on the recovery of solid, protein and carbohydrate were investigated. Autolysis, as a basic method for cell lysis was also used and the results compared. The optimal extraction conditions were investigated. The optimal concentrations of papain and lyticase were found to be 2.5 % and 0.025 %, respectively

An investigation of influence of solvent on the degradation kinetics of carotenoids in oil extracts of Calendula officinalis

The stability of carotenoids was studied in marigold oil extracts prepared with following solvents: Myritol 312®, paraffin oil, almond oil, olive oil, sunflower oil, grape seed oil, and soybean oil. The concentration of the carotenoids was determined by spectroscopic measurement at 450 nm. Degradation rate showed a first order dependence on the concentration of carotenoids with a faster first stage (which lasted 3550 days, depending on the solvent) and a slower second stage. The highest degradation rates were observed in extracts prepared with linoleic acid rich solvents (sunflower oil, soybean oil and grape seed oil), while the lowest were found in oil with saturated fatty acids (Myritol 312®) and paraffin oil. These results confirm the connection between the degradation of carotenoids and lipid autoxidation, and suggest that the influence of the oil solvents on the stability of oil extracts of Calendula officinalis is a factor that must be considered when selecting a solvent for the production of marigold oil extracts

Studies on the specificity of Candida rugosa lipase catalyzed esterification reactions in organic media

In this study, the feasibility of the synthesis of various flavor esters catalyzed by a commercial lipase from Candida rugosa was investigated and the process parameters were optimized. Lipase from C. rugosa successfully catalyzed the synthesis of 19 esters. The highest yields, of more than 90 % after 20 h, were observed in the synthesis of short-chain esters, pentyl propanoate, isopentyl butanoate, and butyl butanoate. Increasing the number of carbon atoms of both substrates above 8 caused a significant decrease of the initial reaction rates and the final yields. The enzyme showed surprisingly low affinity towards pentanoic acid and hexanoic acid, compared with the higher homologues, octanoic acid and decanoic acid. In addition to the number of carbon atoms, the structure of the substrates had a significant influence on the enzyme activity. Namely, the activity of the enzyme towards isopropanol was significantly lower compared with n-propanol. Additionally, cis-9-octadecenoic acid was a better substrate than octadecanoic acid, its saturated analogue

Efficiencies of different methods for determination of organophosphate pesticide residues in fermented wheat substrate

In the present work, the efficiencies of three different sample preparation methods for GC/MS determination of pirimiphos-methyl and chlorpyrifos-methyl residues in wheat (Triticumspelta) samples fermented by Lactobacillus plantarum were compared. All three methods werebased on methanol:acetone=1:1 extraction, while further purification of the obtained sampleswas altered. First purification was through a column containing a mixture of aluminiumoxide and activated charcoal slurry-packed and eluted with dichlormethane, the second waspurification on a florisil column slurry-packed and eluted with ethyl acetate:acetone=4:1, whilethe third was based on a combination of the former two methods, i.e. clean-up through columnsfilled with a mixture of aluminum oxide and activated charcoal slurry-packed and elutedwith ethyl acetate:acetone=4:1. The second method was found the most effective for obtainingsatisfactory recoveries at four fortification levels. For pirimiphos-methyl, recoveries werein the range of 91.3-96.0% and had good reproducibility, i.e. RSD ranging from 2.2-4.1%, whilethe corresponding range for chlorpyrifos-methyl was 81.6-88.2%, and the RSD range 2.5-5.4%.The chosen method was further optimized in order to establish the optimum volume of elutionsolvent used during the clean-up procedures. The highest recoveries of 93.7±3.5% for pirimiphos-methyl and 85.3±2.5% for chlorpyrifos-methyl were obtained after elution with 25 mlvolume of solvent. Considering all, simple, efficient and reliable GC/MS detection of pirimiphosmethyland chlorpyrifos-methyl residues in wheat grain substrate altered by fermentation withL. plantarum was achieved by the two-steps extraction with 25 ml of methanol:acetone=1:1solvent mix for 30 min, followed by a clean-up procedure through a glass column with florisilcoupled with elution by 25 ml of ethyl acetate:acetone=4:1

Efikasnost različitih metoda za određivanje ostataka organofosfata u fermentisanim uzorcima žita

In the present work, the efficiencies of three different sample preparation methods for GC/ MS determination of pirimiphos-methyl and chlorpyrifos-methyl residues in wheat (Triticum spelta) samples fermented by Lactobacillus plantarum were compared. All three methods were based on methanol:acetone=1:1 extraction, while further purification of the obtained samples was altered. First purification was through a column containing a mixture of aluminium oxide and activated charcoal slurry-packed and eluted with dichlormethane, the second was purification on a florisil column slurry-packed and eluted with ethyl acetate:acetone=4:1, while the third was based on a combination of the former two methods, i.e. clean-up through columns filled with a mixture of aluminum oxide and activated charcoal slurry-packed and eluted with ethyl acetate:acetone=4:1. The second method was found the most effective for obtaining satisfactory recoveries at four fortification levels. For pirimiphos-methyl, recoveries were in the range of 91.3-96.0% and had good reproducibility, i.e. RSD ranging from 2.2-4.1%, while the corresponding range for chlorpyrifos-methyl was 81.6-88.2%, and the RSD range 2.5-5.4%. The chosen method was further optimized in order to establish the optimum volume of elution solvent used during the clean-up procedures. The highest recoveries of 93.7±3.5% for pirimiphos- methyl and 85.3±2.5% for chlorpyrifos-methyl were obtained after elution with 25 ml volume of solvent. Considering all, simple, efficient and reliable GC/MS detection of pirimiphosmethyl and chlorpyrifos-methyl residues in wheat grain substrate altered by fermentation with L. plantarum was achieved by the two-steps extraction with 25 ml of methanol:acetone=1:1 solvent mix for 30 min, followed by a clean-up procedure through a glass column with florisil coupled with elution by 25 ml of ethyl acetate:acetone=4:1.Poređene su efikasnosti tri različite metode pripreme uzoraka za određivanje ostataka pirimifos-metila i hlorpirifos-metila u uzorcima pšenice (Triticum spelta) fermentisane sa Lactobacillus plantarum. Sve tri metode zasnivale su se na metanolsko-acetonskoj (1:1) ekstrakciji dok su se procedure prečišćavanja dobijenih ekstrakata razlikovale. Prva metoda bila je prečišćavanje kroz kolonu punjenu smešom aluminijum-oksida i aktivnog uglja u kombinaciji sa dihlormetanom kao eluentom, druga prečišćavanje kroz kolonu punjenu florisilom u kombinaciji sa smešom etil-acetata i acetona (4:1) kao eluentom, dok se treća zasnivala na kombinaciji prve dve, tj. prečišćavanju kroz kolonu punjenu smešom aluminijum-oksida i aktivnog uglja u kombinaciji sa smešom etil-acetata i acetona (4:1) kao eluentom. Druga testirana metoda pokazala se kao najefikasnija, pri čemu su dobijeni prinosi pirimifos-metila u opsegu 91,3-96,0% za četiri koncentraciona nivoa obogaćivanja, sa RSD% u opsegu 2,2-4,1%, dok su za hlorpirifos-metil ovi prinosi bili u opsegu 81,6-88,2% sa RSD% u opsegu 2,5-5,4%. Odabrana metoda je dodatno optimizovana variranjem različitih zapremina korišćenog eluenta. Najveći prinos metode za oba ispitivana pesticida postignut je pri zapremini eluenta od 25 ml (93,7±3.5% za pirimifos-metil i 85,3±2,5% za hlorpirifos-metil). Predložena metoda, bazirana na 30-minutnoj ekstrakciji sa 25 ml smeše metanol:aceton=1:1, prečišćavanju na florisilskoj koloni i eluiranju sa 25 ml smeše etilacetat: aceton=4:1, pokazala se kao efikasna, jednostavna i pouzdana metoda za određivanje ostataka ispitivanih organofosfata u fermentisanom žitnom supstratu

Effect of fermentation conditions on lipase production by Candida utilis

A wild yeast strain isolated from spoiled soybean oil and identified as Candida utilis initially presented rather low lipase activity (approximately 4 IU dm-3) in submerged culture in a universal yeast medium containing 2 % malt extract. Stu­dies were undertaken to improve the lipase production. The best yields of lipase were obtained with a medium supplemented with caprylic and oleic acids as indu­cers, but higher concentrations of the former (> 0.5 %) had a negative effect on the lipase production and cell growth. The type of nitrogen source seemed also to be very important. The highest lipolytic activity of 284 IU dm-3 was achieved after 5 days of fermentation in a medium containing oleic acid and hydrolyzed casein as carbon and nitrogen sources, respectively, and supplemented with Tween 80®. It was shown that optimization of the fermentation conditions can lead to a significant improvement in the lipase production (more than 70-fold higher compared to the initial value obtained in the non-optimized medium)