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Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite (R) A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 +/- 1.41 U g(-1) and 85.71 +/- 1.62 U g(-1) after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (similar to 90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.

Lipases can be used for a variety of biotechnological applications: synthesis of fine chemicals, therapeutics, agrochemicals, cosmetics, flavors, biopolymers and biodiesel. Biodiesel is an alternative fuel for diesel engines that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short chain alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The usage of lipases has several advantages over the conventional chemical methods. It is considered as less energy intensive and environmentally friendly. However, there are two main obstacles associated with the effective utilization of lipases in the production of biodiesel. The main one is the cost of the enzyme and its poor stability in the presence of excess alcohol. Several strategies are proposed to overcome these drawbacks: immobilization of lipases, stepwise addition of alcohol, and the usage of novel acyl acceptors and the usage of whole cell biocatalysts., Lipaze predstavljaju jednu od najvažnijih grupa biokatalizatora koji imaju veliku primenu u biotehnološkim procesima. Zahvaljujući činjenici da mogu katalizovati i hidrolizu i esterifikovanje, često se koriste u reakcijama transesterifikacije. Zbog niza prednosti koje imaju nad klasičnim katalizatorima lipaze postaju sve pogodnije za upotrebu u sintezi biodizela. U ovom radu prikazane su karakteristike lipaza koje se koriste u biotehnologiji, kao i njihova primena u sintezi biodizela.

Monoacylglycerols are non-ionic surfactants widely used in the food industry. They are also important in cosmetic and pharmaceutical industries as drug carriers and for consistency improvements in creams and lotions. The current process for their production is based on the glycerolysis of natural fats and oils in the presence of inorganic catalysts at temperatures higher than 220°C. The major drawbacks of this process include high-energy consumption, low yield, and poor product quality. The use of lipases for monoacylglycerols production offers environmental advantages and a reduction in energy consumption. Besides, the same surfactants prepared by the enzymatic synthesis may be labeled as 'natural'. Recent progress in the application of highly-stable lipases in the organic solvents offers the possibility of employing various methods to the enzyme-catalyzed synthesis of monoacylglycerols, such as selective hydrolysis of fats and oils using 1,3-regiospecific lipases, the esterification of glycerol with fatty acids and the glycerolysis of fats or oils. In this review, different reaction systems such as aqueous-organic two-phase systems, microemulsions and reverse micelles systems, anhydrous organic solvents, solvent-free systems with free or immobilized lipases, as well as the use of two-phase membrane reactor systems are presented. We discuss some of the key factors, such as control of water content, removal of products from reaction system, and the effects of solvent on the lipase activity and selectivity, that must be addressed in order to obtain an efficient reaction system with high yields of monoacylglycerols. Engineering of the enzymatic monoacylglycerols synthesis processes requires also optimization of other factors such as: molar ratio of substrates, temperature, type of lipase immobilization and supports (if any), reactor design and operating regime., Monoacilgliceroli (MAG), zahvaljujući svojoj amfipatičnoj strukturi, imaju veliki značaj i primenu kao emulgatori u prehrambenoj industriji. Koriste se i u kozmetičkoj i farmaceutskoj industriji za pripremu emulzija ulje/voda (U/V) i za poboljšanje konzistencije krema i losiona. Konvencionalni hemijski postupci hidrolize masti imaju značajnih nedostataka jer se odvijaju na visokim temperaturama i pritiscima uz korišćenje neorganskih katalizatora. Pri ovako drastičnim procesnim uslovima, obrazuju se proizvodi promenjene boje i neprijatne arome, koji se moraju naknadno prečišćavati što poskupljuje proizvodnju. Enzimski postupci za dobijanje monoacilglicerola imaju značajnih prednosti, a to su pre svega blagi reakcioni uslovi, usmerenost reakcije kao i znatno manji troškovi za energiju. U ovom radu razmatrani su osnovni načini enzimskog dobijanja monoacilglicerola kao što su parcijalna hidroliza masti i ulja poziciono specifičnim lipazama, esterifikacija masnih kiselina i alkohola i gliceroliza masti i ulja, sa posebnim osvrtom na prednosti i nedostatke svakog od njih.

Response surface methodology was used for the evaluation of the effects of various factors on the synthesis of biodiesel catalyzed with immobilized lipase from Rhizomucor miehei in a solvent-free system. The production of biodiesel was optimized and model response equations were obtained, enabling the prediction of biodiesel production from the values of the four main factors. It would seem that the reaction temperature and the amount of water predominantly determined the conversion process while the methanol/oil molar ratio had no significant influence on the reaction rate. The temperature and amount of water showed negative interactive effects on the observed reaction rate per amount of enzyme. However, there were no significant interactions among the other variables according to the test of statistical significance. The highest yield of 10.15 mol kg-1 enzyme was observed at 45°C with a 6:1 methanol to oil molar ratio and with no added water in the system., Cilj ovog rada bila je optimizacija procesa sinteze biodizela korišćenjem imobilisane lipaze iz Rhizomucor miehei. U ovom radu ispitan je uticaj osnovnih procesnih parametara na prinos biodizela: temperature, molarnog odnosa metanol/ulje, koncentracije enzima i koncentracije vode u sistemu bez organskog rastvarača. Optimizacija procesa enzimske sinteze je izvršena korišćenjem rotatabilnog kompozicionog plana. Za obradu podataka korišćena je metodologija odzivnih površina. Ustanovljeno je da se uticaj faktora na brzinu enzimske reakcije može opisati modelom drugog reda sa tri faktora, bez međusobne interakcije. Statistička analiza pokazuje da je najznačajniji parametar količina enzima u reakcionoj smeši. Povišena temperatura je imala negativan uticaj na brzinu enzimske reakcije, kao i povećana koncentracija vode. Za analizu uticaja različitih faktora na brzinu enzimske reakcije po količini enzima, dobijen je model drugog reda, sa dva značajna parametra sa međusobnom interakcijom. Iz modela se vidi da je model izuzetno stabilan pri malom sadržaju vode, a da reakciji pogoduje velika količina vode samo pri niskim temperaturama. Najveći prinos, od 10,15 mol kg-1 enzima, ostvaren je pri sledećim parametrima reakcije: 45°C, molarniodnos metanol/ulje 6:1 i bez dodate vode u sistem.

Biodiesel is an alternative fuel for diesel engine that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The aim of the present work was to investigate novel acyl acceptors for biodiesel production. 2-Propanol and n-butanol have a less negative effect on lipase stability, and they also improve low temperature properties of the fuel. However, excess alcohol leads to inactivation of the enzyme, and glycerol, a major byproduct, can block the immobilized enzyme, resulting in low enzymatic activity. This problem was solved by using methyl acetate as acyl acceptor. Triacetylglycerol is produced instead of glycerol, and it has no negative effect on the activity of the lipase., U ovom radu ispitana je mogućnost primene različitih acil akceptora kao polaznih reaktanata umesto metanola u sintezi biodizela u cilju smanjenja inhibicije enzima, povećanja njegove stabilnosti i mogućnosti značajnog pojednostavljivanja izvođenja procesa. Prvo je ispitana mogućnost korišćenja različitih alkohola: 2-propanol i n-butanol koji imaju manji uticaj na denaturaciju enzima. Osim toga, primenom ovih alkohola dobija se gorivo boljeg kvaliteta, pošto se sa povećanjem broja ugljenikovih atoma povećava oktanski broj, temperatura paljenja, kao i sadržaj toplote. Drugi deo istraživanja bio je orijentisan ka ispitivanju mogućnosti korišćenja metilacetata kao pogodnog nukleofila. Za razliku od alkohola, primenom ovog acil akceptora omogućen je jednostepeni postupak sinteze biodizela, što značajno smanjuje vreme trajanja reakcije, kao i kompleksnost iste. Takođe, primenom metilacetata povećava se stabilnost biokatalizatora, što omogućava višekratnu upotrebu. Naime, u reakciji sa metilacetatom kao nusprodukt se ne stvara glicerol već triacetilglicerol, koji nema negativan efekat na stabilnost lipaze.

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., Cilj ovog rada je bio ispitivanje mogućnosti sinteze različitih estara pomoću lipaze iz Candida rugosa kao i optimizacija procesnih parametara. Pokazano je da se data lipaza može koristiti kao efikasan biokatalizator za dobijanje devetnaest različitih estara. Najveći prinosi, veći od 90%, dobijeni su u slučaju sinteze nižih estara kao što su pentil-propanoat, 2-metilbutil-butanoat i butil-butanoat. Početna brzina sinteze estara i krajnji prinosi bili su znatno niži sa supstratima koji sadrže više od 8 ugljenikovih atoma. Enzim je pokazao iznenađujuće mali afinitet prema pentanskoj i heksanskoj kiselini, u poređenju sa višim homolozima, oktanskom i dekanskom.Pored broja ugljenikovih atoma, na aktivnost enzima značajno je uticalo prisustvo dvostruke veze u molekulu supstrata i razgranatost supstrata. Naime, lipaza iz C. rugosa ispoljila je mnogo manju aktivnost u reakciji sa izopropanolom nego sa propanolom, dok je pokazala mnogo veći afinitet prema 9-cis-oktadecenskoj kiselini u odnosu na njen zasićeni analog, oktadekansku kiselinu.