Dušan Veličković

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite

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 10mM 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°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+)) and stimulated by 50mM Fe(2+). Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.

Structural Elucidation of Enzymatically Synthesized Galacto-oligosaccharides Using Ion-Mobility Spectrometry–Tandem Mass Spectrometry

Galacto-oligosaccharides (GOS) represent a diverse group of well-characterized prebiotic ingredients derived from lactose in a reaction catalyzed with β-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 β-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 β-galactosidase from Aspergillus oryzae has the highest affinity toward formation of β-(1→3) or β-(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.

Design of biocompatible immobilized Candida rugosa lipase with potential application in food industry.

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.

Mass Spectrometric Imaging of Wheat (Triticum spp.) and Barley (Hordeum vulgare L.) Cultivars: Distribution of Major Cell Wall Polysaccharides According to Their Main Structural Features.

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans (BG) are the main components of cereal cell walls and influence many aspects of their end uses. Important variations in the composition and structure of these polysaccharides have been reported among cereals and cultivars of a given species. In this work, the spatial distribution of AX and BG in the endosperm of mature grains was established for nine wheat varieties and eight barley varieties using enzymatically assisted mass spectrometry imaging (MSI). Important structural features of the AX and BG polymers that were previously shown to influence their physicochemical properties were assessed. Differences in the distribution of AX and BG structures were observed, both within the endosperm of a given cultivar and between wheat and barley cultivars. This study provides a unique picture of the structural heterogeneity of AX and BG polysaccharides at the scale of the whole endosperm in a series of wheat and barley cultivars. Thus, it can participate meaningfully in a strategy aiming at understanding the structure-function relationships of these two polymers.

Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity

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.

Specificity of maltase to maltose in three different directions of reaction: hydrolytic, vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis.

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 Km value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate.

Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker’s yeast maltase

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 α 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.

Dual effect of benzyl alcohol on α-glucosidase activity: efficient substrate for high yield transglucosylation and non-competitive inhibitor of its hydrolytic activity.

Benzyl alcohol, a potent anesthetic and bacteriostatic, can be efficiently glucosylated by α-glucosidase from Saccharomyces cerevisiae to produce benzyl alcohol α-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 α-glucosidases hydrolytic activity (Ki=18mM, 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 α-glucosydase inhibitors binding, since the mechanism of inhibition has not yet been elucidated.

A study of transglucosylation kinetic in an enzymatic synthesis of benzyl alcohol glucoside by α-glucosidase from S. cerevisiae

Abstractα-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.

Synthesis of medium-chain length capsinoids from coconut oil catalyzed by Candida rugosa lipases

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.