Lipaza iz plijesni Mucor griseocyanus: proizvodnja, karakterizacija i istraživanje nekih katalitičkih svojstava imobiliziranog enzima

The aim of this work is to study the production of extracellular lipase by Mucor griseocyanus 55.1.1 strain on different substrates in order to select the ideal one for lipase synthesis. The carbon sources used were: olive oil, glycerol, coconut oil, sunflower oil, glucose, starch and sucrose. The obtained results indicate that the synthesis of the enzyme was possible in the presence of all substrates. Lipase activities in the range of 0.04 to 0.1 IU/mL were obtained. It was found that the most suitable carbon source for the production of the enzyme was a combination of coconut oil and sucrose at 0.5 and 1.5 % (m/V), respectively, and the level of activity reached under this condition was 0.113 IU/mL. The optimum pH and temperature for enzymatic extract activities were identified in a pH range of 4 to 6 and at a temperature of 60 °C. Enzymatic extract was stable for a period of 5 h in neutral and weakly acidic media (pH=6) at moderate temperatures between 20 and 40 °C. Studies on the catalytic properties (stereoselectivity and enantioselectivity) of the immobilized lipase using the esters of methyl phenyl glycinic and (R,S)-methyl mandelic acid showed excellent properties of the enzyme compared to commercial lipases tested. M. griseocyanus lipase exhibited a greater stereoselectivity towards the R-isomer of methyl phenyl glycinic acid ester. However, with the esters of methyl mandelic acid, the enzyme showed a certain preference toward the S-isomer and it was hydrolysed 20 times faster than the R-isomer.Svrha je ovoga rada bila proučiti proizvodnju ekstracelularne lipaze iz soja Mucor griseocyanus 55.1.1. na raznim podlogama radi odabira najboljeg načina sinteze lipaze. Upotrijebljeni su ovi izvori ugljika: maslinovo ulje, glicerol, kokosovo i suncokretovo ulje, te škrob i saharoza. Rezultati su pokazali da se sinteza enzima odvijala na svim podlogama, te da je postignuta aktivnost lipaze u rasponu od 0,04 do 0,1 IU/mL. Utvrđeno je da su najbolji rezultati dobiveni s podlogom koja sadržava 0,5 % (m/V) kokosova ulja i 1,5 % (m/V) saharoze, pri čemu je aktivnost enzima iznosila 0,113 IU/mL. Optimalna pH-vrijednost bila je od 4 do 6, a temperatura 60 ºC. Ekstrakt enzima bio je stabilan tijekom 5 sati u neutralnoj ili slabo kiseloj sredini (pH=6), pri temperaturama od 20 do 40 ºC. Istraživanjem katalitičkih svojstava (stereoselektivnosti i enantioselektivnosti) imobilizirane lipaze pomoću metilfenilnog estera glicinske kiseline i (R,S)-metilnog estera bademove kiseline dokazana su odlična svojstva tog enzima u usporedbi s komercijalnim lipazama. Lipaza iz plijesni M. griseocyanus ima veću stereoselektivnost prema R-oblicima metilfenilnog estera glicinske kiseline i S-izomeru metilnog estera bademove kiseline (kojeg hidrolizira 20 puta brže od R-izomera)

Production of Extracellular Lipase from Aspergillus niger by Solid-State Fermentation

Proizvodnja lipaze u soju plijesni Aspergillus niger J-1 ispitana je submerznim uzgojem i površinskim uzgojem na čvrstoj podlozi od pšeničnih posija. Provedeno je optimiranje podloge za oba postupka uzgoja. Maksimalna aktivnost lipaze od 1,46 IU/mL postignuta je submerznim uzgojem u podlozi s 2 % glukoze i 2 % maslinova ulja pri 1 vvm i 450 m-1. Medutim, 9,14 IU/g suhe tvari ekvivalentne 4,8 IU/mL aktivnosti lipaze postignuto je površinskim uzgojem na čvrstoj podlozi s 0,75 % amonijeva sulfata i 0,34 % uree. Optimalni pH i temperatura za aktivnost enzima bili su pH=6 i 40 °C. Enzim je pokazao 80 % od početne aktivnosti u neutralnom i blago kiselom mediju i pri temperaturi od 20 i 30 °C tijekom 24 sata.Lipase production in Aspergillus niger J-1 was tested using both submerged fermentation (SmF) and solid-state fermentation (SSF) on a mineral culture medium and wheat bran, respectively. The optimization of the culture medium was carried out for both SmF and SSF. The maximum lipase activity, 1.46 IU/mL, was obtained during the submerged fermentation in a medium containing glucose at 2 % and olive oil at 2 % under conditions of 1 vvm and 450 m–1. However, 9.14 IU/g of dry solid substrate equivalent to 4.8 IU/mL of lipase activity was reached using solid-state fermentation process with a medium containing 0.75 % of ammonium sulphate and 0.34 % of urea. The optimum pH and temperature for enzymatic activity were pH=6 and 40 °C, respectively. The enzyme also exhibited 80 % of its initial activity in neutral and mildly acid media and at temperatures between 20 and 30 °C for a period of 24 hours

Mucor griseocyanus Lipase: Production, Characterization and Study of Some Catalytic Properties of the Immobilised Enzyme

The aim of this work is to study the production of extracellular lipase by Mucor griseocyanus 55.1.1 strain on different substrates in order to select the ideal one for lipase synthesis. The carbon sources used were: olive oil, glycerol, coconut oil, sunflower oil, glucose, starch and sucrose. The obtained results indicate that the synthesis of the enzyme was possible in the presence of all substrates. Lipase activities in the range of 0.04 to 0.1 IU/mL were obtained. It was found that the most suitable carbon source for the production of the enzyme was a combination of coconut oil and sucrose at 0.5 and 1.5 % (m/V), respectively, and the level of activity reached under this condition was 0.113 IU/mL. The optimum pH and temperature for enzymatic extract activities were identified in a pH range of 4 to 6 and at a temperature of 60 °C. Enzymatic extract was stable for a period of 5 h in neutral and weakly acidic media (pH=6) at moderate temperatures between 20 and 40 °C. Studies on the catalytic properties (stereoselectivity and enantioselectivity) of the immobilized lipase using the esters of methyl phenyl glycinic and (R,S)-methyl mandelic acid showed excellent properties of the enzyme compared to commercial lipases tested. M. griseocyanus lipase exhibited a greater stereoselectivity towards the R-isomer of methyl phenyl glycinic acid ester. However, with the esters of methyl mandelic acid, the enzyme showed a certain preference toward the S-isomer and it was hydrolysed 20 times faster than the R-isomer

Production of Extracellular Lipase from Aspergillus niger by Solid-State Fermentation

Lipase production in Aspergillus niger J-1 was tested using both submerged fermentation (SmF) and solid-state fermentation (SSF) on a mineral culture medium and wheat bran, respectively. The optimization of the culture medium was carried out for both SmF and SSF. The maximum lipase activity, 1.46 IU/mL, was obtained during the submerged fermentation in a medium containing glucose at 2 % and olive oil at 2 % under conditions of 1 vvm and 450 m–1. However, 9.14 IU/g of dry solid substrate equivalent to 4.8 IU/mL of lipase activity was reached using solid-state fermentation process with a medium containing 0.75 % of ammonium sulphate and 0.34 % of urea. The optimum pH and temperature for enzymatic activity were pH=6 and 40 °C, respectively. The enzyme also exhibited 80 % of its initial activity in neutral and mildly acid media and at temperatures between 20 and 30 °C for a period of 24 hours