Effect of intense pulsed light on the deactivation of lipase: Enzyme-deactivation kinetics and tertiary structural changes by fragmentation.

The effect of intense pulsed light (IPL) irradiation on Chromobacterium viscosum lipase was investigated with a primary focus on catalytic activity and molecular structure. During IPL irradiation, lipase activity decreased significantly with increasing pulse fluence (Fp) and exposure time (te). IPL-induced deactivation kinetics were further elucidated based on a two-step series-type deactivation model (constant deactivation rate k1 >k2). Fp was found to be the dominant variable affecting the degree of lipase deactivation, and residual activity was not associated with increasing te below a certain Fp energy density (2.66 mJ/cm2), implying a critical threshold for IPL-induced deactivation of lipase. From the results of fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we determined that IPL-induced deactivation was caused by fragmentation, leading to lipase tertiary structural changes. Furthermore, the results of FindPept analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the internal sensitive bonds of lipase were cleaved preferentially by IPL, such that IPL irradiation induced site-sensitive fragmentation and peptide bond cleavage.

Effect of visible light on catalytic hydrolysis of p-nitrophenyl palmitate by the Pseudomonas cepacia lipase immobilized on sol-gel support.

This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250-750 nm. The reaction follows Michaelis-Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver-Burk plot K M and V max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.

An efficient enzymatic modification of cordycepin in ionic liquids under ultrasonic irradiation.

A comparative study of the immobilized Candida antarctica lipase B (Novozym 435)-catalyzed acylation of cordycepin with vinyl acetate in ionic liquids (ILs) under ultrasonic irradiation and shaking was conducted. The application of ultrasonic irradiation instead of shaking during acylation resulted in an enhanced reaction rate and a higher level of substrate conversion. Among the various ILs examined, 1-butyl-3-methylimidazolium tetrafluorobrate ([C4MIm][BF4]) was the best medium for the reaction because it produced the highest substrate conversion. In [C4MIm][BF4], the optimal ultrasonic power, water activity, and reaction temperature were 120 W, 0.33, and 50 °C, respectively. The acylation of cordycepin in [C4MIm][BF4] proved to be regioselective under both conditions: the C5'-OH was acylated. Novozym 435 exhibited a much higher operational stability in [C4MIm][BF4], and 58.0% of its original activity was maintained after ten reuse cycles under ultrasonic irradiation. Compared with the cordycepin, the rate of adenosine deaminase-catalyzed deamination was greatly reduced when the 5'-OH was substituted by acetyl group. These results demonstrated that the combined application of ultrasonic irradiation and IL as a medium was an efficient approach for the enzymatic modification of cordycepin.

Ultrasound irradiation promoted enzymatic transesterification of (R/S)-1-chloro-3-(1-naphthyloxy)-2-propanol.

(R)-1-Chloro-3-(1-naphthyloxy)-2-propanol, which is the key intermediate of (S)-propranolol, was successfully prepared via enantioselective transesterification catalyzed by lipase under ultrasound irradiation. Compared with conventional shaking, the enzyme activity and enantioselectivity were dramatically increased under ultrasound exposure. Effects of various reaction conditions on the synthetic activity of enzyme as well as enantioselectivity, including the type of enzyme, ultrasound power, solvent, acyl donor, temperature and substrate molar ratio, were investigated. Pseudomonas sp. lipase (PSL) showed an excellent catalytic performance under optimum conditions (enzyme activity: 78.3 ± 3.2 µmol·g⁻¹·min⁻¹, E value: 98 ± 6).

Kinetics of ultrasound-assisted lipase-catalyzed glycerolysis of olive oil in solvent-free system.

This work reports experimental kinetic data of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. The experiments were performed in a mechanically stirred reactor under ultrasound irradiation, evaluating the effects of temperature (50-70 °C), enzyme concentration (2.5-10 wt%) and glycerol to oil molar ratio (0.8:1-3:1). Results show that ultrasound-assisted lipase-catalyzed glycerolysis might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides, were achieved at mild irradiation power supply (~130 W) and temperature, in a relatively short reaction time (2h) and low enzyme content (7.5 wt%). To completeness, two simplified kinetic modeling approaches, based on the ordered-sequential bi bi mechanism and reaction stoichiometry, were employed to represent the experimental data, thus allowing a better understanding of the reaction kinetics.

Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system.

This work reports the transesterification of soybean oil with ethanol using two commercial immobilized lipases under the influence of ultrasound irradiation. The experiments were performed in an ultrasonic water bath, following a sequence of experimental designs to assess the effects of temperature, enzyme and water concentrations, oil to ethanol molar ratio and output irradiation power on the reaction yield. Results show that ultrasound-assisted lipase-catalyzed transesterification of soybean oil with ethanol might be a potential alternative route to conventional alkali-catalyzed method, as high reaction yields (~90 wt.%) were obtained at mild irradiation power supply (~100 W), and temperature (60 °C) in a relatively short reaction time, 4h, using Lipozyme RM IM as catalyst. The repeated use of the catalyst under the optimum experimental condition resulted in a decay in both enzyme activity and product conversion after two cycles. The use of Novozym 435 led to lower conversions (about 57%) but the enzyme activity was stable after eight cycles of use, showing, however, a reduction in product conversion after the forth cycle.

Ultrasound assisted lipase catalyzed synthesis of poly-6-hydroxyhexanoate.

Ultrasonic irradiation greatly improved the Candida antarctica lipase B mediated ring opening polymerization of ε-caprolactone to poly-6-hydroxyhexanoate in the ionic liquid 1-ethyl-3-methylimidazolium tetraflouroborate. Compared to the conventional nonsonicated reaction, sonication improved the monomer conversion by 63% and afforded a polymer product of a narrower molecular weight distribution and a higher degree of crystallinity. Under sonication, the polydispersity index of the product was ~1.44 compared to a value of ~2.55 for the product of the conventional reaction. With sonication, nearly 75% of the monomer was converted to product, but the conversion was only ~16% for the reaction carried out conventionally. Compared to conventional operation, sonication enhanced the rate of polymer propagation by >2-fold and the turnover number of the lipase by >3-fold.

Enzymatic transesterification of Jatropha curcas oil assisted by ultrasonication.

Ultrasonication used for the production of fatty acid methyl ester from non-edible vegetable oil using immobilized lipase (Chromobacterium viscosum) as a catalyst from Enterobacter aerogenes to make the process fully ecologically and environmental friendly. The optimal conditions for biodiesel production is the molar ratio oil to methanol 1:4, catalyst concentration 5 wt.% of oil, reaction time 30 min, ultrasonic amplitude 50% (100 W/m(3)) and cycle 0.7s. ultrasonication reduce the reaction time comparing to the conventional batch process. The purity and conversion of the biodiesel was 84.5±0.5 analyzed by reversed phase HPLC.

Enzymatic synthesis of ascorbyl palmitate in ultrasound-assisted system: process optimization and kinetic evaluation.

This work is focused on the optimization of reaction parameters for the synthesis of ascorbyl palmitate catalyzed by Candida antarctica lipase in different organic solvents under ultrasound irradiation. The sequential strategy of experimental design proved to be useful in determining the optimal conditions for reaction conversion in tert-butanol system using Novozym 435 as catalyst. The optimum production was achieved at 70°C, ascorbic acid to palmitic acid molar ratio of 1:9, enzyme concentration of 5 wt% at 3h of reaction, resulting in an ascorbyl palmitate conversion of about 27%. Reaction kinetics for ascorbyl palmitate production in ultrasound device showed that satisfactory reaction conversions (∼26%) could be achieved in short reaction times (2h). The empirical kinetic model proposed is able to satisfactorily represent and predict the experimental data.

The effect of ultrasound on lipase-catalyzed regioselective acylation of mangiferin in non-aqueous solvents.

A simple and efficient method for regioselective acylation of mangiferin catalyzed by lipase under ultrasound irradiation is reported. Compared with the conventional methods, its main advantages are shorter reaction time and higher yields. The optimum conditions were screened out. Under the optimal conditions (lipase: PCL, acyl donor: vinyl acetate; reaction solvent: DMSO, reaction temperature: 45 degrees C, ultrasonic power: 200 W; substrate ratio: acyl donor/mangiferin 6/1, enzyme loading: 6 mg/ml), the regioselective acylation yield was up to 84%.

Hydrolytic enzymes in activated sludge: extraction of protease and lipase by stirring and ultrasonication.

Hydrolytic enzymes released by the microorganisms in activated sludge are responsible for the organic matter degradation; however, the optimal extraction procedure of this valuable resource has not been well established until now. The present study evaluates the recovery of protease and lipase from the activated sludge by using stirring and ultrasonication, varying different parameters such as extraction time, concentration of additives (Triton X100, Cation Exchange Resin and Tris buffer), stirring velocity, ultrasonic power and sludge source. Sludge was collected from two urban wastewater treatment plants located in Prague (Czech Republic) and Reus (Spain). It was found that stirring using 2% v/v Triton X100 for 1h was enough to extract 57.4 protease units/g VSS, and that the same method using a combination of 10mM Tris pH 7.5+0.48 g/mL CER+0.5% TX100 as an additive allowed to extract 15.5 lipase units/g VSS from sludge collected from Reus Wastewater Treatment Plant. Ultrasonication allowed reducing the extraction time to 10 min for protease (using 2% v/v Triton X100 yielding 52.9 units/g VSS) and to 20 min for lipase (without any additive yielding nearly 21.4 units/g VSS), which makes this method appropriate for the extraction of enzymes from the activated sludge, and suitable to be scaled up for its application in the industry.

Lipase-catalyzed diacylglycerol production under sonochemical irradiation.

The present paper describes a protocol for production of diacylglycerol by the partial hydrolysis of soybean oil catalyzed by lipase under ultrasound irradiation. Better yields and shorter reaction times were obtained under sonication as compared to the thermal process.

Effect of monopropylene glycol and gamma irradiation on Yarrowia lipolytica lipase stabilization.

This work investigated the effects of monopropylene glycol, protease inhibitor, and gamma irradiation on Yarrowia lipolytica lipase stability during storage. Enzyme liquid stabilization was achieved by addition of monopropylene glycol (MPG) at respective concentrations of 50, 75, and 90%, the protease inhibitors (P2714 and P8215) at 0.1%, and the gamma irradiation with 10kGy, 15kGy, and 25kGy doses. The results showed that monopropylene glycol limited the microorganism growth and decreased the enzymatic activity at high concentration (up to 50%), at two temperatures (20 and 4 degrees C). Enzyme stored at 20 degrees C lost its activity by 80% after two months. This loss was attributed to the protease's effect. At this temperature, the protease's activities have been limited by the specific inhibitors. The gamma irradiations improve microbial safety of liquid enzyme.

The effect of ultrasound on lipase-catalyzed hydrolysis of soy oil in solvent-free system.

Comparative studies of lipase-catalyzed hydrolysis of soy oil in solvent-free system were carried out in shaking bath and in ultrasonic bath. A suitable ultrasonic power of 1.64 W cm(-2) was determined to guarantee satisfactory hydrolysis extent and lipase activity. The influence of temperature, pH, enzyme concentration and water/oil ratio was investigated subsequently. Compared with that in shaking bath, optimum temperature and inactivation temperature of lipase in ultrasonic bath were about 5-10 degrees C higher, while pH effect in ultrasonic bath was similar; ultrasound also led to a smooth increase of reaction rate at relatively higher enzyme loading and less use of water to saturate hydrolysis substrate. In optimum conditions, the overall hydrolysis reaction rate in the ultrasonic bath process was above 2-fold than that in the shaking bath process.

Probing the effects of microwave irradiation on enzyme-catalysed organic transformations: the case of lipase-catalysed transesterification reactions.

The lipase-catalysed transesterification reaction of methyl acetoacetate in toluene as a solvent has been studied using carefully controlled conditions. Results suggest that microwave heating does not have a noticeable effect on reaction rate or product conversion.

Stability improvement of immobilized Candida antarctica lipase B in an organic medium under microwave radiation.

The influence of microwave heating on the stability of immobilized Candida antarctica lipase B was studied at 100 degrees in an organic medium. The microwave radiation was carried out before enzymatic reaction (storage conditions) or during the enzymatic catalysis (use conditions). In both cases, enzymatic stability was higher under microwave heating than under conventional thermal heating, in strictly identical operating conditions. Furthermore, the gain of enzymatic stability under microwave heating appears to be higher in a more polar solvent, which interacts strongly with the microwave field. Our results suggest that microwave radiation has an effect, not related to temperature, on the process of enzymatic inactivation.

Influence of gamma-irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus.

The effect of gamma-irradiation and maize lipids on aflatoxin B1 production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (aw 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B1 were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full-fat maize (FM) and defatted maize (DM) media and no aflatoxin B1 production at 3.0 kGy gamma-irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at aw 0.84 and produce aflatoxin B1 is related to the lipid composition of maize. The enhancement of aflatoxin B1 at low doses was correlated to the enhancement of fungal lipase activity.

Effect of 24 hours light on circadian rhythms of secretory enzymes and morphology of rat von Ebner's glands.

Von Ebner's glands of the rat are minor salivary serous glands in the posterior portion of the tongue. They secrete two digestive enzymes, lingual lipase and amylase. In this investigation, circadian rhythm in feeding was established under a normal 12 h light/12 h dark cycle, with the rats eating primarily during the dark period. At lights on, the size of the acinar cells and the area of the inclusive secretory granules, and the amount of digestive enzyme activity (lingual lipase and amylase) remaining in the gland was significantly less than in the mid-afternoon, after very little daylight food consumption. However, after 7 days of continuous light the circadian rhythm was altered: the food consumption during the normal night-time hours (5 p.m. to 8 a.m.) went from 88% of total 24 h food consumption to 45%, and during normal daylight hours (8 a.m. to 5 p.m.) from 12% to 55%. These changes were correlated with histometric findings of a near reversal of the areas of acinar cells and secretory granules of a.m. and p.m. samples under continuous light. Lingual lipase activity in the glands went from 35% under 12 h light to 61% under continuous light in the a.m. and from 65% to 39% in the p.m. Amylase activity also showed nearly a reversal in activity remaining in the gland, from 36% at 12 h light to 58% at 24 h light in the a.m. and 64% to 41% for the p.m. samples. These results indicate that the von Ebner's glands of the rat have a circadian rhythm of secretion and storage of secretory proteins that is subject to light entrainment similar to that seen in other exocrine glands such as the parotid and pancreas.

Microwave incubation improves lipolytic enzyme preservation for ultrastructural cytochemistry.

Standard methods for the ultrastructural detection of lipase and sphingomyelinase activities in the skin result in considerable loss of structural preservation, often interfering with accurate delineation of enzyme localization in association with specific organelles. Moreover, poor preservation occurs, even after extensive aldehyde prefixation, owing to the prolonged incubation times needed to detect residual enzyme activity, which often require non-physiological conditions. A modified incubation protocol is described here, which uses microwave irradiation in conjunction with drastically shortened incubation times, resulting in both superior ultrastructural preservation and excellent localization in mammalian epidermis. This method should be useful generally not only for the study of lipase localization in skin, but also in conjunction with the cytochemical detection of a variety of enzymes in various types of tissue.