Design of a polyaniline based biosensor electrode for glucose: A comparative study of two immobilized systems

The present study compares the results of two different methods employed for preparation of polyaniline based glucose biosensor with respect to enzyme loading, biosensing efficiency and potential stability. Kinetic analysis of the potentiometric data for two enzyme immobilized electrode systems show that the GOx/PANI electrode is suitable for assaying samples with low analyte concentrations, whereas the GOx/m-ABA/PANI electrode system exhibits a better potential stability. It may therefore be possible to achieve high level of biosensing efficiency by chemical modeling and synthesis combined with careful selection of the immobilization method

Enzimska proizvodnja estara fenolnih kiselina

As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring of cinnamic acid caused slight decline in achieved conversions. Further, the introduction of electron donating groups in para position led to serious decline in achieved conversions. It was concluded that this inhibition was consequence of both electron and steric effects, with electron effect being the dominant one...Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elektron donorskih grupa u para položaj prouzrokovalo drastičan pad u ostvarenim prinosima. Utvrđeno je da je elektronski efekat inhibicije dominantan u odnosu na sterni..

Enzymatic synthesis of phenolic acid esters.

Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elektron donorskih grupa u para položaj prouzrokovalo drastičan pad u ostvarenim prinosima. Utvrđeno je da je elektronski efekat inhibicije dominantan u odnosu na sterni...As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring of cinnamic acid caused slight decline in achieved conversions. Further, the introduction of electron donating groups in para position led to serious decline in achieved conversions. It was concluded that this inhibition was consequence of both electron and steric effects, with electron effect being the dominant one..

Enzymatic synthesis of phenolic acid esters.

Fenolne kiseline su sekundarni metaboliti biljaka i kao takve čine deo svakodnevne čovekove ishrane. Poznato je da ova jedinjenja poseduju niz fizioloških svojstava koja ih čine atraktivnim u naučnom svetu, među kojima je antioksidativna aktivnost najbolje ispitana. Ovi prirodni antioksidansi još više dobijaju na značaju zbog indikacija da razgradnjom komercijalnih sintetskih antioksidanasa nastaju kancerogena jedinjenja. Međutim, zbog hidrofilne prirode njihova efikasnost u nepolarnim sredinama je ograničena, a lipofilizacija esterifikacijom je jedan od načina da se ovaj problem prevaziđe. Enzimska sinteza fenolnih estara predstavlja ekološki prihvatljiv način sinteze estara, a sami proizvodi dobijeni na ovaj način spadaju u zelene proizvode, što je od izuzetnog značaja kod proizvodnje namirnica namenjenih za ljudsku upotrebu. Kako bi enzimska sinteza estara bila konkurentna hemijskoj, odnosno kako bi se sintetisale velike količine estra uz male utroške enzima, neophodno je izvršiti optimizaciju ključnih reakcionih parametara (koncentracije enzima i supstrata, temperature, intenziteta mešanja i reakcionog vremena), što je bio i cilj ove disertacije. Pre svega pristupilo se pronalaženju adekvatnog biokatalizatora, koji bi sintetisao fenolne estre u visokom prinosu. Ispitani su komercijalni imobilisani preparati lipaze B iz Candida antarctica i lipaze iz Rhizomucor miehei, Novozyme 435 i Lipozyme RM IM, redom, kao i nekoliko lipaza izolovanih i prečišćenih na Katedri za Biohemijsko inženjerstvo i biotehnologiju. Od ispitanih enzima, Novozyme 435 se pokazao kao superioran u sintezi svih sintetisanih estara. U nastavku je pokazano da specifičnost ovog preparata zavisi od strukture fenolne kiseline i da najveći afinitet pokazuje prema fenolnim kiselinama sa zasićenim bočnim nizom, p-hidroksifenilpropionskom i dihidrokafenom kiselinom. Dvostruka veza konjugovana sa aromatičnim prstenom kod cimetne kiseline, dovela je do blagog pada prinosa reakcije, dok je uvođenje elektron donorskih grupa u para položaj prouzrokovalo drastičan pad u ostvarenim prinosima. Utvrđeno je da je elektronski efekat inhibicije dominantan u odnosu na sterni...As secondary plant metabolites phenolic acids are common components of daily human diet. It is well established that these compounds have several physiological activities, amongst which the antioxidant activity is the most studied one. Indications that degradation of commercial synthetic antioxidants leads to formation of toxic and carcinogenic compounds have made phenolic compounds even more attractive. Nevertheless, their hydrophilic nature limits their efficiency in non polar environments. One of the ways to overcome this obstacle is lipophilization of these compounds via esterification. Enzymatic synthesis seems to be eco-friendly way of producing lipophilic phenols, and the products obtained on this way are considered as green which is of utter importance for the products intended for human consumption. For enzymatic synthesis to prevail over the chemical synthesis, the process has to be efficient i.e. high ester yields should be produced using minimal amounts of enzyme. In order to achieve this it is necessary to optimize key process parameters (enzyme and substrate concentrations, temperature, mixing intensity and reaction time), that was set as the main goal of this thesis. The first aim was to find adequate biocatalyst which would synthesize phenolic esters with high yields. Commercial immobilized preparations of lipase B from Candida antarctica and lipase from Rhizomucor miehei, Novozyme 435 and Lipozyme RM IM, respectively, were tested along with several lipases isolated at the Department of Biochemical Engineering and Biotechnology, University of Belgrade. Amongst all tested enzymes, Novozyme 435 was proved as the superior biocatalyst for all synthesized esters. Further, it was shown that specificity of Novozyme 435 towards phenolic acids was highly dependent upon their structure. The highest conversions were achieved with p-hydroxyphenyl propionic and dihydrocaffeic acids, which both have saturated side chain. Double bond conjugated with aromatic ring of cinnamic acid caused slight decline in achieved conversions. Further, the introduction of electron donating groups in para position led to serious decline in achieved conversions. It was concluded that this inhibition was consequence of both electron and steric effects, with electron effect being the dominant one..

Synthesis of ethyl cinnamate catalyzed by lipase B from Candida antarctica

Cinnamic acid and its esters are widespread throughout plant kingdom and therefore, they are common components of our daily diet. Ethyl cinnamate has been approved by FDA for direct addition to food for human consumption. Enzyme synthesis is strongly preferred compared to chemically catalyzed esters synthesis when product quality is a main issue, as is the case for food production. The aim of this study was to examine possibilities for enzyme catalyzed synthesis of cinnamic acid esters and to optimize the synthesis of ethyl cinnamate in terms of selected parameters, including the type of the organic solvent and substrate initial molar ratio. All reactions were performed batchwise under pH and temperature control in vessels containing 5 mL of reaction medium, using cinnamic acid as limiting substrate (0.167 M). Each reaction mixture was supplemented with 75 mg of commercial immobilized lipase B from Candida antarctica, Novozyme 435. The antioxidant activity of obtained ester was measured by using chemical and electrochemical techniques. We have proven that ethyl cinnamate can be synthesized using lipase B from Candida antarctica with very high reaction yields in the simple bioreactor system.The best reaction yield (89%) was obtained in isooctane when substrate molar ratio of 1:3 was used. Both, DPPH assay and cyclic voltammetry measurement has shown that ethyl cinnamate has better antioxidant properties than cinnamic acid itself

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

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

Optimization of submerged fermentation conditions for gluten-degrading enzyme production using B. subtilis isolate

In modern times wheat gluten has drawn attention to many research groups. Wheat gluten represents one of the most widely used proteins in the food industry. It is a byproduct of the starch industry and has a higher percentage of protein content compared to other plant-based protein sources. In order to help reduce the allergenicity of wheat gluten, bacterial enzymes have been proven to have beneficial results in wheat gluten treatment. In search for an extracellular peptidase producing strain we have tested Bacillus subtilis TMF-1 isolate, which has previously been proven to have several enzyme activities. B. subtilis TMF-1 isolate has a food grade status, making it safe for application in the food industry. Thus, the aim of this research was to examine the possibility of utilizing mentioned strain in terms of glutendegrading enzyme production. Tested strain was first streaked onto several agar plates in order to detect extracellular peptidase activity. Bacterial isolate has then been sequentially transferred to the same growth medium several times. Conditions varied for the submerged fermentation in 25 mL flasks were pH value of fermentation broth, concentration of gluten powder (0 - 10 g/L) in fermentation broth and concentration of peptone (0 - 1 g/L). Shaking flasks containing the fermentation broth with the bacterial strain were kept for 48 h at 37 0C. The results obtained show that the isolate has the possibility of thriving in low acidic to neutral pH values of the fermentation broth. Varied gluten concentrations showed that even 1 g/L of gluten powder was sufficient for the bacterial strain to manifest extracellular proteolytic enzyme activity. Peptone concentrations were also varied, but even the minimal presence of peptone has proven beneficial for bacterial growth and proteolytic activity. This research show that the B. subtilis TMF-1 isolate has proteolytic activity specific for wheat gluten as substrate and that it may be used in further research in order to utilize its enzymatic production abilities for lowering wheat gluten allergenicity

Improvement of antioxidant properties of egg white protein enzymatic hydrolysates by membrane ultrafiltration

The production of bioactive peptides from egg white proteins (EWPs) and their separation are emerging areas with many new applications. The objective of this study was to compare antioxidant activity of three distinct EWP hydrolysates and their peptide fractions prepared by membrane ultrafiltration using membranes with 30, 10 and 1 kDa molecular weight cut-off. The hydrolysates were obtained by thermal and ultrasound pretreated EWPs hydrolyzed with a bacterial protease, Alcalase. It appeared that the pretreatment significantly affected peptide profiles and antioxidant activity of the hydrolysates measured by ABTS, DPPH and FRAP methods. The hydrolysate prepared using alcalase and ultrasound pretreatment at 40 kHz - 15 min has shown to be the most effective in scavenging both DPPH and ABTS radicals (28.10 +/- 1.38 and 79.44 +/- 2.31%, respectively). It has been noticed that this hydrolysate had a nutritionally more adequate peptide profile than the other hydrolysates with a much lower amount of peptides lt 1 kDa (11.19 +/- 0.53%) and the greatest content of the peptide fraction in the molecular weight (MW) range of 1-10 kDa (28.80 +/- 0.07%). This peptide fraction has shown the highest DPPH and ABTS antioxidant activity compared to all other fractions having a potential to be used as a functional food ingredient