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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 islet β-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating β-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live β-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger β-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In β-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living β-cells within complex mixtures for further manipulation and characterisation.

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]

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%.

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 aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K(m) value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate.

BACKGROUND: A totally new approach has been applied for mathematical modeling of the enzyme activity/pH relationship, for quantification and distribution of enzyme activity in and out of carrier pores. This is a very simple and elegant method for determination of the distribution of enzyme molecules on the surface and inside the particles, simply through measurement of enzyme activity at different pH values. RESULTS: Amyloglucosidase (AG) from Aspergillus niger was covalently immobilized onto poly(GMA-co-EGDMA) by the glutaraldehyde and periodate methods. Mathematical modeling of the pH optima for two types of covalently immobilized AG resulted in higher enzyme amounts on the surface within periodate immobilizate (67%) in comparison with glutaraldehyde immobilizate (53%). These values are modified to 64.25% for periodate immobilizate and 49.95% for glutaraldehyde immobilizate when diffusion effects are taken into account. CONCLUSION: The mathematical model applied enabled observation of the difference between the two types of coupling agents and different immobilization procedures throughout quantification of the immobilized enzyme on the matrix surface and inside pores. Copyright © 2012 Society of Chemical Industry

Yeast Candida antarctica produces two lipase forms, which are widely used as catalysts in variety of organic reactions, many of which are applied on a large scale. In this work, production of two forms of lipase from C. antarctica DSM 70725 (CAL A and CAL B) was monitored during seven days of cultivation in the optimal medium using different electrophoretic and zymographic techniques. According to electrophoresis after silver staining, C. antarctica lipase A (molecular mass 45 kDa) was produced starting from the second day of cultivation. C. antarctica lipase B (CAL B) was also produced starting from the second day, but protein was present in the fermentation broth predominantly as dimer (molecular weight 66 kDa), while presence of monomeric form of CAL B (molecular weight of 33 kDa) was observed starting from the fourth day of cultivation. Both types of zymograms (based on hydrolysis and synthesis reactions) were used for detection of lipase activity in the fermentation broth. C. antarctica lipase A showed activity only in hydrolytic zymogram, when α-naphtyl butyrate was used as substrate. In the same zymogram, with α-naphtyl acetate as substrate no CAL A activity was detected. Similarly, CAL A showed no activity in synthesis based zymograms towards oleic acid and octanol as substrates, indicating that CAL A is not active towards very short or long-chain substrates. As opposite of CAL A, both monomeric and dimeric form of CAL B were detected in the all zymograms, suggesting that CAL B is active towards wide range of substrates, regardless to the chain length. Thus, zymogram based on hydrolysis of α-naphtyl butyrate represents a simple method for monitoring the production of two forms of lipase from C. antarctica, that greatly differ in their characteristics.

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol (R) XP80 and Triton (R) X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1 h) while the protease activity was enhanced by the addition of Triton (R) WR1339 and Tween (R) 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.

In this study, exopolysaccharide (EPS) produced by an extremophylic strain of Pseudomonas aeruginosa san-ai was used as a support for immobilization of lipase produced together with EPS, using new and simple, single step procedure. The highest EPS production (36.5 mg L −1 ) was observed in the Luria Bertani (LB) medium supplemented with sunflower oil and Tween 80. The EPS structure was analyzed by FT-IR and 1 H NMR, and it was found that EPS from P. aeruginosa is of the alginate type. Lipase, produced by the same strain was entrapped in Ca–EPS beads, formed by dripping a concentrated culture supernatant in CaCl 2 solutions. Effects of immobilization conditions on loading efficiency and immobilization yield were investigated, and the potential for reuse of immobilized lipase was monitored. It was shown that the immobilized enzyme could be used for up to three reaction cycles, after which 77% of activity was retained.

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was observed in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the value of 35.0 U cm(-3) in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude lipase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity towards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23 % residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents. Proizvodnja lipaze iz Pseudozyma aphidis utvrđena je u šest različitih medijuma. Najviša proizvodnja uočena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. Praćenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uočeno je da se najviša proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, što je četri puta veća proizvodnja od one do sada prijavljene u literaturi. Utvrđen je efekat različitih polarnih organskih rastvarača, mešljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. Hidrolitička aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaračima utvrđena je upotrebom istog spektrofotometrijskog testa. Pokazano je da lipaza ima aktivnost prema p-NPP-u samo u acetonu i acetonitrilu, dok je enzim stabilan jedino u acetonu i zadržava 23% aktivnosti nakon 24 časa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti korišćena kao biokatalizator za potencijalne primene u acetonu kao medijumu.

This work presents findings, which indicate important role of fructose, fructose 6-phosphate (F6P), and fructose 1,6-bisphosphate (FBP) in preservation of homeostasis in plants under low temperature. Cold combined with light is known to incite increased generation of superoxide in chloroplasts leading to photoinhibition, but also an increased level of soluble sugars. In the present study, oxidative stress in pea leaves provoked by cold/light regime was asserted by the observed decrease of the level of oxidized form of PSI pigment P700 (P700+). Alongside, the increased antioxidative status and the accumulation of fructose were observed. The antioxidative properties of fructose and its phosphorylated forms were evaluated to appraise their potential protective role in plants exposed to chilling stress. Fructose, and particularly F6P and FBP exhibited high capacities for scavenging superoxide and showed to be involved in antioxidative protection in pea leaves. These results combined with previously established links implicate that the increase in level of fructose sugars through various pathways intercalated into physiological mechanisms of homeostasis represents important non-enzymatic antioxidative defense in plants under cold-related stress.

Variations were studied of the activity and isoenzyme patterns of soluble peroxidase, catalase, catechol oxidase and superoxide dismutase, in needles of the Balkan endemic conifer Serbian spruce, Picea omorika (Panc.) Purkinye. The samples were collected from the natural habitat of the species, Mt. Tara. Seasonal changes were found to affect enzymatic activities and isoenzyme profiles. Total protein content was significantly lower in the summer than in other seasons. Several isoforms of peroxidase, catechol oxidase and superoxide dismutase (SOD), as well as two catalase isoenzymes were detected. The number of peroxidase isoenzymes was greatest during the vegetative season. Catalase and catechol oxidase peaked in summer and spring, respectively. Total SOD and Mn-SOD activities were significantly higher in the winter samples than the summer ones. (C) 2006 Elsevier Ltd. All rights reserved.

We succeeded in the immobilization of 190 mg of periodate oxidized glucoamylase per gram of macroporous polymer. The covalently immobilized enzyme had a specific activity of 1100 U/g. The temperature and pH optimum as well as kinetic parameters were determined. The immobilized enzyme was tested in different types of reactors for hydrolysis of concentrated maltose and starch hydrolysate syrups. The DE value of 98.6, obtained the immobilized enzyme, was slightly higher than that for the soluble form, when acting on 20% substrates. During continuous use in a packed bed reactor over a period of 4 weeks the immobilized enzyme produced 1300 kg of glucose per 1 L of reactor volume without any decrease in its activity. (c) 2006 Elsevier Inc. All rights reserved.

Two lines of Picea omorika (Panc.) Purkyn? were compared with respect to germination percentage as well as specific activity and isoenzyme pattern of catalase, superoxide dismutase, and peroxidase (POD) during germination. Line A had a higher germination percentage and higher enzyme activities in dry seeds and seedlings compared to line C. Peroxidase activity was not detected in dry seeds, but measured up to 10 U/g and 28 U/g on the 7th day of germination in lines C and A, respectively. The most abundant POD basic isoform in seedlings of both lines (pI 8.2) was not found previously in needles of adult Serbian spruce trees of the same lines. .

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.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.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 study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 degrees C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t(1/2) values obtained at 75 C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system. (c) 2006 Elsevier B.V. All rights reserved.

The activity of alpha-glucosidase from baker's yeast was determined in various concentrations of dioxan, tetrahydrofuran, tert-butanol, dimethylformamide, methanol and dimethylsulfoxide (DMSO). Higher activities were observed with sucrose than with nitrophenylglucoside as substrate in cosolvent mixtures. In 30% (v/v) DMSO, 25% of the activity obtained in pure water was detected, and in 30% (v/v) methanol 12.5% of the activity in pure water was detected, while in other cosolvents there was almost no activity under these conditions. alpha-glucosidase was immobilized onto a macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), by the glutaraldehyde method. By immobilization, the half-life of the enzyme in 35% (v/v) methanol was increased from 6 to 60min and from 4 to 15min in 45% (v/v) DMSO. The activity of the immobilized enzyme in 30% (v/v) DMSO and 30% (v/v) methanol was 22% and 18% of the activity in pure water, respectively.

Activity and isoenzyme composition of superoxide dismutase in the apoplast and whole-needle extract of Picea omorika (Panc.) Purkyn?, was studied. Total SOD activity of the soluble fraction of the needle extract exceeded markedly that of the apoplastic SOD. Several acidic and two slow-migrating basic isoforms were found in the whole extract. Extracellular SOD had an extremely acidic isoform. Using specific inhibitors, we identified Cu/Zn- and MnSOD forms in the total extract, but only MnSOD in the apoplast. The Feisoform was not present in a detectable amount.

Hydroquinone-α-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with α-glucosidase from baker’s yeast. Only one phenolic –OH group was α-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 °C for 20 h with 50 mM hydroquinone and 1.5 M maltose in 100 mM sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.

Glucoamylase was covalently immobilized through the spacer-arm of the poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) spheres by using a glutaraldehyde as a coupling agent. The influence of the enzyme load applied to the support on immobilization, yield and specific activity, has been determined. Obtained specific activity was 700 U/g with immobilization yield of 35 %. The Km value for immobilized glucoamylase was 1.28 % (w/v), pH and temperature optimum were 4.5 and 70?C, respectively. The conversion of 20 % (w/w) starch hydrolysate achieved with the immobilized glucoamylase was 97 % after 5 hours.

The application of amyloglucosidase immobilized on the macroporous co-polymer of ethylene glycol dimethacrylate and glycidyl methacrylate (poly (GMA-co-EGDMA)) in an enzyme reactor was shown. The higher thermostability of immobilized glucoamylases than the soluble one was demonstrated. Immobilized amyloglucosidase obtained by the periodate method shows two times higher thermo stability than the soluble form. Glucoamylases immobilized on poly (GMA-co-EGDMA) have good mechanical and chemical features in the reactor and when applied in a continuous flow reactor for 28 days no changes are observed. In this period periodate immobilized amyloglucosidase shows no decrease in activity. It showed potential for the continuous production of glucose from starch over a prolonged period of time. U okviru ovoga rada prikazani su rezultati dobijeni izučavanjem termostabilnosti dva kovalentna imobilizata amiloglukozidaze na makroporoznom poli(GMA-co-EGDMA), kao i njihova primena za hidrolizu škroba pri simuliranim industrijskim uslovima. U svim eksperimentima sa reaktorima, bilo šaržnim, bilo da se radi o reaktoru sa napakovanim slojem kao supstrat korišćen je 20 % (m/m) industrijski hidrolizat škroba. Oba imobilizata su pokazala povećanu termostabilnost u odnosu na rastvoreni enzim, ali je ona značajno veća kod imobilizata dobijenog perjodatnom metodom. Prilikom upotrebe u bač reaktoru imobilizati su deset puta brže dali krajnje DE vrednosti slične onoj koju daje rastvorni enzim. Količina i sastav šećera je po završenoj hidrolizi analiziran i HPLC-om. Određena je operativna stabilnost perjodatno imobilizovane amiloglukozidaze u reaktoru sa napakovnim slojem i pri tome je konstatovano da imobilizat ne gubi aktivnost u trajanju od 28 dana.

The optimal conditions for the immobilization of invertase and glucoamylasewere found via their carbohydrate moiety on a macroporous copolymer of ehtyleneglycoldimethacry late and glycidylmethacrylate. Almost all of the added enzyme was bound to the polymer by increasing the time of incubation of the oxidized enzyme with polymer. A specific activity of 5500 U/g for invertase was obtained and 1100 U/g for glucoamylase. The specific productivity for invertase in a packed bed reactor was 3.5 kg/lh and for glucoamylase 1.9 kg/lh. During continuous use in a packed bed the reactor operational half life for invertase was 290 days, while no decrease in activity was observed for glucoamylase. In 50% (v/v) ethanol the immobilized enzymes were five to ten times more stable, and more than 200 times more stable in 25% (v/v) dioxane. The immobilized enzymes retained all activity in petroleum ether after 3 days of incubation. Because of their higher stability over native enzymes, and the large surface area of the polymer immobilized glucoamylase and invertase could be more useful for glycoside synthesis in non-aqueous solvents than native ones.

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.

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.

4-Hydroxyphenyl-beta-isomaltoside has been synthesized by alpha-glucosidase assisted transglycosylation between arbutin as acceptor and sucrose as donor molecules, respectively. Optimum conditions for the transglucosylation reaction were 40 degrees C for 20 h with 10 mM arbutin and 1.5 M sucrose in a 100 mM sodium citrate/phosphate buffer at pH 5.0. The new glucoside was obtained in a 50% molar yield with respect to arbutin. (C) 2007 Elsevier Ltd. All rights reserved.

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.

Benzyl alcohol, a potent anesthetic and bacteriostatic, can be efficiently glucosylated by alpha-glucosidase from Saccharomyces cerevisiae to produce benzyl alcohol alpha-glucoside with a 75% yield. However, while studying the transglucosylation reaction conditions, it was found out that benzyl alcohol is a non-competitive inhibitor of alpha-glucosidase's hydrolytic activity (K-i = 18 mM, toward maltose). Due to its interesting ability to be glycosylated by the enzyme and to inhibit its hydrolytic activity, we proposed a plausible mechanism for the phenolic alpha-glucosydase inhibitor's binding, since the mechanism of inhibition has not yet been elucidated. (C) 2013 Elsevier Ltd. All rights reserved.

Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60 degrees C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4th hour of reaction enabled increase of yield from 16.7 mmol g(-1) to 19.3 mmol g(-1). Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation.

The yeast Candida rugosa is a convenient source of lipases for science and industry. Crude preparation of Candida rugosa lipase (CRL) consists of several extracellular lipases. Isoenzyme profile depends on the culture or fermentation conditions. All isoforms are coded by the lip pseudogene family; they are monomers of 534 amino acids and molecular weight of about 60 kDa. They share the same catalytic mechanism and interfacial mode of activation. Isoenzymes differ in isoelectric points, post-translational modifications, substrate specificity and hydrophobicity. The presence of different lipase isoforms and other substances (i.e., inhibitors) in crude preparation leads to lack of their productivity in biocatalytic reactions. Purification of specific isoform improves its overall performance and stability. This paper provides an overview of different methods for purification of CRL isoenzymes up to date, their advantages and disadvantages. Lipaze (hidrolaze estara glicerola, E.C.3.1.3.3) su važna grupa enzima, široko rasprostranjenih u prirodi. Mogu se izolovati iz materijala biljnog, životinjskog ili mikrobnog porekla. Zahvaljujući svojim karakteristikama, pobuđuju sve više pažnje kao efikasni biokatalizatori u različitim sintetičkim i hidrolitičkim procesima. Među lipazama, poreklom iz mikroorganizama, posebno su značajne one koje produkuje kvasac Candida rugosa. Komercijalni preparat lipaza iz C. rugosa može sadržati 5-7 izoformi ekstracelularnih lipaza. Sve te izoforme kodirane su od strane lip familije pseudogena, a na njihovu ekspresiju utiču uslovi u kojima se mikroorganizam gaji (sastav hranljive podloge je najvažniji). Ekstracelularne lipaze, koje proizvodi C. rugosa su monomerni glikoproteini, molekulske mase od oko 60 kDa, sa 534 aminokiseline. Za sve izoforme je karakterističan isti složeni mehanizam aktivacije na granici faza i mehanizam katalize, kakav se sreće i kod serin-proteaza. Izoenzimi se međusobno razlikuju po post-translacionim modifikacijama (udelu ugljohidratne komponente), supstratnoj specifičnosti, izoelektričnim tačkama i hidrofobnosti. Prisustvo više izoformi lipaza u komercijalnom preparatu utiče na njihovu produktivnost u reakcijama koje katalizuju. Takvi preparati često sadrže i druge supstance koje mogu uticati na aktivnost enzima (na primer inhibitore). Razdvajanjem pojedinačnih izoformi iz komercijalnog preparata poboljšavaju se njihova enantioselektivnost, specifična aktivnost i stabilnost enzima, što je od izuzetnog značaja za njihovu dalju primenu. U ovom radu su predstavljeni različiti pristupi u razdvajanju pojedinačnih izoformi vanćelijskih lipaza iz komercijalnog preparata C. rugosa, njihove prednosti i nedostaci.

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10 mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60 degrees C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn2+, Ca2+, Mg2+, Cu2+, Mn2+) and stimulated by 50 mM Fe2+. Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect. (C) 2011 Elsevier Ltd. All rights reserved.

Novel glucoside of physiological active vanillyl alcohol was synthesized for the first time using maltase from Saccharomyces cerevisiae as catalyst, and established its structure as 4-hydroxy-3-methoxybenzyl-alpha-d-glucopyranoside. The key reaction factors for this transglucosylation reaction were optimized using response surface methodology and the highest yield so far in maltase catalyzed transglucosylation reaction was obtained. It was found out that optimum temperature of reaction was 37 A degrees C, optimal maltose concentration was 60% (w/v), optimal pH was 6.6, and optimal concentration of vanillyl alcohol was 158 mM. Under these conditions, yield of glucoside was 90 mM with no by product formation. It was shown that this compound posses good antioxidant activity as well as stability in gastrointestinal tract. It was demonstrated that it is hydrolyzed on brush border membrane of enterocytes, so it can serve in protecting gastrointestinal system from oxidation, as well as source of anticonvulsive drug after the hydrolysis of glucoside on brush border membrane of small intestine.

In this report, alpha-isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highly efficient chemoselective and diastereoselective reaction, catalyzed by maltase from bakers' yeast (Saccharomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with 50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of the reaction was determined as 36.5 degrees C, the optimal maltose concentration was 40% (w/v), the optimal pH was 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of alpha-isosalicin was obtained, with a yield of 10 g/L, and no by product formation was observed.

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active alpha isosalicin by baker's yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

Diverse recombinant immunoreagents specific for TNT-derivatives were tested in different assay forms in order to analyze their specificity and sensitivity. Performance of immunoassays was based on TNP-protein conjugates immobilization on a solid surface. In this work, the detection limit for TNT-analog TNP-Tris was 250 fmol or 87 pg mL−1 (87 ppt), which represents the most sensitive assay published until now, regarding the detection of recombinant antibodies.

An approach to stable covalent immobilization of chemically modified penicillin G acylase from Escherichia coli on Sepabeads® carriers with high retention of hydrolytic activity and thermal stability is presented. The two amino-activated polymethacrylate particulate polymers with different spacer lengths used in the study were Sepabeads® EC EA and Sepabeads® EC HA. The enzyme was first modified by cross-linking with polyaldehyde derivatives of starch in order to provide it with new useful functions. Such modified enzyme was then covalently immobilized on amino supports. The method seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause the loss of activity. Performances of these immobilized biocatalysts were compared with those obtained by the conventional method with respect to activity and thermal stability. The thermal stability study shows that starch-PGA immobilized on Sepabeads EC-EA was almost 4.5-fold more stable than the conventionally immobilized one and 7-fold more stable than free non-modified PGA. Similarly, starch-PGA immobilized on Sepabeads EC-HA was around 1.5- fold more stable than the conventionally immobilized one and almost 9.5-fold more stable than free non-modified enzyme.

Hydroquinone alpha-isomaltoside and hydroquinone alpha-glucoside were synthesized by transglucosylation in an aqueous system with baker's yeast alpha-glucosidase from hydroquinone and maltose as a glucosyl donor. Only one phenolic group was glucosylated, with alpha-selectivity, and the nature of the reaction products was governed by the concentration of hydroquinone. The optimal conditions for synthesis of glycosides were 9 mM hydroquinone and 1.5 M maltose in a 100 mM sodium citrate/phosphate buffer at pH 5.0 and 30 degrees C for 20 h. Under these conditions both hydroquinone alpha-glycosides were obtained in nearly equimolar amounts with a total molar yield of 28% with respect to hydroquinone and a total glycoside concentration of 1 mg/mL in the reaction mixture. (c) 2005 Elsevier B.V. All rights reserved.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.