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..

Electrochemically synthesized polyaniline as support for lipase immobilization

Electrochemical synthesis of polyaniline support for enzyme immobilization provides easier control over the properties of obtained polymer and reduced risk of biocatalyst inactivation with residues of toxic compounds. In the present study, immobilization of lipase from Candida rugosa on electrochemically synthesized PANI (activated with glutaraldehyde) resulted with high lipase loadings up to 93.7 mg of proteins per gram of dry support. The activation of support and immobilization were optimized, with respect to activity yield. The optimum concentration of glutaraldehyde was 2% (w/v) and optimum concentration of enzyme was 4 mg ml−1. Modification of enzyme surface with carbodiimide and ethylenediamine was performed in order to increase concentration of amino groups. Aminated lipase exhibited higher specific activity (52%) and thermal stability (3 times) after immobilization, compared with non-modified lipase. Also, reusability of immobilized enzyme was significantly increased with amination, especially if immobilization was performed at pH 10, so in such a way obtained derivative retained 91% of activity after 15 reaction cycles

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

ULTRASOUND INDUCED FUNCTIONALIZATION OF SOY PROTEIN CONCENTRATE

Processing conditions for the fabrication of soy protein concentrates (SPCs) have a profound impact on the tightly packed, globular structure of soy proteins, which is reflected in the weakening of structural and functional properties, limiting their use in food systems. Many scholars have investigated the modification of soy protein, but this is the first time that high-intensity ultrasound technology has been used to address its limitations through improvement of the physicochemical properties of SPC. Therefore, the aim of this study was to develop an ultrasound-based method capable of producing SPC with improved functional properties making it a multifunctional ingredient for food systems intended for human consumption. The effects of high-intensity ultrasonication (20 kHz; 30% for 0.5; 2; 5 or 10 min) on the solubility, emulsifying properties, hydrophobicity, oil and water binding capacities and color of commercially available SPC were investigated. Ultrasonic cavitation induced the restructuring of SPC, which was confirmed by significant changes in functional and structural properties. After ultrasonic treatment (30% amplitude for 5 min), the most significant shifts in solubility were observed. The emulsion fabricated with this restructured SPC was firm, stable, without perceptible phase separation, with emulsifying activity and emulsion stability of 1024.4 ± 10.6 m2/g and 836.3 ± 12.2 h, correspondingly. Ultrasonic treatment of 30% amplitude for 2 min enabled SPC with best oil (3.26 ± 0.4 goil/gprotein) and water binding capacity (5.04 ± 0.9 gwater/gprotein). Furthermore, the results additionally revealed that with the increase in sonication time the surface hydrophobicity of SPC increased first and then decreased. The value of a* and b* decreased significantly with the ultrasonic treatment time increment, while lengthened ultrasonic cavitation increased the L* value. In conclusion, the functional and structural improvement of SPC endorsed the adequacy of ultrasonic cavitation in SPC modification