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Partition of pectinases in aqueous two-phase systems

dc.contributor.advisorPeričin, Draginja
dc.contributor.otherPetrović, Suzana
dc.contributor.otherPeričin, Draginja
dc.contributor.otherKevrešan, Slavko
dc.creatorAntov, Mirjana
dc.date.accessioned2019-09-27T10:51:25Z
dc.date.available2019-09-27T10:51:25Z
dc.date.available2020-07-03T13:56:13Z
dc.date.issued2000-12-27
dc.identifier.urihttp://nardus.mpn.gov.rs/handle/123456789/11366
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/Disertacija153787837152879.pdf?controlNumber=(BISIS)71461&fileName=153787837152879.pdf&id=12001&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/record.jsf?recordId=71461&source=NaRDuS&language=srsr
dc.identifier.urihttps://www.cris.uns.ac.rs/DownloadFileServlet/IzvestajKomisije156887590636748.pdf?controlNumber=(BISIS)71461&fileName=156887590636748.pdf&id=13453&source=NaRDuS&language=srsr
dc.description.abstractU radu je ispitana raspodela pektinaza u vodenim dvofaznim sistemima sastavljenim od polietilen glikola 4000 i jednog od dva dekstrana, sirovog i dekstrana 40000. Koncepcija rada je podrazumevala definisanje sistema u kojima su vršeni eksperimenti raspodele, ispitivanje parametara raspodele pektinaza komercijalnog enzimskog preparata u model sistemima i, konačno, kultivaciju Polyporus squamosus u dvofaznim sistemima. Ispitivanje uticaja masenih udela polietilen glikola na raspodelu pektinaza u model sistemu, pri konstantnom udelu sirovog dekstrana od 7.5% (w/w), pokazalo je negativan uticaj na parametre raspodele, te su najveće vrednosti koeficijenta raspodele (K) dobijene pri najmanjem ispitivanom udelu polietilen glikola, 5% (w/w), i iznosili su za endo-pektinazu (endo-p) 1.76, odnosno za egzo- pektinazu (egzo-p) 1.22. Istu zavisnost je pokazalo i ispitivanje u model sistemu sa dekstranom 40000, ali je vrednost Kendo bila mnogo niža (0.22). Povećanje udela sirovog dekstrana, pri stalnom udelu, 5% (w/w), polietilen glikola, negativno je uticalo na vrednosti parametara raspodele egzo-p, te su maksimalne vrednosti dobijene pri najmanjem udelu, 3% (w/w), dekstrana (Kegzo 0.79, prinos u gornjoj fazi (Y) 62.04%, stepen prečišćenosti u gornjoj fazi (st.preč.) 5.5). Sa povećanjem dužine veznih linija u model sistemu sa sirovim dekstranom opadale su vrednosti parametara raspodele pektinaza. Najbolji rezultati (Kendo 1.66, Yendo 60.85%, Kegzo 0.95, Yegzo 47.07%) dobijeni su najkraćoj veznoj liniji, dužine 7.44%. Povećanje odnosa zapremina u istom sistemu smanjilo je koeficijente raspodele endo- i egzo-p, kao i njihove stepene prečišćenosti u gornjoj fazi, a u isto vreme povećalo prinose u toj fazi. Dodatak amonijum sulfata i natrijum sulfata u model sistem polietilen glikol/sirovi dekstran u koncentraciji od po 15 mmoI/l povećao je Kendo 1-25, odnosno 1.2 puta, dok je Kegzo u prisustvu 15 mmol/l amonijum sulfata ili 5 mmol/l natrijum sulfata bio za 60% veći. Povećanje rN vrednosti KH2PO4-Na2HPO4 pufera povećalo je koeficijente raspodele oba tipa pektinaza, te je Kendo, na rN 7.5, bio 0.9, a Kegzo 0.76, na rN 7.0. Najbolja raspodela endo-p u gornju fazu model sistema ostvarena je pri 0.4 mol/l fosfatnog pufera (K 4.21, Y 85.78%, st.preč. 10). Vrednosti iznad 0.1 mol/l fosfatnog pufera nisu imale značajnijeg uticaja na raspodelu egzo-p. Kultivacija Polyporus squamosus je bila moguća u dvofaznom sistemu polietilen glikol/sirovi dekstran, sa pektinom. kao inducerom, pri čemu je ostvareno odvajanje biomase od pektinaza njihovom raspodelom u suprotne faze. U sistemu sastava 5% (w/w) polietilen glikol/4% (w/w) sirovi dekstran, količina produkovane biomase je bila 4.5 puta veća od one u homogenom medijumu, ukupne egzo-p aktivnosti 1.82 puta veća, a endo-p aktivnosti jednaka onoj u homogenom medijumu. Drugog dana kultivacije ostvarene su najveće vrednosti parametara raspodele (Kendo 2.45, Yendo 80.22%, st.preč.endo 7; Kegzo 0.6, Yegzo 49.83%, st.preč.egzo 5.19). Povećanje koncentracije amonijum sulfata za 15 mmol/l u podlozi za kultivaciju u dvofaznom sistemu sastava 5% (w/w) polietilen glikol/4% (w/w) sirovi dekstran, povećalo je koeficijent raspodele endo-p za 81%, vrednost ove aktivnost u grnjoj fazi za 37%, a da nije uticalo na raspodelu biomase P. squamosus. Prisustvo različitih koncentracija fosfata nije uticalo na raspodelu biomase P. squamosus u dvofaznom sistemu. Pri 0.2 mol/l KH2PO4 postignuto je idealno odvajanje biomase od endo-p, uz Kegzo 112. Najveća vrednost ukupnih produkovanih aktivnosti oba tipa ostvarena je pri 0,1 mol/l fosfata, uz koeficijente raspodele za endo-p i egzo-p 3.9 i 0.95 i visoke stepene njihove prečišćenosti u gornjoj fazi (5.89 i 14.36, redom). Kultivacija P. squamosus je ostvarena i u dvofaznom sistemu koji je sadržao suve izlužene repine rezance, sa raspodelom biomase u donju fazu. Koeficijenti raspodele su iznosili 4.70, odnosno 2.78, za endo-p, odnosno egzo-p. Prinosi oba enzima u gornjoj fazi su bili 90.71% za endo-p i 85.24% za egzo-p, uz najveće stepene njihove prečišćenosti, 4.26 i 7.98, redom.sr
dc.description.abstractPartition of pectinases in aqueous two-phase systems composed of polyethylene glycol 4,000 and crude dextran or dextran 40,000 was studied in this work. As the first step, phase diagrams were constructed, than partition of commercial pectinases was studied in model systems and finally, cultivation of Polyporus squamosus in aqueous two-phase systems was conducted. Increasing of the concentration of polyethylene glycol in model system at fixed 7.5% (w/w) crude dextran decreased partition parameters. Maximal values for partition coefficient (К) of endo-pectinase (endo-p), 1.76, and exo-pectinase (ехо-р), 1.22, were obtained at 5% (w/w) polyethylene glycol. The same was revealed in investigations in model system with dextran 40,000, but Kendo was lower (0.22). Increasing of concentration of crude dextran at fixed 5% (w/w) polyethylene glycol decreased partition parameters of ехо-р, so their maximal values (Кexo 0.79, top phase yield (Y) 62.04% and purification factor in top phase 5.5) were obtained at 3% (w/w) crude dextran. Increasing of the tie-line length caused decreasing of partition parameters and maximal results were achieved at the shortest tie-line 7.44% (Kendo 1-66, Yendo 60.85%, Кехо 0.95, Yexo 47.07%) in model system with crude dextran. With increasing of the volume ratio, partition coefficients of endo-p and ехо-р were decreased, as well as their purification factor in top phase, while top phase yield for both enzymes had higher values. The addition of 15 mmol ammonium sulphate/l and 15 mmol sodium sulphate/l in model system, composed of polyethylene glycol and crude dextran, increascd Kendo 1.25 and 1.2 times, respectively, while Кехо in the presence of 15 mmol ammonium sulphate/l or 5 mmol sodium sulphate/l was 60% higher. Increasing of рН of KH2PO4- Na2HPO4 buffer favoured partition of both endo-p and ехо- p in top phase, so Kendo was 0.9 at рН 7.5 and Кехо was 0.76 at рН 7.0. The favourable partition of endo-p in top phase was achieved in the presence of 0.4 mol phosphate buffer/l (К 4.21, Y 85.78% and purification factor in top phase 10). Concentrations of phosphate buffer above 0.1 mol/l didn’t have significant influence on partition of ехо-р. Cultivation of Роlуроrus squamosus in polyethylene glycol/crude dextran aqueous two-phase system was accomplished with pectin as inducer and biomass was separated from pectinases by their partition into opposite phases. In system composed of 5% (w/w) polyethylene glycol and 4% (w/w) crude dextran, amounts of produced biomass and ехо-р activity were 4.5 and 1.82 times higher, respectively, and endo-p activity equal to those obtained in homogeneous cultivation. At the second day of cultivation the highest values of partition parameters were obtained (Kendo 2.45, Yendo 80.22%, purification factor for endo-p 7; Кeхо 0.6, Yexo 49.83%, purification factor for ехо-р 5.19). Increasing of concentration of ammonium sulphate in two-phase medium for 15 mmol/l increased Kendo for 81% and endo-p activity in top phase for 37%, but didn’t influence partition of biomass. Presence of phosphate at various concentrations didn’t affect partition of biomass in two-phase system. At 0.2 mol KH2PO4/I tlie onesided partition of endo-p was accomplished while Кeхо was 1.12. The highest amounts of produced activities of endo-p and ехо-р were obtained at 0.1 mol phosphate/l and partition coefficients were 3.9 and 0.95, respectively, followed by high values of purification factors in top phase (5.89 and 14.36, rcspectively). Cultivation of P. squamosus was accomplished in two-phase system containing dry sugar beet extraction waste and fungal growth was restrected to bottom phase. Partition coefficients were 4.70 and 2.78 for endo-p and ехо-р, respectively. Тор phase yields amounted 90.71% for endo-p and 85.24% for ехо-р followed by the highest obtained purification factors in top phase (4.26 and 7.98, respectively).en
dc.languagesr (latin script)
dc.publisherУниверзитет у Новом Саду, Технолошки факултетsr
dc.rightsopenAccessen
dc.sourceУниверзитет у Новом Садуsr
dc.subjectVodeni dvofazni sistemisr
dc.subjectAqueous two-phase systemsen
dc.subjectfenomeni raspodelesr
dc.subjectpektinazesr
dc.subjectpartition phenomenonsen
dc.subjectpectinasesen
dc.titleRaspodela pektinaza u vodenim dvofaznim sistemimasr
dc.title.alternativePartition of pectinases in aqueous two-phase systemsen
dc.typedoctoralThesissr
dc.rights.licenseBY-NC-ND
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/40828/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/40829/IzvestajKomisije.pdf


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