Kinetic study on the effect of ultrasound on lipase-catalyzed hydrolysis of soy oil: Study of the interfacial area and the initial rates

Comparative studies of lipase-catalyzed hydrolysis of soy oil takes place at the interface between the oil and the aqueous solution in solvent-free system were carried out in shaking bath and in ultrasonic bath. The interfacial area between the oil phase and the aqueous phase influences the rate of hydrolysis. Compared to shaking bath, ultrasonic shaking was found to be a more effective procedure to disperse the oil in water. Larger interfacial area and smaller drop size could be obtained in ultrasonic bath. The initial rate of hydrolysis was increased with the increasing of interfacial area. In ultrasonic bath, the highest initial rate of reaction was obtained with the oil volume fraction of 0.7, which was in accord to the highest interfacial area obtained with the oil volume fraction of 0.7, when the ultrasonic power was 1.64 W/cm². The higher initial rate of hydrolysis was 161 mol/m³ min. The ultrasonic bath was a useful way to disperse soy oil in water to obtain a larger interfacial area, which caused the higher initial rate of soy oil hydrolysis in the solvent-free system.     

形态硒的研究过程中硒代胱氨酸的稳定性

运用高效液相色谱-电感耦合等离子体-质谱法（HPLC-ICP-MS）研究了硒代胱氨酸（SeCys2）在目前广泛应用的水浴振荡提取和方便快速的超声提取过程中的稳定性。在水溶性SeCys2的提取过程中,不管是37℃水浴振荡过程还是超声提取过程对SeCys2的稳定性影响都不大。在酶溶性SeCys2的提取过程中,由于SeCys2分子很容易在Se—Se间断裂,而在水浴振荡或超声作用下酶容易发生多肽链断裂或空间构型变化,游离出来的硒代半胱氨酸（SeCys）和酶分子中断裂出来的小分子结合形成新的存在形式,从而导致了SeCys2在C18柱的洗脱时间发生迁移。     

Ultrasound assisted enzyme catalyzed hydrolysis of waste cooking oil under solvent free condition

The present work demonstrates the hydrolysis of waste cooking oil (WCO) under solvent free condition using commercial available immobilized lipase (Novozyme 435) under the influence of ultrasound irradiation. The process parameters were optimized using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound intensity. It has been observed that ultrasound-assisted lipase-catalyzed hydrolysis of WCO would be a promising alternative for conventional methods. A maximum conversion of 75.19% was obtained at mild operating parameters: molar ratio of oil to water (buffer pH 7) 3:1, catalyst loading of 1.25% (w/w), lower ultrasound power 100 W (ultrasound intensity – 7356.68 W m⁻²), duty cycle 50% and temperature (50 °C) in a relatively short reaction time (2 h). The activation energy and thermodynamic study shows that the hydrolysis reaction is more feasible when ultrasound is combined with mechanical agitation as compared with the ultrasound alone and simple conventional stirring technique. Application of ultrasound considerably reduced the reaction time as compared to conventional reaction. The successive use of the catalyst for repetitive cycles under the optimum experimental conditions resulted in a loss of enzymatic activity and also minimized the product conversion.     

Ultrasound assisted enzymatic conversion of non edible oil to methyl esters

Conventional and ultrasound-assisted hydrolysis and subsequent esterification of Nagchampa oil under mild operating conditions have been investigated with an objective of intensification of methyl esters production using a sustainable approach. The effect of ratio of reactants, temperature, enzyme loading, pretreatment of enzyme (using ultrasonic irradiations) on the hydrolysis and esterification reaction has been studied. Optimum conditions for hydrolysis were observed to be 1:1 weight ratio of oil: water for Lip Z and 1:3 for Lip 2 enzymes, enzyme loading of 400 units for Lip Z and 800 mg for Lip 2 enzymes and reaction time of 6 h. In the case of esterification reaction, optimum conditions obtained were oil to methanol molar ratio of 1:2, enzyme loading of 1000 mg and reaction time of 20 h. Use of pretreated enzyme (using ultrasonic irradiations) was found to increase the extent of esterification reaction from 75% to 92.5%. It was observed that use of ultrasound in the reaction significantly intensified the esterification reaction with time requirement reducing from 20 h for conventional stirring based approach to only about 7.5 h in the presence of ultrasound. The extent of esterification obtained with sonicated enzyme also increased to 96% from 75% with unsonicated enzyme.     

Ultrasound assisted enzyme catalyzed transesterification of waste cooking oil with dimethyl carbonate

This work reports the production of biodiesel with waste cooking oil and dimethyl carbonate in solvent free system through transesterification by immobilized enzyme (Novozym 435) under the influence of ultrasound irradiation. The experiments were conducted in an ultrasonic water bath under three different conditions i.e. ultrasonic irradiation (UI) without stirring, UI coupled with stirring and only stirring to compare their overall effects on fatty acid methyl esters (FAME) conversion. As compared with the conventional stirring method, where FAME conversion was 38.69% at 4 h, the UI without stirring significantly enhanced the conversion of enzymatic transesterification to 57.68% for the same reaction time. However the reaction rate was further increased under the condition of ultrasonication coupled with stirring and resulted into higher conversion of 86.61% for the same reaction time. Effects of reaction parameters, such as temperature, ratio of DMC/oil, speed of agitation and enzyme loading on the conversion were investigated. Furthermore, repeated use of Novozym 435 showed gradual decline in both conversion as well as enzyme activity.     

Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors

This study is focused on the enzymatic esterification of phytosterols with different acyl donors to produce the corresponding phytosterol esters catalyzed by Canadia sp. 99-125 lipase under ultrasound irradiation. An ultrasonic frequency of 35 kHz, power of 200 W and time of 1h was determined to guarantee satisfactory degree of esterification and lipase activity. The influence of temperature, substrates concentration and molar ratio was investigated subsequently. The optimum production was achieved in isooctane system at 60°C with phytosterol concentration of 150 μmol/mL and phytosterol to fatty acid molar ratio of 1:1.5, resulting in a phytosterol esters conversion of above 85.7% in short reaction time (8h). Phytosterols esters could also be converted in high yields to the corresponding long-chain acyl esters via transesterification with triacylglycerols (above 90.3%) under ultrasound irradiation. In optimum conditions, the overall esterification reaction rate using the ultrasonic pretreatment process was above 2-fold than that of mechanical stirring process without damage the lipase activity.     

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, 4 h, 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 irradiation promoted efficient solvent-free lipase-catalyzed production of mono- and diacylglycerols from olive oil

This work reports the enzymatic production of mono- and diacylglycerols under the influence of ultrasound irradiation, in a solvent-free system, with and without the presence of surfactants at a constant temperature of 65°C, glycerol to oil molar ratio of 2:1 and a commercial immobilized lipase (Novozym 435) as catalyst. For this purpose, two operation modes were adopted: the use of a sonotrode (ultrasonic probe), without agitation, varying reaction time, irradiation amplitude (25-45% of the total power) and type of surfactant, and a mechanically stirred reactor (600 rpm) under ultrasound irradiation in a water bath, testing different surfactants. Results show that very satisfactory MAG and DAG yields, above 50 wt.%, can be obtained without the use of surfactant, at mild irradiation power supply (～130 W), with no important enzyme activity losses verified, in a relatively short reaction time (2h), and low enzyme content (7.5 wt.%). Also, reaction kinetic results show that contents of MAG+DAG as high as ～65 wt.% can be achieved at longer times (6h), indicating a promising route for producing MAG and DAG using ultrasound irradiation.     

The effect of ultrasound on enzymatic degumming process of rapeseed oil by the use of phospholipase A1

Comparative studies of enzymatic degumming process of rapeseed oil were carried out in mechanical-stirring and ultrasonic-assisted mechanical-stirring systems. The influences of enzyme dosage (10–50 mg/kg), pH (4.5–6), temperature (45–65 °C), water amount (1–3%), ultrasonic power (0.06–0.09 W/cm³) and reaction time were investigated subsequently. A suitable ultrasonic power of 0.07 W/cm³ was determined to guarantee satisfactory degumming efficiency and enzyme activity. Compared to the mechanical-stirring system, optimum temperature of phospholipase A (PLA) in the ultrasonic-assisted mechanical-stirring system was about 5 °C higher, while the effects of pH on both of the two systems were quite similar. Less time and water were used in the ultrasonic-assisted mechanical-stirring system for enzymatic degumming. The study on the quality changes of degummed oils showed that ultrasound could accelerate the oxidation of edible oils due to the effect of cavitation, thus more attention should be paid on the oxidative stability in the further application.     

苦瓜果实中盐酸小檗碱的提取工艺

以新鲜苦瓜为原料,用盐酸小檗碱为对照品,正交设计优化苦瓜中盐酸小檗碱提取条件,采用超声波和水浴提取法等2种工艺提取苦瓜中盐酸小檗碱,研究单因素对盐酸小檗碱提取率的影响,确定提取盐酸小檗碱的最佳条件.水浴提取苦瓜盐酸小檗碱以A2C1B1组合为最佳提取条件,提取率为5.1642mg/g.超声波提取苦瓜盐酸小檗碱以A3B2C2组合为最佳提取条件,提取率为6.3267mg/g.实验结果表明,超声波法提取效果优于水浴提取法.     

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.     

Degradation of 4-chloro 2-aminophenol using combined strategies based on ultrasound,photolysis and ozone

The present work investigates the degradation of 4-chloro 2-aminophenol (4C2AP), a highly toxic organic compound, using ultrasonic reactors and combination of ultrasound with photolysis and ozonation for the first time. Two types of ultrasonic reactors viz. ultrasonic horn and ultrasonic bath operating at frequency of 20 kHz and 36 kHz respectively have been used in the work. The effect of initial pH, temperature and power dissipation of the ultrasonic horn on the degradation rate has been investigated. The established optimum parameters of initial pH as 6 (natural pH of the aqueous solution) and temperature as 30 ± 2 °C were then used in the degradation studies using the combined approaches. Kinetic study revealed that degradation of 4C2AP followed first order kinetics for all the treatment approaches investigated in the present work. It has been established that US + UV + O₃ combined process was the most promising method giving maximum degradation of 4C2AP in both ultrasonic horn (complete removal) and bath (89.9%) with synergistic index as 1.98 and 1.29 respectively. The cavitational yield of ultrasonic bath was found to be eighteen times higher as compared to ultrasonic horn implying that configurations with higher overall areas of transducers would be better selection for large scale treatment. Overall, the work has clearly demonstrated that combined approaches could synergistically remove the toxic pollutant (4C2AP).     

Ultrasound assisted enzyme catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate

The present work illustrates the transesterification of glycerol to glycerol carbonate (GlyC) from dimethyl carbonate (DMC) using commercial immobilized lipase (Novozym 435) under ultrasonic irradiation. The experiments were performed in a batch reactor placed in an ultrasonic water bath using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound power on the conversion of glycerol to GlyC. It has been found that ultrasound-assisted lipase-catalyzed transesterification of glycerol would be a potential alternative to conventional alkali-catalyzed method, as high conversion (99.75%) was obtained at mild operating conditions: molar ratio of DMC to glycerol 3:1, catalyst amount of 13% (w/w), lower power input (100 W), duty cycle 50% and temperature (60 °C) in a relatively short reaction time (4 h) using Novozym 435 as catalyst. Ultrasound reduces the reaction time up to 4 h as compared to conventional stirring method (14 h) catalyzed by Novozym 435. The repeated use of the catalyst under the optimum experimental condition resulted in decay in both enzyme activity and product conversion.     

Ultrasound-assisted dextranase entrapment onto Ca-alginate gel beads

In this research work, dextranase has immobilized onto calcium alginate beads using a novel ultrasound method. The process of immobilization of the enzyme was carried out in a one-step ultrasound process. Effects of ultrasound conditions on loading efficiency and immobilization yield of the enzyme onto calcium alginate beads were investigated. Furthermore, the activity of the free and immobilized enzymes prepared with and without ultrasound treatment, as a function of pH, temperature, recyclability and enzyme kinetic parameters, was compared. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with an ultrasonic irradiation at 25 kHz, 40 W for 15 min, under which the loading efficiency and the immobilization yield increased by 27.21% and 18.77%, respectively, compared with the immobilized enzymes prepared without ultrasonic irradiation. On the other hand, immobilized enzyme prepared with ultrasonic irradiation showed V_max and K_M value higher than that for the immobilized enzyme prepared without ultrasonic irradiation, likewise, both the catalytic and specificity constants of immobilized enzyme prepared with ultrasonic irradiation were higher than that for immobilized enzyme prepared without ultrasound, indicating that, this new ultrasonic method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared with immobilized enzyme prepared without ultrasound treatment, the immobilized enzymes prepared with ultrasound irradiation exhibited: a higher pH optimum, optimal reaction temperature, activation energy, and thermal stability, as well as, a higher recyclability, which, illustrating the effectiveness of the sonochemical method. To the best of our knowledge, this is the first report on the effect of ultrasound treatments on the immobilization of dextranase.     

Enzymatic catalyzed palm oil hydrolysis under ultrasound irradiation: Diacylglycerol synthesis

Diacylglycerol (DAG) rich oils have an organoleptic property like that of regular edible oils, but these oils do not tend to be accumulated as fat. Palm oil ranks first in the world in terms of edible oil production owing to its low cost. The aim of this study was to propose a new methodology to produce diacylglycerol by hydrolysis of palm oil using Lipozyme RM IM commercial lipase as a catalyst under ultrasound irradiation. The reactions were carried out at 55 °C with two different methods. First, the reaction system was exposed to ultrasonic waves for the whole reaction time, which led to enzymatic inactivation and water evaporation. Ultrasound was then used to promote emulsification of the water/oil system before the hydrolysis reaction, avoiding contact between the probe and the enzymes. An experimental design was used to optimize the ultrasound-related parameters and maximize the hydrolysis rate, and in these conditions, with a change in equilibrium, DAG production was evaluated.Better reaction conditions were achieved for the second method: 11.20 wt.% (water + oil mass) water content, 1.36 wt.% (water + oil mass) enzyme load, 12 h of reaction time, 1.2 min and 200 W of exposure to ultrasound. In these conditions diacylglycerol yield was 34.17 wt.%.     

Ultrasound assisted enzymatic degumming of crude soybean oil

The present work deals with ultrasound assisted enzymatic degumming (UAED) of crude soybean oil quantifying the extent of degumming (EOD), cavitational yield and synergistic index (f) for the combination approaches. The effect of different operating parameters such as enzyme loading, pH, presence of water, temperature and ultrasonic power on the EOD has been investigated. Ultrasound combined with enzyme at loading of 2.0 ml/L resulted in EOD as 92.2% under ambient conditions. Addition of water (5%) in combination with ultrasound and enzyme at 2.0 ml/L loading and pH of 5 resulted in maximum EOD (98.4%) in 120 min of treatment. The extent of phospholipid separation was also observed to be dependent on the power dissipation and maximum phospholipids separation was obtained at 100 W. Scale-up studies were performed at 500 ml and 1 L operating volume under optimized conditions of 2.0 ml/L as the enzyme loading, pH of 5, 5% water addition and ultrasonic power of 100 W where 93.63% and 91.15% phospholipid separation respectively was obtained. The effects of ultrasonic treatment were also quantified in terms of the acid value reduction and oxidative stability for the processed oil. It was demonstrated that suitable reduction in acid value (final value less than 1) and oxidative stability (TOTOX less than 4) is effectively obtained using UAED. Overall the approach of UAED was established to show much higher efficacy for soybean oil processing as compared to only ultrasound or only enzymatic treatment.     

Ultrasound assisted intensification of biodiesel production using enzymatic interesterification

Ultrasound assisted intensification of synthesis of biodiesel from waste cooking oil using methyl acetate and immobilized lipase obtained from Thermomyces lanuginosus (Lipozyme TLIM) as a catalyst has been investigated in the present work. The reaction has also been investigated using the conventional approach based on stirring so as to establish the beneficial effects obtained due to the use of ultrasound. Effect of operating conditions such as reactant molar ratio (oil and methyl acetate), temperature and enzyme loading on the yield of biodiesel has been investigated. Optimum conditions for the conventional approach (without ultrasound) were established as reactant molar ratio of 1:12 (oil:methyl acetate), enzyme loading of 6% (w/v), temperature of 40 °C and reaction time of 24 h and under these conditions, 90.1% biodiesel yield was obtained. The optimum conditions for the ultrasound assisted approach were oil to methyl acetate molar ratio of 1:9, enzyme loading of 3% (w/v), and reaction time of 3 h and the biodiesel yield obtained under these conditions was 96.1%. Use of ultrasound resulted in significant reduction in the reaction time with higher yields and lower requirement of the enzyme loading. The obtained results have clearly established that ultrasound assisted interesterification was a fast and efficient approach for biodiesel production giving significant benefits, which can help in reducing the costs of production. Reusability studies for the enzyme were also performed but it was observed that reuse of the catalyst under the optimum experimental condition resulted in reduced enzyme activity and biodiesel yield.     

An investigation of lipase catalysed sonochemical synthesis: A review

Ultrasonic irradiation has recently gained attention of researchers for its process intensification in numerous reactions. Earlier ultrasound was known for its application either to deactivate enzyme activity or to disrupt the cell. However, in recent years, practice of ultrasonic irradiation began to emerge as a tool for the activation of the enzymes under mild frequency conditions. The incorporation of ultrasound in any of enzymatic reactions not only increases yield but also accelerates the rate of reaction in the presence of mild conditions with better yield and less side-products. To attain maximum yield, it is crucial to understand the mechanism and effect of sonication on reaction especially for the lipase enzyme. Thus, the influence of ultrasound irradiation on reaction yield for different parameters including temperature, enzyme concentration, mole ratio of substrates, solvents ultrasonic frequency and power was reviewed and discussed. The physical effect of cavitation determined by bubble dynamics and rate of reaction through kinetic modelling also needs to be assessed for complete investigation and scale up of synthesis. Thus, prudish utilisation of ultrasound for enzymatic synthesis can serve better future for sustainable and green chemistry.     

几种荧光素酯的自然水解和脂酶催化水解反应的研究

本文研究了荧光素的发光条件以及浓度、温度、pH对四种荧光素酯(荧光素二乙酸酯、荧光素二丙酸酯、荧光素二丁酸酯、荧光素二己酸酯)水解反应的影响,讨论了在脂酶存在下荧光素酯的分解反应机理,确定了每种荧光素酯的最佳酶反应条件.实验结果表明,脂酶对荧光素二丙酸酯(FDP)的酶反应效率最好,有应用前景.     

Effect of operational parameters on degradation of Malachite Green by ultrasonic irradiation

The aim of the present study was to apply ultrasonic technique to remove Malachite Oxalate Green (MG) from aqueous solution. An ultrasonic bath with frequency of 35 kHz was used to investigate the effect of different operational parameters such as MG concentration, power density, temperature, mechanical agitation and addition of EtOH, 2-PrOH and iso-BuOH. Decolorization of MG follows a first order kinetics and hydroxyl radicals have an important role in degradation of MG. The apparent reaction rate constant (k(ap)) was influenced by variation of operational parameters. The activation energy was 30.95 kJ/mol in temperature range of 21-34 degrees C, suggesting a diffusion-controlled reaction. Alcohols act as hydroxyl radicals scavengers having undesirable contribution. UV-vis spectral change of MG showed hypsochromic shift occurred with increasing sonication time, proposing N-demethylation process of MG.