Production of microbial lipases and proteases as additives in detergent formulations.

U okviru ove teze, ispitan je veliki broj mikroorganizama sa aspekta produkcije ekstracelularnih lipaza i proteaza koje bi se potencijalno koristile kao aditivi u detergentima. Selekcija proizvodnih mikroorganizama obuhvatila je nekoliko faza kako bi se identifikovali mikroorganizmi koji proizvode lipaze i proteaze dobrih svojstava i u dobrom prinosu. Ove studije su izdvojile dva mikrobna producenta traženih enzima, koji pokazuju neophodnu funkcionalnost u alkalnim uslovima i u širokom spektru temperatura. Dobijene lipaze iz Pseudomonas putida B-21 i lipaze i proteaze iz Pseudomonas aeruginosa san-ai su zatim ispitane sa aspekta stabilnosti u prisustvu površinski aktivnih materija, oksidacionih agenasa i komercijalnih detergenata kako bi se utvrdila primenljivost dobijenih enzima kao aditiva u formulacijama detergenata. Uzevši u obzir pokazanu stabinost enzima koje produkuje P. aeruginosa san-ai oni su primenjeni u formulaciji nekoliko novih proizvoda koji pokazuju zadovoljavajuća svojstva sa stanovišta efikasnosti čišćenja, stabilnosti pri skladištenju i sastava otpadnih voda od pranja. Enzimska formulacija detergenata je optimizovana metodom odzivnih površina (RSM). Uzimajući u obzir ekološke aspekte kao i pokazanu efikasnost, pokazano je da se najbolji efekti odmašćivanja postižu u formulaciji koja pored enzima sadrži nisku dozu Lutensol®-a XP 80 (0,4%). Dalja optimizacija produkcije enzima RSM metodom definisala je sastave podloga i procesne parametre kojima se modifikuju regulatorni putevi biosinteze enzima sa ciljem povećanja prinosa lipaza i proteaza. Visoka proteolitička aktivnost se tako može ostvariti fermentacijom u medijumu koji je obogaćen Triton®-om X-100 (0.12% w/v) pri čemu proces fermentacije treba voditi na 32 °C. Niže temperature (20 °C) i suplementacija suncokretovim uljem pogoduju biosiontezi lipaze.In this thesis, a vast number of microorganisms was tested in terms of the production of extracellular proteases and lipases with the ability to be used as detergent additives. These studies have marked two microbial strains which produce enzymes that show the necessary functionalities in alkaline conditions and a wide range of temperatures. A lipase derived from Pseudomonas putida B-21, and protease and a lipase from Pseudomonas aeruginosa san-ai were further tested with respect to stability in the presence of surfactants, oxidizing agents and commercial detergents in order to determine the applicability of the obtained enzymes in detergent formulations. Taking into the account demonstrated properties of lipase and protease produced by P. aeruginosa san-ai, these enzymes were applied in the formulation of several new products that demonstrate satisfactory performance in terms of cleaning efficiency, storage stability and wastewater composition. Enzyme detergent formulation was optimized using respond surface methodology (RSM). Considering the environmental aspects and demonstrated degreasing efficacy, detergent formulation which contains the produced enzymes should be based on low-dose Lutensol® XP 80 (0.4 %). Further optimization of the enzymes production using RSM defined medium composition and process parameters in order to increase the yield of lipases and proteases produced. The high proteolytic activity can be accomplished by fermentation in a medium enriched with Triton® X-100 (0.12% w/v) and the process should be run at 32 °C. Lower temperature (20 °C) and sunflower oil supplementation will favor lipase biosynthesis

Production of microbial lipases and proteases as additives in detergent formulations.

U okviru ove teze, ispitan je veliki broj mikroorganizama sa aspekta produkcije ekstracelularnih lipaza i proteaza koje bi se potencijalno koristile kao aditivi u detergentima. Selekcija proizvodnih mikroorganizama obuhvatila je nekoliko faza kako bi se identifikovali mikroorganizmi koji proizvode lipaze i proteaze dobrih svojstava i u dobrom prinosu. Ove studije su izdvojile dva mikrobna producenta traženih enzima, koji pokazuju neophodnu funkcionalnost u alkalnim uslovima i u širokom spektru temperatura. Dobijene lipaze iz Pseudomonas putida B-21 i lipaze i proteaze iz Pseudomonas aeruginosa san-ai su zatim ispitane sa aspekta stabilnosti u prisustvu površinski aktivnih materija, oksidacionih agenasa i komercijalnih detergenata kako bi se utvrdila primenljivost dobijenih enzima kao aditiva u formulacijama detergenata. Uzevši u obzir pokazanu stabinost enzima koje produkuje P. aeruginosa san-ai oni su primenjeni u formulaciji nekoliko novih proizvoda koji pokazuju zadovoljavajuća svojstva sa stanovišta efikasnosti čišćenja, stabilnosti pri skladištenju i sastava otpadnih voda od pranja. Enzimska formulacija detergenata je optimizovana metodom odzivnih površina (RSM). Uzimajući u obzir ekološke aspekte kao i pokazanu efikasnost, pokazano je da se najbolji efekti odmašćivanja postižu u formulaciji koja pored enzima sadrži nisku dozu Lutensol®-a XP 80 (0,4%). Dalja optimizacija produkcije enzima RSM metodom definisala je sastave podloga i procesne parametre kojima se modifikuju regulatorni putevi biosinteze enzima sa ciljem povećanja prinosa lipaza i proteaza. Visoka proteolitička aktivnost se tako može ostvariti fermentacijom u medijumu koji je obogaćen Triton®-om X-100 (0.12% w/v) pri čemu proces fermentacije treba voditi na 32 °C. Niže temperature (20 °C) i suplementacija suncokretovim uljem pogoduju biosiontezi lipaze.In this thesis, a vast number of microorganisms was tested in terms of the production of extracellular proteases and lipases with the ability to be used as detergent additives. These studies have marked two microbial strains which produce enzymes that show the necessary functionalities in alkaline conditions and a wide range of temperatures. A lipase derived from Pseudomonas putida B-21, and protease and a lipase from Pseudomonas aeruginosa san-ai were further tested with respect to stability in the presence of surfactants, oxidizing agents and commercial detergents in order to determine the applicability of the obtained enzymes in detergent formulations. Taking into the account demonstrated properties of lipase and protease produced by P. aeruginosa san-ai, these enzymes were applied in the formulation of several new products that demonstrate satisfactory performance in terms of cleaning efficiency, storage stability and wastewater composition. Enzyme detergent formulation was optimized using respond surface methodology (RSM). Considering the environmental aspects and demonstrated degreasing efficacy, detergent formulation which contains the produced enzymes should be based on low-dose Lutensol® XP 80 (0.4 %). Further optimization of the enzymes production using RSM defined medium composition and process parameters in order to increase the yield of lipases and proteases produced. The high proteolytic activity can be accomplished by fermentation in a medium enriched with Triton® X-100 (0.12% w/v) and the process should be run at 32 °C. Lower temperature (20 °C) and sunflower oil supplementation will favor lipase biosynthesis

Production of microbial lipases and proteases as additives in detergent formulations.

U okviru ove teze, ispitan je veliki broj mikroorganizama sa aspekta produkcije ekstracelularnih lipaza i proteaza koje bi se potencijalno koristile kao aditivi u detergentima. Selekcija proizvodnih mikroorganizama obuhvatila je nekoliko faza kako bi se identifikovali mikroorganizmi koji proizvode lipaze i proteaze dobrih svojstava i u dobrom prinosu. Ove studije su izdvojile dva mikrobna producenta traženih enzima, koji pokazuju neophodnu funkcionalnost u alkalnim uslovima i u širokom spektru temperatura. Dobijene lipaze iz Pseudomonas putida B-21 i lipaze i proteaze iz Pseudomonas aeruginosa san-ai su zatim ispitane sa aspekta stabilnosti u prisustvu površinski aktivnih materija, oksidacionih agenasa i komercijalnih detergenata kako bi se utvrdila primenljivost dobijenih enzima kao aditiva u formulacijama detergenata. Uzevši u obzir pokazanu stabinost enzima koje produkuje P. aeruginosa san-ai oni su primenjeni u formulaciji nekoliko novih proizvoda koji pokazuju zadovoljavajuća svojstva sa stanovišta efikasnosti čišćenja, stabilnosti pri skladištenju i sastava otpadnih voda od pranja. Enzimska formulacija detergenata je optimizovana metodom odzivnih površina (RSM). Uzimajući u obzir ekološke aspekte kao i pokazanu efikasnost, pokazano je da se najbolji efekti odmašćivanja postižu u formulaciji koja pored enzima sadrži nisku dozu Lutensol®-a XP 80 (0,4%). Dalja optimizacija produkcije enzima RSM metodom definisala je sastave podloga i procesne parametre kojima se modifikuju regulatorni putevi biosinteze enzima sa ciljem povećanja prinosa lipaza i proteaza. Visoka proteolitička aktivnost se tako može ostvariti fermentacijom u medijumu koji je obogaćen Triton®-om X-100 (0.12% w/v) pri čemu proces fermentacije treba voditi na 32 °C. Niže temperature (20 °C) i suplementacija suncokretovim uljem pogoduju biosiontezi lipaze.In this thesis, a vast number of microorganisms was tested in terms of the production of extracellular proteases and lipases with the ability to be used as detergent additives. These studies have marked two microbial strains which produce enzymes that show the necessary functionalities in alkaline conditions and a wide range of temperatures. A lipase derived from Pseudomonas putida B-21, and protease and a lipase from Pseudomonas aeruginosa san-ai were further tested with respect to stability in the presence of surfactants, oxidizing agents and commercial detergents in order to determine the applicability of the obtained enzymes in detergent formulations. Taking into the account demonstrated properties of lipase and protease produced by P. aeruginosa san-ai, these enzymes were applied in the formulation of several new products that demonstrate satisfactory performance in terms of cleaning efficiency, storage stability and wastewater composition. Enzyme detergent formulation was optimized using respond surface methodology (RSM). Considering the environmental aspects and demonstrated degreasing efficacy, detergent formulation which contains the produced enzymes should be based on low-dose Lutensol® XP 80 (0.4 %). Further optimization of the enzymes production using RSM defined medium composition and process parameters in order to increase the yield of lipases and proteases produced. The high proteolytic activity can be accomplished by fermentation in a medium enriched with Triton® X-100 (0.12% w/v) and the process should be run at 32 °C. Lower temperature (20 °C) and sunflower oil supplementation will favor lipase biosynthesis

Lipaze i proteaze dobijene iz ekstremofilne Pseudomonas aeruginosa vrste kao aditivi u formulacijama deterdženata

Enzymes produced by indigenous Pseudomonas aeruginosa strain have been subjected to research considering their potential application as detergent additives. As previously noted, lipase produced by Pseudomonas aeruginosa is highly alkaline, thermostable and solvent tolerant. Furthermore, same strain exhibits both lipase and protease activity establishing this lipase as potentially desirable component of enzyme-containing detergents. Further research was carried out to investigate insusceptibility of this lipase against coexisting native protease, several commercial surfactants, oxidizing agents and commercial detergents. Lipases and proteases remained highly active when incubated with several different surfactants and oxidizing agents under washing conditions. Moreover, presence of surfactants and oxidizing agents such as Tween® 20 and Triton® X-100 initially augment lipase and protease activity. Additionally, crude lipase preparation was insusceptible to coexisting native protease hence indicating possible storage stability. Overall, the remarkable properties of these enzymes make them potential detergent additives.Enzimi iz ekstremofilne Pseudomonas aeruginosa vrste pokazuju visoku stabilnost u alkalnoj sredini što ih čini pogodnim za primenu u proizvodnji deterdženata. U ovom radu ispitana je aktivnost lipaza i proteaza dobijenih iz ove vrste u uslovima od značaja za industriju deterdženata. Ustanovljeno je da u realnim uslovima pranja, oba enzima zadržavaju visoku aktivnost u prisustvu različitih površinski aktivnih materija i oksidacionih agenasa. Takođe, prisustvo nekih aditiva kao što su Tween® 20, Triton® X-100, SDS i NaClO u početnoj fazi povećava lipolitičku aktivnost dok je povećanje proteolitičke aktivnosti primećeno u formulacijama koje sadrže Tween® 20 i Triton® X-100. Nadalje, sama lipaza je očuvala aktivnost u prisustvu nativnih proteaza što upućuje na zaključak da bi ovi enzimi zadržali visoku efikasnost u formulacijama deterdženata i tokom procesa pranja/čišćenja

Uticaj sastava fermentacione podloge na prinos produkovanih lipaza pomoću kvasca Candida utilis

In this work lipolytic activity of various microorganisms isolated from spoiled soybean oil was studied. Wild yeast strain identified as Candida utilis was chosen for the producing microorganism based on the activity shown on tributyrin agar plate. Studies were undertaken to improve lipase production by optimizing fermentation medium composition. A response surface approach has been used to optimize medium constituents namely maltose, oleic acid, Tween 80Ò and hydrolyzed casein due to the preliminary investigations in which they had positive effect on lipase production. Dependence of lipase production of medium constituents was explained with second order regression equation. Based on the coefficient values comparison, Tween 80Ò concentration has strongest influence on lipase production, followed by oleic acid concentration and then maltose concentration while the influence of hydrolyzed casein concentration was negligible. It was shown that optimization of fermentation conditions can lead to a significant improvement on the lipase production. .U radu je ispitana lipolitička aktivnost kvasca identifikovanog kao Candida utilis koji je izabran kao lipolitički najaktivniji soj među nekoliko različitih mikroorganizama izolovanih iz pokvarenog sojinog ulja. Ispitan je uticaj sastava fermentacione podloge na mogućnost povećanja produkcije lipaza u odnosu na pokazanu lipolitičku aktivnost u sladnom bujonu kao univerzalnom kvaščevom medijumu. Optimizacija sastava hranljive podloge izvršena je prema rotatabilnom četvorofaktornim eksperimentalnim planom na pet nivoa prema kojem su varirane koncentracije maltoze, oleinske kiseline, Tweena 80Ò i hidrolizata kazeina za koje je u preliminarnim ispitivanjima pokazano da stimulišuće deluju na produkciju lipaza. Zavisnost produkcije lipaza od sastava fermentacione podloge opisana je regresionim modelom drugog reda, a na osnovu vrednosti signifikantnih koeficijenata regresije utvrđeno je da na povećanje produkcije lipaza najveći uticaj ima emulgator Tween 80Ò, zatim oleinska kiselina i na kraju maltoza dok je uticaj koncentracije hidrolizata kazeina zanemarljiv. Pokazano je da se pažljivim izborom sastava hranljive podloge prinos produkovanih lipaza može višestruko uvećati.

Proizvodnja ramnolipida i lipaze iz Pseudomonas aeruginosa san-ai - optimizacija procesa primenom metode odzivnih površina

Pseudomonas aeruginosa has been repeatedly reported as a powerful producer of rhamnolipid biosurfactants as well as hydrolytic enzymes. In this study, the effects of four fermentation factors were evaluated using response surface methodology and experiments were performed in accordance with a four-factor and five-level central composite experimental design. The investigated factors were: fermentation temperature, time of fermentation, concentration of sunflower oil and concentration of Tween® 80. The most important finding was that regression coefficients of the highest values were those that describe interactions between factors and that they differ for lipase and rhamnolipid production, which were both investigated in this study. Production of both metabolites was optimized and response equations were obtained, making it possible to predict rhamnolipid concentration or lipase activity from known values of the four factors. The highest achieved rhamnolipid concentration and lipase activity were 138 mg dm-3 (sunflower oil concentration: 0.8%, Tween® 80 concentration: 0.05%, temperature: 30 °C and fermentation time: 72 h) and 11111 IU dm-3 (sunflower concentration: of 0.4%, Tween® 80 concentration: 0.05%, temperature: 30 °C and fermentation time: 120 h), respectively.Pseudomonas aeruginosa san-ai, izolovan je iz izrazito alkalne emulzije koja je korišćena kao mazivo u industriji pri obradi metala. Sposobnost da preživi u visoko alkalnoj sredini (pH 10) učinila je ovaj mikroorganizam veoma interesantnim za istraživanje, budući da je za preživljavanje u tako ekstremnim uslovima neophodno da mikroorganizam proizvodi enzime specifičnih karakteristika. Prethodna istraživanja su pokazala da ovaj ekstremofilni mikroorganizam ekstracelularno produkuje hidrolitičke enzime, koji zbog izuzetno atraktivnih karakteristika imaju potencijal za primenu u nizu biotehnoloških postupaka. Ipak, iako je pokazano da ovaj atraktivni soj produkuje industrijski veoma interesantne biomolekule (proteaze i lipaze), produkcija ramnolipida, jedinjenja čija oblast primene svakodnevno raste, pomoću ovog soja je malo ispitana. Ramnolipidi su amfifilna jedinjenja, koja se sastoje iz hidrofilne šećerne komponente i hidrofobne komponente koju čine β-hidroksi masne kiseline. Spadaju u grupu mikrobioloških surfaktanata ili biosurfaktanata, koji bi trebalo u budućnosti da se koriste kao zamena za sintetičke surfaktante koji nisu biodegradabilni i kao takvi predstavljaju opasnost za životnu sredinu. Sve veće interesovanje za industrijsku primenu ramnolipida, dovelo je do potrebe za optimizacijom njihove proizvodnje. Cilj ovog rada bila je optimizacija produkcije ramnolipida kao i lipaze pomoću Pseudomonas aeruginosa san-ai. Ispitan je uticaj četri fermentaciona faktora: koncentracije suncokretovog ulja u intervalu: 0,2-1,0 % (w/v), Tween® 80 u intervalu: 0-0,2 % (v/v), temperature: 20­60 °C i vremena trajanja fermentacije: 48-144 h. Uticaj fermentacionih faktora na prinos navedenih metabolita ispitan je pomoću centralnog kompozitnog rotatabilnog eksperimentalnog plana, na pet nivoa vrednosti ispitivanih faktora. Analizom dobijenih regresionih koeficijenata ustanovljeno je da su vrlo izražena interaktivna dejstva nekoliko parova faktora. Kod produkcije ramnolipida, najveća je vrednost koeficijenta koji opisuje negativnu interakciju između koncentracije suncokretovog ulja i temperature, a kao bitne pokazale su se i pozitivna interakcija između koncentracije Tween® 80 i temperature, kao i negativna interakcija između koncentracija suncokretovog ulja i Tween® 80. Interesantno je da su se kod produkcije lipaze kao značajni faktori pokazali samo temperatura i vreme fermentacije. Najveći prinos ramnolipida, 138 mg dm-3, postignut je pri niskoj koncentraciji Tween® 80 (0,05 %) i visokoj koncentraciji ulja (0,8 %) na 30 °C posle 72 h, dok je najveća lipolitička aktivnost, 11111 IU dm-3, ostvarena pri istoj koncentraciji Tween® 80 (0,05 %) i istoj temperaturi od 30 °C, nešto nižoj koncentraciji suncokretovog ulja (0,4 %) i dužem vremenu fermentacije od 120 h

Uticaj uslova fermentacije na produkciju lipaze iz 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 ( gt 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). .U radu je ispitana mogućnost proizvodnje lipaze pomoću divljeg soja kvasca izolovanog iz pokvarenog sojinog ulja i identifikovanog kao Candida utilis. Ovaj mikroorganizam je po- kazao malu lipolitičku aktivnost kada je uzgajan u 2 % sladnom bujonu kao univerzalnom kvaščevom medijumu (oko 4 IU dm-3). Ispitani su uticaji različitih faktora kao što su sastav hranljive podloge i dodatak određenih induktora i stimulatora produkcije lipaza na povećanje lipolitičke aktivnosti. Oleinska i kaprilna kiselina su se pokazale kao najefikasniji induktori produkcije lipaze. U malim koncentracijama (do 0,5 %) kaprilna kiselina je pokazala veći uticaj na proizvodnju lipaze od oleinske kiseline, da bi pri većim koncentracijama imala toksičan uticaj na ćelijski rast. Proces je optimizovan i sa aspekta izvora azota. Najveća lipolitička aktivnost od 284 IU dm-3 ostvarena je nakon 5 dana fermentacije u optimizovanoj podlozi koja je sadržala oleinsku kiselinu i hidrolizat kazeina kao izvore ugljenika i azota i Tween 80® kao stimulator produkcije enzima. Pokazano je da se pažljivim izborom sastava hranljive podloge može značajno povećati produkcija lipaza (više od 70 puta u poređenju sa početnom vrednošću dobijenom u neoptimizovanoj podlozi).

Enzymatic hydrolysis of egg-white proteins in a membrane reactor

The objective of this research was to improve antioxidative properties of egg-white proteins by means of enzymatic hydrolysis. For this purpose a continuous stirred tank reactor including polyethersulfone ultrafiltration module with a molecular weight cut-off of 10 kDa was employed. Several proteolytic enzymes have been tested in order to obtain best quality of peptide-based formulations intended for human consumption. Amongst proteases form Bacillus licheniformis (Alcalase), protease form Bacillus amyloliquefaciens (Neutrase) and protease from papaya latex (papain), the highest degree of hydrolysis (DH), as well as the best antioxidative properties of obtained hydrolysates, were achieved with Alcalase. Further optimization included finding of the optimal enzyme concentration and residence time. Results showed that the DH was directly dependent upon the enzyme concentration, while the permeate flow did not show any influence on the DH. The reactor was maintained in operation for 3 h at 50 °C and pH 8 with permeate flow of 2 cm3 min-1. Degree of hydrolysis reached steady value of 60 % after 75 min. Antioxidative properties were analyzed with DPPH method and confirmed with linear sweep voltammetry. Results undoubtedly show that the obtained products have improved antioxidative properties compared to untreated egg-white proteins

Immobilization of horseradish peroxidase onto kaolin by glutaraldehyde method and its application in decolorization of anthraquinone dye

The problem of environmental pollution becomes more worrisome day by day, primarily due to the large amounts of wastewater contaminated with various harmful organic compounds, discharged untreated or partially clean into the environment. Feasibility of use of horseradish peroxidase (Amoracia rusticana) in the synthetic dyes decolorization was approved by many researchers. Among a number of supports used for the immobilization, it was found that natural clay, kaolin, has excellent features which are a precondition for obtaining biocatalysts with the excellent performances. For this reason, a horseradish peroxidase was immobilized onto kaolin using glutaraldehyde as a cross-linking agent. Obtained biocatalyst was applied in the decolorization of anthraquinone dye C.I. acid violet 109. Under determined optimal conditions (pH 4.0, hydrogen peroxide concentration 0.6 mM, dye concentration 30 mg L-1, temperature 24 degrees C) around 76% of dye decolorization was achieved. Reusability study showed that resulting biocatalyst was possible to apply four times in the desired reaction with relatively high decolorization percentage