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Within this study, the investigation of potential selective immobilization of fructosyltransferase (FTase) derived from Aspergillus aculeatus (from commercial enzyme preparation Pectinex® Ultra SP-L) onto the polystyrenic macroporous resin Purolite® A109 and its further utilization for the synthesis of a highly valuable group of prebiotic compounds named fructo-oligosaccharides (FOS), was performed. The obtained results suggest that partial purification of fructosyltransferase from complex enzyme mixture with predominant pectinase activity (ratio of activity immobilization yields around 20.0) occurred by performing a simple immobilization protocol, provided that immobilization lasted for 5 h using an enzyme concentration of 50 mg g−1 of support under slightly acidic conditions (pH 6.0). Immobilized preparation was thereafter successfully utilized for FOS synthesis (56–59 % FOS of total sugars) in both a batch and air-lift reactor system. Operational stability study proved the exceptionally high potential of immobilized preparation for reproducible and cost-effective application, since 90 % of initial activity was preserved after ten consecutive reaction cycles. Thereafter, a prospectus for continuous production of FOS in the air-lift reactor was assayed. Achieved yields, 53 % FOS of total sugars for at least 9 h, indicated the great suitability of the obtained immobilized preparation for application in large-scale systems.

A new micro-kinetic model is proposed for galacto-oligosaccharide (GOS) synthesis with β-galactosidase from Aspergillus oryzae. Several kinetic models with similar reaction mechanisms, i.e., steps and kinetic constants, have been investigated covering a range of initial lactose (20-40 wt %) and enzyme concentrations (0.5-2 mg/mL). The kinetic parameters were estimated simultaneously for all experimental results using a hybrid genetic algorithm. A detailed analysis of the effect of process parameters on the concentrations of lactose and GOS is performed. The proposed model has a very good quantitative fit of the experimental data, with an average error of 9.34%. More importantly, qualitative trends for the change in the concentration of lactose and GOS are predicted excellently, especially if one takes into account the variation of both concentrations of enzyme and lactose in a range wider than reported in the literature to date. Also, the analysis between the two comparative reactions of transglycosylation and lactose hydrolysis confirmed the positive effect of increasing lactose concentration on the production of GOS. On the other hand, a higher enzyme concentration resulted in a faster production of GOS but with more intensive decomposition at longer reaction times.

A new micro-kinetic model of the enzyme-catalyzed synthesis of fructo-oligosaccharides (FOS) was developed. A commercial enzyme mixture Pectinex® Ultra SP-L derived from Aspergillus aculeatus was used. A variety of initial enzyme concentrations (1–5 vol%) and sucrose concentrations (400–600 g/L) were experimentally investigated and included in kinetic modeling. Several variations of kinetic mechanisms and corresponding models have been examined. A hybrid genetic algorithm was used to predict the kinetic parameters simultaneously for all experimental data. The best fitting model has been adopted, and with an average error of 13.34%, it describes the experimental data very well. The influence of initial concentrations on the conversion of sucrose and production of FOS is being carefully investigated. It was shown that the initial sucrose concentration significantly affects the highest level of FOS concentration, but the enzyme concentration controls the time at which maximum is reached as well as the rate of FOS decomposition.

In this study, a heterofunctional carrier was obtained by modification of a macroporous polystyrene ion exchange resin with primary amino groups (Purolite® A109) with epichlorohydrin, in view of achieving covalent immobilization of fructosyltransferase (FTase) from a complex enzyme mixture, Pectinex® Ultra SP-L, that is responsible for the synthesis of functionally active fructo-oligosaccharides (FOS). A two-step immobilization protocol, comprising the physical adsorption of FTase performed at pH 4 and subsequent buffer exchange to promote the establishment of covalent bonds at pH 9, was proposed for obtaining a highly active immobilized preparation (243 IU g−1 of support). Additionally, this protocol provided development of a preparation with 20 times more prominent expressed activity of FTase compared to the commercial preparation with predominant pectinase activity. The obtained immobilized preparation was further tested in batch and air-lift reactor systems for FOS synthesis, yielding 52.8% and 54.7% of FOS in total carbohydrates, respectively. Finally, the continuous production of FOS in the air-lift reactor was established for 7 days, with an average FOS yield of 52.5%. Accordingly, it is demonstrated that the immobilization process enabled the development of preparations with exceptional potential for industrial implementation.

The novel lignin-based microspheres (LMS-DEGDMA) were produced through a two-step modification procedure consisting of kraft lignin (KL) modification with acrylic acid synthesized from fructose; and suspension and copolymerization of acrylic acid modified KL (KL-A) with diethylene glycol dimethacrylate (DEGDMA). The applied procedure provided LMS-DEGDMA microspheres in the size range of 50-90 μm with specific surface area of 39 m2/g, porosity ϵp 62, pore diameters of 4-20 nm and total pore volume of 0.14 cm3/g. Immobilization of laccase from Myceliophthora thermophilia expressed in Aspergillus oryzae (Novozyme® 51003) on LMS-DEGDMA was explored. By optimization of the immobilization process, it was demonstrated that laccase immobilization on LMS-DEGDMA exhibited the best results (protein immobilization yield of 70 %, activity yield of 27 %, and catalytic activity of 262 IU/g of support) when the immobilization was carried out at pH 5.0 and 100 mg of offered protein per g support during 1 h. Additionally, the LMS-DEGDMA-laccase preparation showed efficiency (>80 %, 5-7 cycles) in decolorization of anthraquinone dyes (Lanaset® violet B, Lanaset® blue 2 R and C.I. Acid Green 40) and their mixture. In case of Lanaset® violet B, LMS-DEGDMA improved laccase's kinetics characteristics (90 % over 24 h) along with operational stability (> 63 % catalytic activity, 7 cycles).

In this study, the suitability of fumed silica nanoparticles (FNS) and its derivatives (amino-modified FNS (AFNS), cyanuric chloride-activated AFNS (CCAFNS) and epoxy-modified FNS (GFNS)), for covalent immobilisation of two commercial protease preparations Alcalase(R) and Flavourzyme(R) was investigated. The highest hydrolytic activities of immobilised preparations were 25 IU g(-1) support (Alcalase-GFNS) and 2.95 IU g(-1) support (Flavourzyme-CCAFNS). Furthermore, the immobilised preparations showed 43% and 20% of initial specific activities of commercial protease preparations, respectively. Flavourzyme-CCAFNS also exhibited the highest exopeptidase activity of 22.83 L-pNAU g(-1) support. Finally, these two nanobiocatalysts were successfully applied for hydrolysis of sunflower meal protein isolate (SMPI), providing two times higher hydrolysis yields in comparison to free enzymes, justifying the applied immobilisation process. Namely, the highest hydrolysis yield (30%) was gained by the sequential hydrolysis with Alcalase-GFNS and Flavourzyme-CCAFNS, which resulted in the formation of small hydrophobic and hydrophilic peptides, lt = 5 kDa, confirmed by HPLC analysis and electrophoretic separation.

Glycoderivatives are an important class of molecules with enormous relevance in numerous biological phenomena; therefore, they have a key role in the learning, understanding, and assessment of different diseases. Nanotechnology, and in particular the design of new nanomaterials, is one of the areas of greatest interest today. In this case, graphene nanomaterials represent very interesting platforms for studying glycosystems, glyconanomaterials that combine the biomolecular recognition and the characteristics of nanoscale objects in the development of early diagnosis systems, and efficient specific therapeutic modalities. In this mini-review, we discuss some results recently described in the literature on the conjugation of graphene materials and carbohydrates through the selective interaction of glycoenzymes in graphene to create new materials with biosensing applications, the development and application of sugar-graphene composites, and finally biosystems combining the properties of graphene with metallic nanoparticles and sugars for the creation of excellent glyconanomaterials as novel systems for the therapy or diagnosis of important diseases such as cancer or diabetes.

Recently, prebiotics are attracting plenty of attention in the field of skin care, since it is found that they are able to support the balance of beneficial and harmful microorganisms on the skin, and accordingly prevent several skin conditions associated with microbial imbalance. Topical application of prebiotics, although insufficiently investigated, holds great promise in improving skin health. The purpose of this research was to determine the prebiotic potential of galacto-oligosaccharides (GOS) for skin microbiota and suitability for incorporation in different topical formulations, and finally, provide insights into their diffusion properties.The prebiotic effect of GOS was evaluated through the influence on the growth of Staphylococcus epidermidis and Staphylococcus aureus, the most common resident and pathogenic bacterium of the skin microbiota, respectively. Also, with the future use of GOS in cosmetic products in mind, the diffusion of GOS molecules from two different topical formulations, hydrogel and oil-in-water (O/W) gel emulsion, was monitored employing Franz diffusion cell and two systems-with cellulose acetate membrane and transdermal diffusion test model, Strat-M® membrane. Course of fermentation and the amount of diffused GOS molecules were monitored using high-performance liquid chromatography (HPLC).The in vitro results revealed that GOS at a concentration of 5% (w/v) has a pronounced stimulatory effect on S. epidermidis, while simultaneously showing an inhibitory effect on S. aureus, both in nutrient broth and cosmetic formulations. GOS trisaccharide and tetrasaccharide diffusion coefficients from O/W gel emulsion were calculated to be 5.61·10When applied in adequate concentration, GOS has the potential to be used as a skin prebiotic. Novel GOS enriched formulations, Aristoflex® AVC-based hydrogel and Heliogel™-based O/W gel emulsion, provided efficient diffusion and delivery of prebiotic GOS molecules to the skin surface.Récemment, les prébiotiques attirent beaucoup d'attention dans le domaine des soins de la peau, car il a été constaté qu'ils sont capables de soutenir l'équilibre des micro-organismes bénéfiques et nocifs sur la peau et, par conséquent, de prévenir plusieurs affections cutanées associées à un déséquilibre microbien. L'application topique de prébiotiques, bien qu'insuffisamment étudiée, est très prometteuse pour améliorer la santé de la peau. Le but de cette recherche était de déterminer le potentiel prébiotique des galacto-oligosaccharides (GOS) pour le microbiote cutané et leur aptitude à être incorporés dans différentes formulations topiques, et enfin, de fournir des informations sur leurs propriétés de diffusion. MÉTHODES: L'effet prébiotique du GOS a été évalué à travers l'influence sur la croissance de Staphylococcus epidermidis et de Staphylococcus aureus, les bactéries résidentes et pathogènes les plus courantes du microbiote cutané, respectivement. De plus, en gardant à l'esprit l'utilisation future du GOS dans les produits cosmétiques, la diffusion des molécules de GOS à partir de deux formulations topiques différentes, l'hydrogel et l'émulsion de gel huile-dans-eau (H/E), a été surveillée à l'aide d'une cellule de diffusion de Franz et de deux systèmes - avec de la cellulose membrane en acétate et modèle de test de diffusion transdermique, membrane Strat-M®. Le cours de la fermentation et la quantité de molécules de GOS diffusées ont été surveillés en utilisant la chromatographie liquide à haute performance (HPLC).Les résultats in vitro ont révélé que le GOS à une concentration de 5% (p/v) a un effet stimulant prononcé sur S. epidermidis, tout en montrant simultanément un effet inhibiteur sur S. aureus, à la fois dans les bouillons nutritifs et les formulations cosmétiques. Les coefficients de diffusion GOS trisaccharide et tétrasaccharide de l'émulsion de gel H/E ont été calculés comme étant de 5,61·10Lorsqu'il est appliqué à une concentration adéquate, le GOS a le potentiel d'être utilisé comme prébiotique cutané. De nouvelles formulations enrichies en GOS, l'hydrogel à base d'Aristoflex® AVC et l'émulsion de gel H/E à base d'Heliogel™, ont permis une diffusion et une délivrance efficaces des molécules GOS prébiotiques à la surface de la peau.

In this study, immobilization of laccase from Trametes versicolor on eight Lifetech™ supports, with different characteristics (pore size, length of the spacer arm and functional groups), was studied and optimized for intended use in bioremediation for decolorization of industrial wastewaters. Out of six tested amino-functionalized supports, the most promising carrier was proved to be porous Lifetech™ ECR8309F with primary amino groups and a C2 spacer arm. Onto this support, laccase is attached by forming electrostatic interactions so that the most active preparation has shown the activity of 66876 U/g support. On the other hand, during immobilization of laccase on epoxy-functionalized Lifetech™ ECR8285F, via hydrophobic interactions and covalent bonding confirmed by a desorption assay, immobilization yield of 60 % and the activity of 118929 U/g were accomplished. Furthermore, immobilized enzyme on this support showed high capacity for decolorization of dyes (Lanaset® Violet B, Lanaset® Blue 2R, bromothymol blue and bromocresol green), by combination of both adsorption and enzyme degradation. Decolorization was in the range of 88 to 96 % after 4 h, with more than 80 % achieved after only 45 min. Also, this preparation demonstrated high operational stability during seven consecutive reuses in all examined dye reaction systems.[Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. 451-03-68/2020-14/200135 and Grant no. 451-03-68/2020-14/ 200287]

This work aims to evaluate the effect of cosmetic formulations on the skin permeation of phloridzin acetates, therefore in vitro transdermal delivery of these bioactive compounds across Strat-M membrane is investigated. The formulations tested are gel emulsion, oil in water emulsion (O/W emulsion), gel in oil emulsion, liquid crystal emulsion, and liposomes dispersed in gel. The release of esters from the O/W emulsion is diffusion-controlled (n = 0.5), while release kinetics from gel-based formulations depends on coupled diffusion and polymer relaxations effect (0.5 < n < 1). The highest values of effective diffusion coefficient are obtained after release from O/W emulsion (24.64 × 10−10 m2 min−1 for phloridzin acetate and 6.55 × 10−10 m2 min−1 for phloridzin diacetate). The lag phase lasting from 1 h (gel emulsion) to 5 h (liquid crystal emulsion) indicates that esters permeation is significantly affected by the formulation used. Formulations in which the water is continuous phase are the most appropriate carrier system for transdermal delivery of phloridzin esters. Additionally, liposomes are successfully applied as carrier systems for the controlled release of esters, although it should be noted that liposomes without cholesterol show remarkable stability (ζ = −43 mV) and homogeneity (polydispersity index = 0.198). Practical application: Phloridzin is a flavonoid glycoside that possesses many beneficial activities such as antioxidant, UV-protecting, and anti-inflammatory activity, so the use of this flavonoid and its derivatives in numerous cosmetic and pharmaceutical formulations seems very attractive. Results obtained in this study give a comprehensive insight into the release mechanism of phloridzin acetates from emulsions and liposomes, enable the selection of optimum cosmetic formulations depending on the application, and provide significant scientific contribution in the field of topical administration of bioactive flavonoid derivatives.

The obvious benefits of employing prebiotics as functional components in many foods and feed products have resulted in higher demand for their industrial production, necessitating the development of more efficient and cost-effective manufacturing procedures. As a result, the goal of this study was to synthesize confirmed prebiotics, namely fructo-oligosaccharides (FOS), using sucrose as a substrate, since it allows the synthesis of oligosaccharides with lower polymerization degree, and consequently, a more pronounced prebiotic effect. Due to its availability, low market price, and high stability under industrial conditions, a commercial enzymatic mixture, Pectinex® Ultra SP-L, is used as a source of enzyme - fructosyltransferase (FTase). By varying key experimental conditions such as pH, temperature, enzyme and substrate concentrations, as well as the duration of the process, the composition of the FOS mixture can be adjusted to fit the potential applications. It was found that by performing the reaction in an aqueous medium (pH 7), at a temperature of 50 °C using an enzyme concentration of 1% (v/v) and any sucrose concentration in the range of 200-700 g/L, it was possible to achieve maximum FOS yield of 60% of total carbohydrates within a 24 h. The produced syrup with a high content of FOS can be further used as an adequate food additive, or else, developed processes should be used for the transformation of various food products (such as juices, jams, fillings, candies, cakes, etc.) in which sucrose dominates, creating products with lower caloric and higher functional value.

Cellulases are enzymes which catalyse cellulose hydrolysis and are widely used in various industry branches. Lately, their application in treatment of different agroindustrial waste materials which could serve for fuel production is being extensively explored. In order to increase their stability and cost-effectiveness of their usage, application of their immobilized forms are preferred over free enzymes. Hereby, we tested eight different LifetechTM immobilization supports differing in polarity, porosity and functional groups as carriers for Asspergillus Niger cellulase immobilization. Most promising carrier was methacrylate based, with primary amino groups, C6 "space arm" and pores with diameter of 60-120 nm - LifetechTM ECR8409F. For this support, most important immobilization parameters were investigated and after 3 h at pH 6 with initial protein concentration of 23.3 mg/g support immobilized cellulase with 406 IU/g (with carboxymethyl cellulose as a substrate) was obtained. This preparation was successfully applied in the hydrolysis of lignocellulosic fraction of sunflower seed meal, which is widely available byproduct of sunflower seed meal fractionation for protein-rich fractions production. Initial reaction rates and yields of reducing sugars were unchanged comparing to free enzyme, indicating that there were no significant diffusion limitations for substrate to approach active sites of A. Niger cellulase molecules immobilized onto LifetechTM ECR8409F support. Celulaze su enzimi koji katalizuju hidrolizu celuloze i u širokoj su primeni u brojnim granama industrije. U poslednje vreme, mogućnost njihove primene u tretiranju različitih opadnih agroindustrijskih sirovina u cilju dobijanja biogoriva intenzivno se istražuje. Da bi se povećale stabilnost i ekonomičnost njihove primene, potrebno je umesto slobodnog enzima koristiti imobilisane forme. U ovom istraživanju osam LifetechTM nosača različitih polarnosti, poroznosti i funkcionalnih grupa. testirano je za imobilizaciju celulaze producenta Aspergillus Niger. Najpogodniji nosač bio je na bazi metakrilata, sa primarnim amino grupama, C6 "dugom nožicom" i porama prečnika 60-120 nm - LifetechTM ECR8409F. Za ovaj nosač određeni su najznačajniji uslovi imobilizacije i nakon 3 sata na pH 6, pri početnoj koncentraciji proteina od 23,3 mg/g nosača dobijena je imobilisana celulaza aktivnosti 406 IU/g (sa karboksi metil celulozom kao supstratom). Ovaj preparat uspešno je primenjen u reakciji hidrolize lignocelulozne frakcije suncokretove sačme, koja prestavlja nusproizvod frakcionisanja suncokretove sačme pri dobijanju frakcija bogatih proteinima. Početne brzine reakcije i prinosi redukujućih šećera bili su nepromenjeni u odnosu na slobodan enzim, ukazujući da nije bilo značajnog uticaja difuzionih limitacija pri prilasku supstrata aktivnim centrima molekula A. Niger celulaze imobilisane na LifetechTM ECR8409F nosač.

In this research, it has been demonstrated that amino-modified microspheres (A-LMS) based on bio-waste derived material, such as kraft lignin, have good prospects in usage as a support for enzyme immobilization, since active biocatalyst systems were prepared by immobilizing beta-galactosidase fromA. oryzaeand laccase fromM. thermophilaexpressed inA. oryzae(Novozym (R) 51003) onto A-LMS. Two types of A-LMS were investigated, with different emulsifier concentrations (5 wt% and 10 wt%), and microspheres produced using 5 wt% of emulsifier (A-LMS_5) showed adequate pore shape, size and distribution for enzyme attachment. The type of interactions formed between enzymes (beta-galactosidase and laccase) and A-LMS_5 microspheres demonstrated that beta-galactosidase is predominantly attachedviaelectrostatic interactions while attachment of laccase is equally governed by electrostatic and hydrophobic interactions. Furthermore, the A-LMS_5-beta-galactosidase exhibited specificity towards recognized prebiotics (galacto-oligosaccharides (GOS)) synthesis with 1.5-times higher GOS production than glucose production, while for environmental pollutant lindane degradation, the immobilized laccase preparation exhibited high activity with a minimum remaining lindane concentration of 22.4% after 6 days. Thus, this novel enzyme immobilization support A-LMS_5 has potential for use in green biotechnologies.

Solubility and stability of flavonoid glycosides, valuable natural constituents of cosmetics and pharmaceuticals, could be improved by lipase-catalyzed acylation. Focus of this study was on development of eco-friendly process for the production of flavonoid acetates. By using phloridzin as model compound and triacetin as acetyl donor and solvent, 100% conversion and high productivity (23.32 gl(-1) day(-1)) were accomplished. Complete conversions of two other glycosylated flavonoids, naringin and esculin, in solvent-free system were achieved, as well. Comprehensive kinetic mechanism based on two con-secutive mono-substrate reactions was established where first one represents formation of flavonoid monoacetate and within second reaction diacetate is being produced from monoacetate. Both steps were regarded as reversible Michaelis-Menten reactions without inhibition. Apparent kinetic parameters for two consecutive reactions (V-m constants for substrates and products and Km constants for forward and reverse reactions) were estimated for three examined acetyl acceptors and excellent fitting of experimental data (R-2 gt 0.97) was achieved. Obtained results showed that derived kinetic model could be applicable for solvent-free esterifications of different flavonoid glycosides. It was valid for entire transesterification course (72 h of reaction) which, combined with complete conversions and green character of synthesis, represents firm basis for further process development. (C) 2017 Elsevier B.V. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2974]

l -Ascorbyl oleate is recognized as efficient liposoluble antioxidant and biosurfactant which can be synthesized by using immobilized lipases as catalysts in organic media. However, development of economical production process is still a challenge, particularly in (semi)continuous systems. Hereby, we present application of novel immobilized lipase preparation, lipase B from Candida antarctica hydrophobically adsorbed onto styrene divinylbenzene based support, for the synthesis of l -ascorbyl oleate in batch bioreactor (BB) and fluidized bed bioreactor (FBB) with recirculation of reaction medium. Reaction kinetics was described by Ping Pong bi bi mechanism with low acyl donor inhibition. Considerably higher reaction rates (∼40%) were accomplished in FBB. Hydrodynamic study revealed that reaction is kinetically controlled and unhindered external and internal mass transfer was enabled in both systems. However, much higher shear stress occurred in BB (10.46 Pa) comparing to FBB (5 × 10 −2 Pa). After 12 consecutive reaction cycles lasting 24 h, 23.13 g of product per gram of biocatalyst was synthesized in a FBB. Within this study, usage of new immobilized enzyme in semicontinuous system enabled high reaction rates and satisfying operational stability in the synthesis of l -ascorbyl oleate, offering good prospect for further process development.

In order to enhance productivity and cost-effectiveness of current lipase-catalyzed processes of flavonoid esters synthesis, different LifeTech™ ECR carriers varying in functional groups, polarity and porosity were hereby screened as immobilization supports for free lipase B from Candida antarctica. The most prospective ones were examined in esterification of three flavonoid glycoside representatives - phloridzin, naringin and esculin using different acyl donors. Highly active preparations were obtained by using hydrophobic C18 (ECR 8806M), epoxy/butyl (ECR 8285F) and primary amino (ECR 8409F) functionalized resins. Interestingly, lipase immobilized via hydrophobic interactions and covalent bonds exhibited high catalytic activity in esterification reactions using oleic acid as well as natural oils as acyl donors, yielding more than 70 % and 50 % conversion degree, respectively. On the other hand, lipase immobilized onto amino-support by electrostatic interactions, which demonstrated the highest hydrolytic activity, showed poor efficiency in ester syntheses. Operational stability study proved exceptionally high potential of lipases immobilized on epoxy-support or hydrophobic support for reproducible and cost-effective industrial application, since 90 % of initial activity was preserved after six consecutive reaction cycles of oleyl ester synthesis, while within following nine reuses, only moderate activity loss occurred. Moreover, besides very good esterifying activity, lipase immobilized via hydrophobic interactions showed outstanding operational stability during ten reuses in reaction of phloridzin transesterification with coconut oil.

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae (Novozym 51003® laccase) on amino modified fumed nano-silica (AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003® laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity (13.1 IU·mg−1 proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching 68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.

Galacto-oligosaccharides (GOS) are important lactose-derived compounds, considered to be a prebiotics, based on abundant scientific evidence about their unique physical properties and physiological effects. This consequently allows their widespread application as supplement in food and feed industry. They are preferably produced by the enzymatic transgalactosylation action of β-galactosidase. However, this enzyme simultaneously performs its primary biological function of lactose hydrolysis, and it is of crucial importance to gain an insight into the influence of different reaction conditions, and provide favorization of transgalactosylation, particularly GOS synthesis reaction. In this study, the response surface methodology (RSM) was applied in terms of individual experimental factors effect estimation, their mutual interaction identification and finally, the determination of optimum conditions for highest GOS yield achievement. Having said that, it can be observed that the temperature and pH have no significant impact on the GOS yield, while on the other hand, the lactose concentration of 400 g/l, enzyme concentration of 13.5 g/l and reaction time of 13 min represent the optimum conditions for achieving the highest GOS yields. Galakto-oligosaharidi (GOS) obuhvataju raznorodnu grupu nesvarljivih ugljenih hidrata, derivata laktoze, koji se smatraju prebioticima zahvaljujući brojnim naučnim studijama u kojima su dokazana njihova izuzetna fizičko-hemijska, kao i fiziološka svojstva. Ova saznanja omogućila su njihovu široku primenu u prehrambenoj i farmaceutskoj industriji, kao i u industriji stočne hrane. Galakto-oligosaharidi se skoro isključivo proizvode enzimskom sintezom pomoću enzima β-galaktozidaze. Međutim, poznato je da ovaj enzim istovremeno obavlja i svoju osnovnu biološku funkciju, a to je hidroliza laktoze, pa je od presudnog značaja steći uvid u uticaj različitih reakcionih faktora, i na taj način omogućiti favorizovanje reakcije transgalaktozilacije, odnosno sinteze galakto-oligosaharida. U ovoj studiji, u cilju ispitivanja uticaja pojedinačnih reakcionih para-metara, uočavanja i razumevanja njihovih međusobnih efekata i na kraju određivanja optimalnih uslova za povećan prinos galakto-oligosaharida, primenjeni su metodi statističkog planiranja eksperimenata i metod odzivnih površina (RSM). Konačno, nakon analize rezultata primećeno je da temperatura i rN nemaju značajan uticaj na prinos galakto-oligosaharida, dok su sa druge strane optimalne vrednosti signifikantnih faktora: koncentracija laktoze 400 g/l, koncentracija enzima 13,5 g/l i vreme 13 minuta.

β-Galactosidase is the industrially important enzyme that catalyzes both, lactose hydrolysis and synthesis of different bioactive galactosides. In this study, optimal conditions (fermentation temperature, inoculum concentration and lactose concentration) for accomplishing high yields of β-galactosidase activity from Lactobacillus acidophilus ATCC 4356, bacteria regarded as safe for human consumption, were investigated. Using the response surface methodology (RSM), it was concluded that the highest activity and specific activity were obtained by 2-day shake-flask culture fermentation at 28°C, provided that the lactose content in the fermentation medium was 1.48%, and the inoculum concentration was 2.8%. The optimum temperature and pH for the obtained enzyme were 45°C and 6.5, respectively. More importantly, these conditions simultaneously ensure a high enzyme stability. The Km and Vmax values were 0.44 mM and 25.64 mM/h (for o-nitrophenyl-β-D-galactopyranoside), and 3.79 mM and 3.10 mM/h (for lactose), and the substrate excess inhibition was not observed. On the other hand, the enzyme was inactivated in the presence of Ca2+, Ba2+, and Cu2+. β-Galaktozidaza je značajan industrijski enzim koji se tradicionalno koristi za katalizovanje reakcije hidroloze laktoze, ali poslednjih godina nalazi i sve značajniju primenu u reakcijama sinteze različitih bioaktivnih galaktozida. U ovoj studiji, ispitani su optimalni uslovi za postizanje visokih prinosa β-galaktozidaze iz Lactobacillus acidophilus ATCC 4356, bakterije mlečne kiseline koja se smatra bezbednom za korišćenje u prehrambenim proizvodima. Statističkim planiranjem eksperimenata i metodom odzivnih površina, zaključeno je da se najveća aktivnost i specifična aktivnost dobijaju nakon dvodnevne fermentacije na temperaturi od 28°S, kada je koncentracija laktoze 1,48%, a koncentracija inokuluma 2,80%. Temperaturni i rN optimum dobijenog enzima iznose 45°S i 6,8, redom, pri čemu ovi uslovi istovremeno omogućuju i izuzetnu stabilnost enzima. U kinetičkoj studiji određene su vrednosti kinetičkih parametara, Km i Vmax: 0,44 mM i 25,64 mM/h (za o-nitrofenil- β-D-galaktopiranozid), odnosno 3,79 mM i 3,10 mM/h (za laktozu). Takođe, nije primećena inhibicija supstratom u višku, dok se enzim inaktivira u prisustvu jona Ca2+, Ba2+, i Cu2+. PR Projekat Ministarstva nauke Republike Srbije, br. III 46010 .

Background Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity. Results Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively. Conclusion This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.

The present work describes application of natural triglycerides for lipase-catalyzed esterification of structurally diverse flavonoids-esculin, naringin, and phloridzin. Ester mixtures with laurate, linolenate, or linoleate as prevailing products were obtained in reactions with coconut, linseed, and sunflower oils, respectively. By using response surface methodology, the influences of key experimental factors-reaction time, substrates, and enzyme concentrations-for transesterification of esculin with linseed oil were investigated. High molar conversion (78.5%), product yield (19.13 mg/mL), and specific yield (2.83 mmol/g(enzyme)) were achieved at optimized conditions. Determined optimums were successfully applied in esculin transesterification with other two oils. Furthermore, esculin ester mixtures were incorporated in gel emulsions, and their effect on transepidermal water loss (TEWL) as well as skin sensitivity was examined. Obtained results have shown significant hydration effect and good dermatological compatibility without irritating effects of all examined preparations containing oil-derived esculin esters, making them very prospective for application in skin care products.

Whey represents a significant dairy industry by-product that has recently received due attention based on the rich nutrient composition and significant transformation potential. Hereby, we investigated a possibility of whey lactose bioconversion into prebiotic compounds, galacto-oligosaccharides (GOS) using beta-galactosidase transgalactosylation activity. The results showed that whey could be successfully used for GOS synthesis, since the highest GOS concentration (around 62 g L-1) was obtained batchwise using 40% (w/w) sweet whey powder solution under optimum conditions (50 degrees C, pH 4.5). Nevertheless, an efficient immobilized preparation using methacrylic Lifetech ECR8409 immobilization carrier was developed, enabling additional process improvement and ensuring at least 4 reaction cycles with unchanged yields and 2.5- fold enhanced productivity in comparison to the soluble enzyme. Therefore, this study provides a valuable contribution to the efficient and economical valorization of whey, which can be further on utilized as functional food and feed constituent.

BACKGROUND In this study, in order to obtain the best support for the immobilization of lipase from Candida rugosa (CRL), the procedure for chemical modification of fumed silica nanoparticles (FNS) with (3-Glycidyloxypropyl)trimethoxysilane (GOPTMS) was optimized by varying the amount of GOPTMS used and the duration of support modification. To evaluate the prospects of chemical modification of FNS surface on the immobilization process, the activity and thermal stability of lipase immobilized on GFNS was compared with lipase immobilized on FNS and cyanuric chloride functionalized amino silica (CCAFNS). RESULTS The epoxy functionalized silica nanoparticles (GFNS) with 400–500 µmol of introduced epoxy groups per g of support were the most efficient, since immobilized preparations in 1 mol L−1 buffer had the highest activity of approximately 1495 IU g−1 and a loading capacity of 294 mg g−1. Lipase immobilized on GFNS exhibited 1.5-fold higher hydrolytic activity and 2-fold higher esterification activity in the synthesis of aroma esters than lipase immobilized on CCAFNS, while its thermal stability was similar to CCAFNS preparations. CONCLUSION These results indicated that the type of modification of the FNS surface has an influence on the specificity of the immobilized lipase, since lipase immobilized on GFNS showed improved properties for ester synthesis which is a promising area for immobilized lipase application. © 2015 Society of Chemical Industry

Galactosidases are group of enzymes gaining increasing attention in recent times due to the fact they catalyse hydrolysis of galatose-containing oligosaccharide, which reduce digestibility of various food and feed products. Microbial production of galactosidases is the most efficient and widely used. The aim of this study was to selects strain of lactic acid bacteria which can be used as producer of both, α- and β-galactosidases, since bacteria with such properties can be used for improving digestibility of products containing soybean and milk derivatives. In preliminary selection only intracellular activity of α- and β-galactosidases was detected and Lactobacillus reuteri was selected as the most adequate producer of both enzymes. In subsequent experiments, fermentation was optimized with respect to produced intracellular activity of both enzymes using spectrophotometric enzyme assays. Enzymes were characterized with respect to their temperature and pH optima. It was revealed that the most effective factor for production of enzymes is the selection of inducer. The addition of lactose induced production of β-galactosidase, while raffinose was the most efficient inducer of α-galactosidase production. Further improvement of enzyme production was achieved by optimization of inoculum concentration. Galaktozidaze obuhvataju grupu enzima koji poslednjih godina privlače sve veću pažnju zahvaljujući činjenici da katalizuju reakciju hidrolize različitih galakto-oligosaharida prisutnih u širokom spektru proizvoda za prehrambenu i stočnu industriju, čime omogućavaju njihovu bolju svarljivost i iskoristivost. Najefikasnije i najčešće korišćene galaktozidaze vode poreklo iz mikrobnih izvora. Cilj ove studije bio je odabir soja bakterije mlečne kiseline koja se može koristiti za proizvodnju α- i β-galaktozidaza, imajući u vidu da se ove bakterije bez ograničenja mogu koristiti za poboljšavanje svarljivosti proizvoda na bazi soje i mleka. U preliminarnim eksperimentima detektovana je intracelularna aktivnost α- i β- galaktozidaza, a bakterija Lactobacillus reuteri je izabrana kao najadekvatniji proizvođač oba enzima. U narednim eksperimentima, fermentacija je optimizovana u cilju poboljšanja proizvodnje intracelularnih enzima, praćenjem aktivnosti primenom spektrofotometrijskih enzimskih testova. Enzimi su potom okarakterisani sa aspekta optimalnih vrednosti temperature i pH za njihovu maksimalnu hidrolitičku aktivnost. Otkriveno je da najvažniji faktor za proizvodnju enzima predstavlja izbor induktora. Naime, dodavanje laktoze u fermentacionu podlogu dovelo je da indukuje proizvodnju β-galaktozidaze, dok se rafinoza pokazala kao najefikasniji induktor proizvodnje α-galaktozidaza. Dalje unapređenje proizvodnje ovih enzima postignuto je optimizacijom koncentracije inokuluma.

In this study, two commercial supports (Eupergit® C and Purolite® A109) were chemically modified in order to introduce thiosulfonate groups, which could subsequently exclusively react with cysteine residues on enzyme surface. Thereafter, the immobilization of maltase from Saccharomyces cerevisiae onto obtained thiosulfonate-activated supports was performed, resulting in high expressed enzymatic activities (around 50%), while on the other hand, immobilization on unmodified supports yielded expressed activities less than 5%. Moreover, protein loadings up to 12.3 mg g-1 and immobilized activities up to 3580 IU g-1 were achieved by employment of theses thiosulfonate supports. Desorption experiments, performed on samples taken during immobilization, proved that immobilization on thiosulfonate supports encompass first step of fast adsorption on support and second slower step of the covalent bond formation between thiosulfonate groups and thiol groups of cysteine. More importantly, although enzyme coupling occurs via covalent bond formation, performed immobilization proved to be reversible, since it was shown that 95% of immobilized activity can be detached from support after treatment with thiol reagent (β-mercaptoethanol), thus support can be reused after enzyme inactivation. [Projekat Ministarstva nauke Republike Srbije, br. III 46010]

In this study, unmodified, amino modified and cyanuric chloride functionalized amino modified nonporous fumed nano-silica particles (FNS, AFNS and CCAFNS, respectively) were used for the development of efficient nanobiocatalysts for application in the biosynthesis of bioactive galactosides, galacto-oligosaccharides (GOS). Hence, in an attempt to elucidate the mechanism of immobilization, based on available enzyme conformation, the effects of immobilization parameters (initial enzyme concentration, immobilization time and pH) were analyzed. Among all three used nano-sized supports, the one with amino groups (AFNS) exhibited the best beta-galactosidase binding capacity of 220 mg g(-1) support with the efficiency of similar to 90% at pH 4.5 and immobilization time 3 h. The highest hydrolytic activity of similar to 2200 IU g(-1) was achieved, which is far higher than previously reported. Meanwhile, beta-galactosidases covalently immobilized on CCAFNS and adsorbed on FNS nanoparticles were found to have similar trends with respect to immobilization efficiency (58-71%) and hydrolytic activity (similar to 600 IU g(-1)). Furthermore, thermal stability at 60 degrees C was increased by beta-galactosidases immobilized on AFNS and CCAFNS (4 and 1.4-fold, respectively) due to the protective effect of interactions formed between enzyme molecules and the surfaces of nanoparticles. Since b-galactosidase immobilized on AFNS was the nanobiocatalyst with the highest activity and stability, it was applied in GOS production. With AFNS- beta-galactosidase GOS production of 90 g L-1 h(-1) was achieved as compared to 30 g L-1 h(-1) by free beta-beta-galactosidase, meaning that AFNS enhanced the selectivity of b-galactosidase for transgalactosylation, which is a crucial advantage for its application in GOS production.

Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco-friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase-catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short-chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L(-1) of lipase, 50 mM of vitamin C, 10-fold molar excess of oleic acid, and 0.7 mL L(-1) of initial water. Obtained results showed that even short- and medium-chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l-ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

The TiO2 based hybrid supports with different functional groups (amino, glutaraldehyde or epoxy) were prepared and their influence on immobilization of dextransucrase (DS) was studied. Novel synthetic route for surface modification of TiO2 with amino and glutaraldehyde groups was developed taking advantage of charge transfer complex (CTC) formation between surface Ti atoms and salicylate-type of ligand (5 aminosalicylic acid (5-ASA)). The proposed coordination of 5-ASA to the surface of TiO2 powder and optical properties of CTC was presented. The pristine TiO2 and amino functionalized TiO2 have higher sorption capacity for DS (12.6 and 12.0 mg g(-1), respectively) compared to glutaraldehyde and epoxy activated supports (9.6 and 9.8 mg g(-1) respectively). However, immobilized enzyme to either glutaraldehyde or epoxy functionalized TiO2 have almost two times higher expressed activities compared to pristine TiO2 support (258, 235 and 142 IU g(-1), respectively). Thermal stability of enzyme immobilized on glutaraldehyde and epoxy functionalized supports was studied at 40 degrees C, as well as operational stability under long-run working conditions in repeated cycles. After five cycles, DS imobilized on glutaraldehyde activated support retained almost 70% of its initial expressed activity, while, after five cycles, performance of DS immobilized on epoxy activated support was significantly lower (15%).

beta-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude beta-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that beta-galactosidase from L acidophilus shows a particular specificity towards the formation of beta-(1 - gt 6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 WI GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50 degrees C).

Lipophilic derivatives of vitamin C are additives with antioxidant properties, attractive for application in food, cosmetics and pharmaceutics. They could be synthesized in lipase-catalyzed processes by using various acyl donors. Hereby, we present application of linoleic acid, which is polyunsaturated fatty acid essential in human nutrition, for esterification of vitamin C catalyzed by immobilized enzyme preparation Novozym® 435 in acetone. Highest specific ester yield, 9.7 mmol/g of immobilized lipase, was accomplished with 0.15 M of vitamin C, 0.6 M of linoleic acid, 3 g/l of enzyme and 0.07% (v/v) of water, at 60°C. NMR analyses of purified product proved that synthesized molecule was identical to 6-O-ascorbyl linoleate. Capacity of ester for scavenging 2,2-diphenyl-1-picrylhydrazyl radicals was two times higher comparing to parent molecule, vitamin C. Its diffusion coefficient, determined using Franz cell and cellulose acetate membrane, was 40% higher than palmitate and 62% higher than oleate. Obtained results showed that L-ascorbyl linoleate could be successfully synthesized in biocatalyzed processes. Furthermore, it was demonstrated that it possess high potential for application in different lipophilic products due to its liposolubility, high antioxidant efficiency and good diffusion properties. Lipofilni derivati vitamina C su aditivi sa antioksidativnim dejstvom pogodni za primenu u prehrambenim, kozmetičkim i farmaceutskim proizvodima. Mogu biti sintetisani u procesima katalizovanim lipazama korišćenjem različitih acil-donora. U ovom radu, opisana je primena linolne kiseline, polinezasićene masne kiseline esencijalne u ljudskoj ishrani, u esterifikaciji vitamina C katalizovanoj imobilisanim enzimskim preparatom Novozym® 435 u acetonu. Najviši specifični prinos estra od 9,7 mmol/g imobilisane lipaze, ostvaren je sa 0,15 M vitamina C, 0,6 M linolne kiseline, 3 g/l enzima i 0,07 zapr. % vode, na 60°C. NMR analize prečišćenog proizvoda dokazale su da je sintetisani molekul identičan 6-O-askorbil-linolatu. Kapacitet estra za vezivanje 2,2- difenil-1-pikrilhidrazil radikala bio je dva puta viši u odnosu na sam vitamin C. Njegov koeficijent difuzije, određen korišćenjem Franz-ove ćelije i celuloza-acetatne membrane, bio je za 40% viši u odnosu na palmitat i za 62% u odnosu na oleat. Ostvareni rezultati pokazali su da L-askorbil-linolat može uspešno biti sintetisan u biokatalizovanom procesu. Pored toga, dokazano je da ovaj estar poseduje značajan potencijal za primenu u različitim lipofilnim proizvodima zbog svoje liposolubilnosti, snažnog antioksidativnog dejstva i pogodnih difuzionih karakteristika.

(-galactosidase, commonly known as lactase, represents commercially important enzyme that is prevalently used for lactose hydrolysis in milk and whey. To the date, it has been isolated from various sources. In this study different strains of lactic acid bacteria were assessed for their (-galactosidase productivity, and Lactobacillus acidophilus ATCC 4356 resulted with the highest production potential. Thereafter, optimal conditions for accomplishing high yields of (-galactosidase activity were determined. Maximal specific activity (1.01 IU mL-1) was accomplished after 2 days shake flask culture fermentation (150 rpm) at 37°C, with modified Man Rogosa Sharpe culture broth using lactose (2.5%) as sole carbon source. Finally, in order to intensify release of intracellular (-galactosidase different mechanical and chemical methods were conducted. Nevertheless, vortexing with quartz sand (150 μm) as abrasive was proven to be the most efficient method of cell disruption. The optimum temperature of obtained (-galactosidase was 45°C and the optimum range pH 6.5-7.5. Enzim (-galaktozidaza, poznatija kao laktaza, predstavlja industrijski izuzetno važan enzim, koji ima primarnu ulogu u hidrolizi disaharida laktoze. Upotrebom ovog enzima u industriji mleka i mlečnih proizvoda dolazi do poboljšanja fizičkih i senzornih karakteristika proizvoda, kao i do povećanja svarljivosti proizvoda, a samim tim i prevazilaženja problema netolerancije na laktozu. Takođe, hidrolizom laktoze surutke, rešava se pitanje njenog ekološki prihvatljivog odlaganja. Sa druge strane, enzim pod kontrolisanim reakcionim uslovima može katalizovatii proces transgalaktozilacije, odnosno prenošenja galaktozil jedinica na druge šećere prisutne u sistemu (najčešće laktozu), pri čemu nastaju izuzetno važna funkcionalna jedinjenja galaktooligosaharidi. Enzim (-galaktozidaza može biti različitog porekla - biljnog, životinjskog ili mikrobnog. Međutim, najznačajniji među njima su mikrobni postupci proizvodnje, zbog lake fermentacije, velike brzine rasta i razmnožavanja ćelija, visokih prinosa, visoke aktivnosti, kao i stabilnosti enzima. Kao posledica velikog komercijalnog interesa za ovaj enzim, opisane su različite metode dobijanja i prečišćavanja dobijenih enzima iz različitih mikroorganizama. Poslednjih godina bakterije mlečne kiseline privlače sve više pažnje kao potencijalni izvori (-galaktozidaza najviše zahvaljujući svom statusu bezbednih za upotrebu u prehrambenoj i farmaceutskoj industriji, čime se omogućava neometano korišćenje enzima bez primene komplikovanih metoda prečišćavanja. U ovom radu ispitana je mogućnost proizvodnje (-galaktozidaza pomoću nekoliko vrsta bakterija mlečne kiseline. Kao najbolji producent među ispitanim bakterijama pokazala se bakterija Lactobacillus acidophilus. Najveća aktivnost (-galaktozidaze, dobijena je mikroaerofilnom fermentacijom u modifikovanoj komercijalnoj MRS podlozi, sa 2,5% laktoze kao jedinim izvorom ugljenika. Fermentacija je vršena na tresilici (150 rpm) u trajanju od 48 h i na temperaturi od 37°C. Kako je enzim intracelularan, u cilju razaranja ćelija i oslobađanja enzima, primenjeno je više različitih fizičkih i hemijskih metoda, a daleko najboljom pokazala se metoda vorteksiranja sa kvarcnim peskom (150 μm) kao abrazivnim sredstvom. Ovako dobijen enzim pokazao je maksimalnu aktivnost pri temperaturi od 45°C i pH u opsegu 6.5-7.5.

BACKGROUND In this work, fumed nano-silica (FNS) was chemically modified to amino (AFNS) and subsequently to cyanuric chloride (CCAFNS) modified silica and tested for the immobilization of lipase from Candida rugosa (CRL). The effects of the initial enzyme concentration, immobilization time and buffer ionic strength on immobilization were investigated in order to optimize utilization of the support and determine the mechanism of immobilization. The most active preparations were used to examine thermal and operational stability. RESULTS The amount of immobilized enzyme increased with increasing enzyme concentration, achieving loadings of 121.3, 104.8 and 61.2 mg per g of FNS, CCAFNS and AFNS, respectively. Lipase immobilized on CCAFNS carrier in 0.1 mol L−1 buffer expressed the highest lipolytic activity (1320 IU g−1 support), while more stable preparations were obtained in 1 mol L−1 buffer. CONCLUSION Successful modification of silica was confirmed with Fourier transform infrared spectroscopy and thermogravimetric analysis. Activity results showed that, depending on the support, immobilization was governed by different interactions. On FNS and AFNS immobilization was exclusively by adsorption, while on CCAFNS after the initial adsorption lipase molecules reoriented and amino groups of the enzyme formed a covalent bond with the chlorine atom of the modified carrier. Improved thermal and operational stability of lipase immobilized on CCAFNS in 1 mol L−1 buffer led to the conclusion that electrostatic interactions have a great role in the immobilization. © 2014 Society of Chemical Industry

In this study, Purolite (R) A109, polystyrenic macroporous resin, was used as immobilization support due to its good mechanical properties and high particle diameter (400 mu m), which enables efficient application in enzyme reactors due to lower pressure drops. The surface of support had been modified with epichlorhydrine and was tested in lipase immobilization. Optimized procedure for support modification proved to be more efficient than conventional procedure for hydroxy groups (at 22 degrees C for 18 h), since duration of procedure was shortened to 40 min by performing modification at 52 degrees C resulting with almost doubled concentration of epoxy groups (563 mu mol g(-1)). Lipase immobilized on epoxy-modified support showed significantly improved thermal stability comparing to both, free form and commercial immobilized preparation (Novozym (R) 435). The highest activity (47.5 IU g(-1)) and thermal stability (2.5 times higher half-life than at low ionic strength) were obtained with lipase immobilized in high ionic strength. Thermal stability of immobilized lipase was further improved by blocking unreacted epoxy groups on supports surface with amino acids. The most efficient was treatment with phenylalanine, since in such a way blocked immobilized enzyme retained 65% of initial activity after 8 h incubation at 65 degrees C, while non-blocked derivative retained 12%.

alpha-1,4-Glucosidase from Saccharomyces cerevisiae is an enzyme which is widely used in synthesis of different drugs. Glucosidase inhibitors are studied as potential drugs for prevention of HIV and diabetes. For understanding of these processes it is very important to have insights in the transglucosylation activity of this enzyme. In this paper the kinetics of transglucosylation reaction catalyzed by this enzyme in the synthesis of benzyl alcohol glucoside was studied and all relevant kinetic constants for this system are found. It was shown one additional property of transglycosylation reactions catalyzed by glycosidases-inhibition by both, glucose acceptor and glucose donor, and mechanisms for these inhibitions were proposed.

The kinetics of l -ascorbyl oleate synthesis catalyzed by immobilized lipase from Candida antarctica in acetone was investigated. Significant inhibition of synthesis with an excess of ascorbic acid was observed. Experimental data were successfully fitted with a ping–pong bi–bi kinetic model with substrate inhibition, and related kinetic constants were determined. The kinetic study was performed at optimum experimental factors (temperature, initial water content, and enzyme concentration), which were determined using response surface methodology. Then, a model for predicting product–time progress curves was developed by expanding the obtained ping–pong model with terms describing ester hydrolysis. Kinetic constants of the reverse reaction were determined, and good congruence between the model and experimental data was achieved. Calculated kinetic constants revealed that lipase has the highest affinity for ascorbyl oleate, slightly lower activity with ascorbic acid, and the lowest activity with oleic acid. The obtained results are valuable for elucidating the reaction mechanism and represent an important contribution for reaction optimization and creating strategies to increase the productivity of vitamin C ester synthesis.

A commercial preparation of Candida rugosa lipases (CRL) was tested for the production of capsinoids by esterification of vanillyl alcohol (VA) with free fatty acids (FA) and coconut oil (CO) as acyl donors. Screening of FA chain length indicated that C8-C12 FA (the most common FA found in CO triglycerides) are the best acyl-donors, yielding 80-85% of their specific capsinoids. Hence, when CO, which is rich in these FA, was used as the substrate, a mixture of capsinoids (vanillyl caprylate, vanillyl decanoate and vanillyl laurate) was obtained. The findings presented here suggest that our experimental method can be applied for the enrichment of CO with capsinoids, thus giving it additional health promoting properties. This is the peer-reviewed version of the following article: Trbojević Ivić, J.; Milosavić, N.; Dimitrijević, A.; Gavrović Jankulović, M.; Bezbradica, D.; Kolarski, D.; Veličković, D. Synthesis of Medium-Chain Length Capsinoids from Coconut Oil Catalyzed by Candida Rugosa Lipases. Food Chemistry 2017, 218, 505–508. [https://doi.org/10.1016/j.foodchem.2016.09.049]

The esterification is well-established type of reaction in organic synthesis used for manufacturing various valuable esters used prevalently as ingredients of food, skincare products, pharmaceuticals and fuels. In recent decades biocatalytic synthetic routes based on using enzymes as catalysts have received a lot of attention in scientific researches and industry. Main advantages of enzymatic esterifications are high selectivity (regioselectivity and enantioselectivity), performing reaction at milder conditions (moderate temperature and atmospheric pressure) and the fact that enzymatically synthesized product gain label of natural product, which increases its market appeal. The purpose of this review is to point out the most important advances in lipase-catalyzed ester synthesis in non-conventional reaction media, with special focus on several most important types of ester. This part of review starts with outcomes in synthesis of aroma esters, usually obtained using monofunctional aliphatic alcohols and acids, which have been investigated for decades and industrial processes are already established. However, later sections are dedicated to enzymatic esterification of more complex molecules (carbohydrates, vitamin C, flavonoids) having more than one functional groups, which are subject of numerous studies in recent times because in these reactions regioselectivity of biocatalysts can be fully explored and processes are still subject of optimization, especially with respect to selection of reaction media and media engineering.