Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
Regulation and maximization of lipase production in a mutant derivative of R. oligosporus has been investigated using different substrates, inoculum sizes, pH of the medium, temperature, and nitrogen sources in shake flask experiments and batch fermentation in a fermentor. The production of intracellular lipase was improved 3 times following medium optimization involving one-at-a-time approach and aeration in the fermentor. Interestingly, intracellular lipase was poorly induced by oils, instead its production was induced by sugars, mainly starch, lactose, sucrose, xylose, glucose and glycerol. Dependent variables studied were cell mass, lipase activity, lipase yield, lipase specific and volumetric rate of formation. It was confirmed that lipase production in the derepressed mutant is sufficiently uncoupled from catabolite repression. The results of average specific productivities at various temperatures worked out according to the Arrhenius equation revealed that mutation decreased the magnitude of enthalpy and entropy demand in the inactivation equilibrium during product formation, suggesting that mutation made the metabolic network of the organism thermally more stable. The highest magnitudes of volumetric productivity (QP=490 IU/(L·h)) and other product attributes of lipase formation occurring on optimized medium in the fermentor are greater than the values reported by other workers. The purified enzyme is monomeric in nature and exhibits stability up to 80 °C and pH=6.0–8.0. Activation energy, enthalpy and entropy of catalysis at 50 °C, and magnitudes of Gibbs free energy for substrate binding, transition state stabilization and melting point indicated that this lipase is highly thermostable.Primjenom različitih supstrata, veličina inokuluma, pH-vrijednosti podloge, temperature i izvora dušika regulirana je i maksimizirana proizvodnja lipaze s pomoću mutanta Rhizopus oligosporus DGM 31 u pokusima na tresilici i šaržnim procesom u reaktoru. Proizvodnja intracelularne lipaze povećala se 3 puta optimiranjem podloge i prozračivanjem reaktora. Dodatak ulja malo je poboljšao proizvodnju lipaze, a znatno je poboljšana dodatkom šećera, osobito škroba, laktoze, saharoze, ksiloze, glukoze i glicerola. Ispitane su ove zavisne varijable: masa stanica, aktivnost i prinos lipaze, te specifična i volumetrijska brzina nastajanja enzima. Dokazano je da proizvodnja lipaze s pomoću mutanta ne ovisi o represiji s katabolitima. Vrijednosti prosječne specifične produktivnosti pri različitim temperaturama, izračunate prema Arrheniusovoj jednadžbi, pokazuju da mutacija smanjuje količinu entalpije i entropije potrebne za postizanje ravnotežne inaktivacije tijekom formiranja produkta, te da je omogućila odvijanje toplinski puno stabilnijeg metaboličkog procesa u organizmu. Korištenjem optimirane podloge u reaktoru postignuta je dosad najveća volumetrijska produktivnost lipaze (QP=7,3 IU/g škroba/h). Pročišćeni je enzim monomeran i stabilan čak i do 80 °C i pri pH-vrijednosti od 6,0 do 8,0. Veliku termostabilnost lipaze dokazuju: energija aktivacije, entalpija i entropija potrebne za provedbu katalize pri 50 °C, Gibbsova slobodna energija utrošena za vezanje supstrata, stabilnost prijelaznog stanja i točka taljenja
