Enzymatic synthesis of vitamin B6 precursor

3-Cyano-4-ethoxymethyl-6-methyl-2-pyridone is an important precursor in the synthesis of vitamin B6, obtained in the addition reaction between 2-cyanoacetamide and 1-ethoxy-2,4-pentanedione catalyzed by lipase from Candida rugosa (triacylglycerol ester hydrolases, EC 3.1.1.3). This work shows new experimental data and mathematical modeling of lipase catalyzed synthesis of 3-cyano-4-ethoxymethyl-6-methyl-2-pyridone, starting from 1-ethoxy-2,4-pentanedione and 2-cyanoacetamide. Kinetic measurements were done at 50 oC with enzyme concentration of 1.2 % w/v. Experimental results were fitted with two kinetic models: the ordered bi-ter and ping-pong bi-ter model, and the initial rates of the reaction were found to correlate best with a ping-pong bi-ter mechanism with inhibition by 2-cyanoacetamide. Obtained specificity constants indicated that lipase from C. rugosa had higher affinity towards 1-ethoxy-2,4-pentanedione and less bulky substrates. [Projekat Ministarstva nauke Republike Srbije, br. 172013, br. III 46010 and br. 172049

Immobilization of alginate-PAC on Sepabeads EC-HA support

Penicillin acylase (PAC) is an important industrial enzyme for the production of many β-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization was presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA, were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50 ºC for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2,32; 50,65 and 1,68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC

Immobilization of lipase on sepabeads and its application in pentyl octanoate synthesis in a low aqueous system

The object of the study was to investigate the process conditions relevant for the pentyl octanoate production with the lipase from Candida rugosa immobilized on Sepabeads EC-EP carrier. This is an epoxide-containing commercial polymethacrylic carrier with suitable characteristics for enzyme immobilization. The immobilized lipase suitable for pentyl octanoate synthesis has been prepared by a direct lipase binding to polymers via their epoxide groups. The enzymatic activity was determined by both hydrolysis of olive oil in an aqueous system and esterification of n-pentanol with octanoic acid in a low aqueous system. The influence of several important reaction parameters such as temperature, initial water content, initial substrate molar ratio, enzyme loading and time of adding of molecular sieves in the system is carefully analyzed by means of an experimental design. Production of the ester was optimized and an ester production response equation was obtained, making it possible to predict ester yields from known values of the five main factors. Almost complete conversion (>99%) of the substrate to ester could be realized, using lipase loading as low as 37 mg/g dry support and in a relatively short time (24 h) at 45ºC, when high initial substrate molar ratio of 2.2 is used

Optimization of esterification activity of lipase from Candida rugosa immobilized using microwave irradiation

Lipases are very efficient biocatalysts with wide application in synthesis of important ingredients of food, cosmetics and pharmaceutical products, due to their capacity to catalyze both, ester synthesis and ester hydrolysis. The preparation of stable and active immobilized derivatives of lipases is necessity for their application in industrial enzymatic processes. In this work, the optimization of lipase from C. rugosa immobilization by microwave irradiation was performed, since it was previously reported that immobilization process can be drastically accelerated by means of microwave irradiation, even resulting with slight increase of lipase activity. Eupergit®, commercial support with active epoxy groups, was used as immobilization support. In first stage of our study, the immobilization time and ionic strength of immobilization buffer were optimized. It was found out that the highest immobilized activity can be achieved at high ionic strengths (1 M buffer) after 3 min, while further increase of immobilization time led to decrease of lipase activity. Then, the immobilized derivative obtained at optimum conditions was applied in synthesis of amyl isobutyrate in organic solvent. Key reaction factors (temperature, water concentration, immobilized lipase concentration, and substrate molar ratio) were optimized using response surface methodology. The substrate conversion higher above 85% was achieved in our study. The statistical analysis revealed that each of analyzed factors had significant effect on yield of ester, with initial enzyme concentration and substrate molar ratio being the most prominent factors. The second-order regression model that describes the effect of all four factors on substrate conversion was established. The optimum values of factors were: temperature 50ºC, initial immobilized enzyme concentration 220 mg ml-1, added water concentration 0.1% (v/v), and molar ratio acid/alcohol 2.5

Enzymatic synthesis and application of fatty acid ascorbyl esters

Fatty acid ascorbyl esters are liposoluble substances that possess good antioxidative properties. These compounds could be synthesized by using various acyl donors for acylation of vitamin C in reaction catalyzed by chemical means or lipases. Enzymatic process is preferred since it is regioselective, performed under mild reaction conditions, with the obtained product being environmentally friendly. Polar organic solvents, ionic liquids, and supercritical fluids has been successfully used as a reaction medium, since commonly used solvents with high Log P values are inapplicable due to ascorbic acid high polarity. Acylation of vitamin C using fatty acids, their methyl-, ethyl-, and vinyl esters, as well as triglycerides has been performed, whereas application of the activated acyl donors enabled higher molar conversions. In each case, majority of authors reported that using excessive amount of the acyl donor had positive effect on yield of product. Furthermore, several strategies have been employed for shifting the equilibrium towards the product by water content control. These include adjusting the initial water activity by pre-equilibration of reaction mixture, enzyme preparation with water vapor of saturated salt solutions, and the removal of formed water by the addition of molecular sieves or salt hydrate pairs. The aim of this article is to provide a brief overview of the procedures described so far for the lipase-catalyzed synthesis of fatty acid ascorbyl esters with emphasis on the potential application in food, cosmetics, and pharmaceutics. Furthermore, it has been pointed out that the main obstacles for process commercialization are long reaction times, lack of adequate purification methods, and high costs of lipases. Thus, future challenges in this area are testing new catalysts, developing continuous processes for esters production, finding cheaper acyl donors and reaction mediums, as well as identifying standard procedures for purification of products which will not require consumption of large amounts of non-biocompatible organic solvents

ANTIOXIDANT and ANTIMICROBIAL CAPACITY of ENCAPSULATED THYME ESSENTIAL OIL in ALGINATE and SOY PROTEIN-BASED CARRIERS

The aim of this study was to develop a stable hydrogel carrier system for thyme essential oil (TEO) that could protect its sensitive polyphenol compounds. The impact of wall material (soy protein and alginate) on encapsulation efficiency and thymol release in simulated gastrointestinal conditions, was investigated. The release of thymol was ~ 80 % and 20 % in simulated gastric and pancreatic solutions, respectively. Thyme essential oil plays an important role as an antimicrobial and antioxidant agent. Results indicated that encapsulated TEO inside the hydrogel matrix exhibited antioxidant activity demonstrated by CUPRAC and ABTS analysis, even after thermal treatment of the beads, indicating the metal chelate effect as dominant. In vitro antimicrobial activity of encapsulated TEO has been studied against several pathogenic microorganisms such as Escherichia coli, Staphylococcus aureus, Bacillus cereus and Candida albicans. Beads coded as Ca-A1.5/SP1.5 showed anti-Candida albicans activity, while modified bead formulations Ca-A1.5/SP1.5* and Ca-A1.5/SP0.25** showed bactericidal activity against Escherichia coli and Staphylococcus aureus

Ultrasound-emerging technology for valorization of pumpkin leaf biomass: impact of sonication parameters on protein recovery, structure, functionalities, and bioactivities

The potential of food biodiversity and its production streams to generate additional features for isolating high-value products has been underutilized. Even so, the recovery of valuable nutritional compounds like proteins from waste streams and by-products has been identified as a key strategy for enhancing production sustainability in order to open up new market potential. In accordance with those facts, the primary aim of this research was to utilize a relatively novel extraction technique, high-intensity ultrasound, in order to achieve a reduction in time, energy, and extraction solvent consumption while at the same time improving the extraction yield and nutritional value of extracted leaf proteins. For this purpose, an ultrasound probe system with a low frequency (20±0.2 kHz) was utilized to extract the white proteins from the pumpkin leaf biomass, and the effects of different sonication amplitudes (20, 30, 40, 50, 60 and 70%) and duration periods (0, 1, 3, 5, 7.5, 10, 12.5, 15, 17.5, and 20 min) on the yield, solubility and emulsifying qualities, antioxidant properties, and structural characteristics of proteins were studied. In the functional properties evaluated, leaf proteins isolated using ultrasound outperformed those extracted using the conventional extraction method, maceration. High-intensity ultrasound resulted in a slight but gradual decline in solubility with an increase in amplitude, but a significant increase in solubility in an acidic environment was observed with the decrease of cavitation periods. Ultrasound-extracted proteins exhibit nearly 12, 1.5, and 3-fold greater solubility compared to the maceration-extracted sample at pH 3, 4, and 5, respectively. The emulsifying activity diminishes with increasing sonication amplitude and duration but increases in emulsifying stability as sonication periods are extended. The ultrasound extraction provided pumpkin proteins with high radical scavenging activities (i.e., good electron donors) and chelating activity, with half maximal inhibitory concentrations (IC50) in the range of 0.9 to 1.5 mg/ml, and 0.3 to 0.6, respectively, especially at 20 and 40% amplitude. Raman spectroscopy, surface charge, surface hydrophobicity, and sulfhydryl group contents were employed to characterize the structural changes brought on by ultrasound cavitation, and the achieved changes were more influenced by the treatment periods and amplitudes applied. The experimental findings show that the use of ultrasound-emerging technology for protein extraction can significantly increase the yield of pumpkin leaf protein by up to 70%. The utilization of leaf proteins in food products and dietary supplements can be augmented by combining the ultrasound periods and amplitudes to create high-value samples with better capabilities

Encapsulation of α-lipoic acid intochitosan and alginate/gelatin hydrogel microparticles and its in vitro antioxidant activity

Alpha-lipoic acidis an organosulphur compound well-known for its therapeutic potential and antioxidant properties. However, the effective use of α-lipoic acid depends on biological plasma half-life and its preserving stability, which could be improved by encapsulation. In this study, α-lipoic acid was incorporated into chitosan microparticles obtained by reverse emulsion crosslinking technique, as well as into microparticles of alginate/gelatin crosslinked with zinc ions. Encapsulation of α-lipoic acid in both cases was carried out by swelling of synthesized dried microparticles by their dipping in a solution of the active substance under strictly controlled conditions. Encapsulation efficiency of α-lipoic acid obtained in this study was up to 53.9 %. The structural interaction of α-lipoic acid with the carriers was revealed by Fourier transform infrared spectroscopy. In vitro released studies showed that controlled release of α-lipoic acid was achieved through its encapsulation into chitosan microparticles. The results of in vitro antioxidative activity assays of released α-lipoic acid indicated that antioxidant activity was preserved at a satisfactory level. These obtained results suggested that chitosan microparticles could be suitable for modeling the controlled release of α-lipoic acid. [Projekat Ministartsva nauke Republike Srbije, br. III 46010 i br. III46001

Production of Antioxidant Egg White Hydrolysates in a Continuous Stirred Tank Enzyme Reactor Coupled with Membrane Separation Unit

The objective of this research was to design an efficient continuously operated membrane reactor with a separation unit for egg white protein (EWP) hydrolysis and production of hydrolysates with improved antioxidant properties. For this purpose, a mechanically stirred tank reactor coupled with the polyethersulfone ultrafiltration module with a molecular weight cutoff of 10 kDa was employed. Several proteolytic enzymes have been tested in order to obtain the best quality of peptide-based formulations intended for human consumption. Among protease from Bacillus licheniformis (Alcalase), protease from Bacillus amyloliquefaciens (Neutrase), and protease from papaya latex (papain), the highest degree of hydrolysis (DH), as well as the best antioxidant properties of obtained hydrolysates, was achieved with Alcalase. The effects of operating variables such as enzyme/substrate ([E]/[S]) ratio, impeller speed, and permeate flow rate were further studied using response surface methodology (RSM) and Box-Behnken experimental design. Results obtained in RSM analysis confirmed that over the studied range [E]/[S] ratio, impeller speed and permeate flow rate had the significant effect on the DH and reactor capacity. The effects of different impeller geometries were also studied and four-bladed propeller stirrer enabled the highest DH. Antioxidant properties were analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), by the 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical scavenging activity, and by the linear voltammetry methods. Results show that the use of Alcalase in the membrane reactor system is of potential interest for the EWP hydrolysis and obtaining value-added egg products

Design and characterization of alcalase-chitosan conjugates as potential biocatalysts

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6 IU/mg protein and 340.2 mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85 +/- 0.967% after 180 min and five successive reuses obtained under real conditions (50 A degrees C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process