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Damaged grains that are not useful for food or feed are abundant and cheap. The main objective of this paper was to valorize damaged rice grains for bioethanol production using presacchararification step followed by simultaneous saccharification and fermentation (SSF) by using waste brewer’s yeast. Presacchararification step was carried out using crude amylases that produce naturale isolate Paenibacillus chitinolyticus CKS1. Maltose, a fermentable sugar, was the main product during presaccharification. Statistical design, central composite design (CCD), was used for optimization of process parameters for ethanol production, yeast cell yield and utilized sugars. Under optimal conditions 0.5% malt extract, 3.84% inoculum and 2.76 days of fermentation maximum ethanol production of 4.69% was achieved. The obtained ethanol concentration was in a good correlation with yeast cell yield and utilized reducing sugars. The results obtained in this study indicate that the economical production of ethanol could be achieved using cheap waste materials also using low cost enzymes derivated from natural bacterial isolates.

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.

Sugar beet pulp (SBP) and molasses, as an agro industrial waste material, are produced in large amounts annually. Thus, a major challenge nowadays is to develop procedures that could increase the value of the generated waste. In this study, SBP as a support for cell immobilization and molasses as a source of nutrients were used for a dextransucrase (DS) production by Leuconostoc mesenteroides T3. The influence of SBP in native form (SBP-N) and after treatment with NaOH (SBP-NaOH) on DS production was investigated. The optimal medium composition for the maximum DS production was determined by varying the concentration of molasses, SBP, and sucrose. The maximum DS yield of 2.02 U/ml was obtained in the medium with 2.5 % of molasses, 2.5 % SBP-NaOH, and 4 % of sucrose concentration. Scanning electron microscopy (SEM) showed immobilization of Lc. mesenteroides T3 cells onto SBP-NaOH. According to the obtained results, the production of DS on molasses could be improved by using NaOH-treated SBP as a carrier for whole-cell immobilization. Our study reveals the basis for the development of process for DS production with additional reduction of expenses by using waste materials for obtaining the valuable biotechnological product.

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.

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).

(-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.

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%.

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.

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.

We report the successful, low temperature synthesis of pure and Fe-doped titania nanoparticle (NP) powders, using organic Ti- and Fe-precursors. The properties of the synthesized titania based materials were obtained using XRD, UV/vis and EPR spectroscopy and BET measurements. The doping of TiO2 induced a shift in the absorption threshold towards the visible spectral range, compared to pure titania. The process of annealing induced an increase in crystallinity and particle sizes in the obtained materials. The photocatalytic activity, using simulated Solar light (Vitalux lamp), of our samples was checked by following the photodegradation reaction of Rhodamine B and trichlorophenole as model compounds. This is the peer-reviewed version of the article: Carević, M., Abazović, N. D., Savić, T. D., Novaković, T., Mojović, M.,& Čomor, M. (2016). Structural, optical and photodegradation properties of pure and Fe-doped titania nanoparticles probed using simulated Solar light. Ceramics International, Elsevier Sci Ltd, Oxford., 42(1), 1521-1529. [https://doi.org/10.1016/j.ceramint.2015.09.100] The published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/1982]

International audience; Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with beta-galactosidases. Enzymatic transgalactosylation results in a mixture of compounds of various degrees of polymerization and types of linkages. Because structure plays an important role in terms of prebiotic activity, it is of crucial importance to provide an insight into the mechanism of transgalactosylation reaction and occurrence of different types of beta-linkages during GOS synthesis. Our study proved that a novel one-step method, based on ion-mobility spectrometry tandem mass spectrometry (IMS-MS/MS), enables complete elucidation of GOS structure. It has been shown that) beta-galactosidase from Aspergillus oryzae has the highest affinity toward formation of beta-(1 -> 3) or beta-(1 -> 6) linkages. Additionally, it was observed that the occurrence of different linkages varies during the reaction course, indicating that tailoring favorable GOS structures with improved prebiotic activity can be achieved by adequate control of enzymatic synthesis.

International audience; In this study, enzymatic synthesis of galactoside of salicin, compound with potential physiological activity due to structural resemblance with galectin inhibitors, and analgesic and antipyretic properties of salicin, was performed using beta-galactosidase from Aspergillus oryzae. It was determined, using HPLC and ion mobility mass spectrometry, that enzymatic synthesis was highly selective since only one isomer was formed via primary hydroxyl group on glucose moiety of salicin. The optimization of key experimental factors using response surface methodology enabled galactosyl salicin concentration up to 30.8 mM obtained at lactose concentration 40 mM, salicin concentration 110 mM, enzyme amount 360 IU and reaction time 12 h.

A novel strain of Paenibacillus chitinolyticus CKS1 was isolated from forest soil and identified as a potent cellulase producer. The strain was able to grow on various commercial substrates including microcrystalline cellulose (Avicel), carboxymethylcellulose (CMC), and cellobiose but also on lignocellulosic waste material such as medicinal herbs waste and sawdust. On all these substrates the strain produced cellulase composed of two subunits (similar to 70 and similar to 45 kDa) that was active on CMC, Avicel and filter paper. The maximal Avicelase activity (1.94 U/ml) was reached in a medium that contained 0.1% (w/v) of medicinal herbs waste, 3 g l(-1) of yeast extract and 5.0 g l(-1) of casein hydrolysate in 0.1 M phosphate buffer pH 7, after 48 h of incubation at 30 degrees C. The Avicelase performed optimally at 80 degrees C and at pH 4.8. Addition of K+ increased the Avicelase activity almost three fold and the enzyme retained 48.39% of the initial activity after 60 min. The product of Avicel and CMC hydrolysis was glucose with traces of other soluble sugars, indicating that the crude cellulase produced on waste material using the novel P. chitinolyticus strain CKS 1 could be used in eco-friendly processes of cellulose bioconversion, such as enzymatic saccharification of lignocellulosic materials in processes performed under acidophilic conditions and high temperatures.

Lipase-catalyzed acylation of phloridzin with a wide range of fatty acids and antioxidant activities of synthesized esters were investigated in this study. Polar solvents were suitable reaction media for esterification, and the highest yield was achieved in acetonitrile. By using statistically designed optimization of key experimental factors for the synthesis of phloridzil oleate as the model reaction, great improvements in achieved conversion and yield per enzyme mass were enabled. The most significant progress has been made in the cost-effectiveness of the reaction, since the specific yield of 5.45 mmol g(-1) was obtained at 58 degrees C, with 0.09 M phloridzin, 1.17 M oleic acid, and only 0.5% (w/v) biocatalyst. All examined fatty acids were suitable acyl donors, since the chain length and unsaturation degree had slight effects on esterification and all products yielded over 70%. Phloridzil caprate, myristate, and oleate exhibited the highest antioxidant activities among the obtained esters.