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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.

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

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.

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.