Influence of sodium alginate pretreated by ultrasound on papain properties: Activity,structure, conformation and molecular weight and distribution

The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm²), 135 kHz, 50 °C for 20 min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25 W/cm², compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25 W/cm². It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25 W/cm². The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm², under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (M_w) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.     

Synergistic effect of ultrasonication and co-immobilized enzymes on tomato peels for lycopene extraction

In the present work, tomato peels were pre-treated using combination of ultrasound and enzyme co-immobilized amino-functionalized magnetic nanoparticles (AMNPs) for the efficient release of lycopene. To achieve maximum activity of enzymes in the co-immobilized form, optimization of several parameters were carried out. Moreover, the influence of ultrasound and enzyme co-immobilized magnetic nanoparticles on lycopene release was studied. Maximum lycopene release was obtained at 3% (w/w) enzyme co-immobilized AMNPs, pH 5.0, temperature of 50 °C, at 10 W ultrasound power and 20 min incubation time. After enzymatic pre-treatment, lycopene from the pre-treated mixture was extracted and separated using tri-solvent extraction method. Maximum recovery of lycopene using solvent extraction was obtained at 50 °C, 90 min of incubation time and agitation speed of 150 rpm. The presence of lycopene in the extract was confirmed by FT-IR, UV–vis spectroscopy and HPLC analysis. The co-immobilized bio-catalyst showed excellent reusability giving more than 50% lycopene yield even after 6th cycles of reuse.     

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.     

Low intensity ultrasound as a pretreatment to drying of daylilies: Impact on enzyme inactivation,color changes and nutrition quality parameters

Effects of thermal and low intensity ultrasound combined with heat (LIUH) pretreatment prior to microwave vacuum drying on enzyme inactivation, color changes and nutrition quality properties of dried daylilies were investigated. The peroxidase (POD), ascorbic acid oxidase (AAO) and polyphenoloxidase (PPO) thermal and LIUH (0.2 and 0.4 W/cm²) inactivation were determined and compared at 70, 80 and 90 °C. Significant reduction in the POD, AAO and PPO activity was seen in daylilies after an ambient LIUH pretreatment than thermal pretreatment. POD, AAO and PPO thermal and LIUH inactivation followed the first order kinetics. LIUH pretreatment had a more positive influence on maintaining color of dried daylilies than thermal pretreatment. Furthermore, LIUH pretreatment resulted in a significant increase in chlorophylls, carotenoids (lutein, zeaxanthin and β-carotene), and a decrease in degree of browning and 5-hydroxymethylfurfural (HMF) when compared with thermal pretreatment. The antioxidant activity and contents of several nutritional components of dried daylilies pretreated by LIUH were also higher than that of dried daylilies pretreated by thermal pretreatment. This study provides a basis for the design of LIUH conditions to control vegetables browning and color changes prior to drying processing.     

Evaluation on the air-borne ultrasound-assisted hot air convection thin-layer drying performance of municipal sewage sludge

The thin-layer drying behavior of the municipal sewage sludge in a laboratory-scale hot air forced convective dryer assisted with air-borne ultrasound was investigated in between 70 and 130 °C hot air temperatures. The drying kinetics in the convective process alone were compared to that for ultrasound-assist process at three ultrasound powers (30, 90, 150 W). The average drying rates within whole drying temperature range at ultrasound powers of 30, 90 and 150 W increased by about 22.6%, 27.8% and 32.2% compared with the convective drying alone (without ultrasound). As the temperature increasing from 70 °C to 130 °C, there were maximum increasing ratios for the effective moisture diffusivities of the sewage sludge in both falling rate periods at ultrasonic power of 30 W in comparison with other two high powers. In between the ultrasound powers of 0 and 30 W, the effect of the power on the drying rate was significant, while its effect was not obvious over 30 W. Therefore, the low ultrasonic power can be just set in the drying process. The values of the apparent activation energy in the first falling rate period were down from 13.52 to 12.78 kJ mol⁻¹, and from 17.21 to 15.10 kJ mol⁻¹ for the second falling rate period with increasing the ultrasonic power from 30 to 150 W. The values of the apparent activation energy in two falling rate periods with the ultrasound-assist were less than that for the hot air convective drying alone.     

A chemometric approach to evaluate the effects of probe-type ultrasonication on the enzyme inactivation and quality attributes of fresh amla juice

The enzymatic browning induced in amla juice due to the high activity of polyphenol oxidase (PPO) and peroxidase (POD) is one of the critical issues faced by the industry. The present study assessed the suitability of non-thermal, high-intensity ultrasound (US) on the inactivation of PPO and POD in fresh Indian Gooseberry juice. Ultrasonic waves, using a 6 mm titanium alloy probe were irradiated in the juice at a maximum power of 455 W and frequency of 20 kHz. The subsequent effects on biochemical attributes were studied using response surface methodology. Inactivation rates of 90.72 % and 73.18 %, respectively, for PPO and POD enzymes, were observed at the highest US intensity and exposure time. Numerical optimisation using the three-factor, three-level Box-Behnken design suggested that an optimum process at 70 % (energy density: 1610 Wcm⁻²) pulsed at 5 s on and 5 s off for 7 min 30 s resulted in PPO and POD inactivation of the order of 76.42 % and 64.57 % respectively. At these experimental conditions, the optimized levels of biochemical attributes i.e., ascorbic acid (738.50 mg/100 mL), total phenols (17.10 mg/mL), DPPH antioxidant activity (58.47 %), tannins (7.11 µg/mL), colour change (ΔE = 9.04) and flavonoids (6.14 mg/mL) were achieved. The overall statistical models were significant for all the responses except for reducing sugars. Furthermore, the approximation equations for individual responses indicated that the goodness of fit was adequate (R² > 0.90). The results suggested that ultrasound is a suitable processing technique for amla juice stabilisation compared to thermal treatments that result in the loss of quality.     

Influence of low ultrasound intensity on the degradation of dextran catalyzed by dextranase

In our current research work, the effect of ultrasound irradiation on the enzymatic activity and enzymatic hydrolysis kinetic parameters of dextran catalysis by dextranase were investigated. Furthermore, the effects of ultrasound irradiation on the structure of dextranase were investigated with the aid of fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The maximum activity of dextranase was observed when the sample was treated with ultrasound at 25 kHz, 40 W for 15 min, under which the enzyme activity increased by 13.43% compared the routine thermal incubation at 50 °C. Experimental Kinetics results, demonstrated that, both the V_max and K_M values of dextranase increased with ultrasound-treated compared with the incubation at 50 °C. Likewise, both the catalytic and specificity constants were higher under the effects of an ultrasonic field, indicating that, the substrate is converted into the product at an increased rate when compared with the incubation at 50 °C.On the other hand, fluorescence and CD spectra reflected that the ultrasound irradiation had increased the number of tryptophan on dextranase surface with increased α-helix by 15.74% and reduced random coil by 5.41% upon ultrasound-treated dextranase protein compared to the control, which were helpful for the improvement of its activity.     

Liquid-phase adsorption and desorption of phenol onto activated carbons with ultrasound

The effect of 48-kHz ultrasound on the adsorption and desorption of phenol from aqueous solutions onto coconut shell-based granular activated carbons was studied at 25 degrees C. Experiments were performed at different carbon particle sizes (1.15, 2.5, 4.0 mm), initial phenol concentrations (1.06-10.6 mol/m3), and ultrasonic powers (46-133 W). Regardless of the absence and presence of ultrasound, the adsorption isotherms were well obeyed by the Langmuir equation. When ultrasound was applied in the whole adsorption process, the adsorption capacity decreased but the Langmuir constant increased with increasing ultrasonic power. According to the analysis of kinetic data by the Elovich equation, it was shown that the initial rate of adsorption was enhanced after sonication and the number of sites available for adsorption was reduced. The effect of ultrasonic intensity on the initial rate and final amount of desorption of phenol from the loaded carbons using 0.1 mol/dm3 of NaOH were also evaluated and compared.     

超高压和酶抑制剂联合处理对荔枝果肉中过氧化物酶和果胶甲基酯酶的影响

 为了研究超高压与酶抑制剂联合处理对荔枝果肉中过氧化物酶（POD）和果胶甲基酯酶（PME）的影响，将荔枝（“淮枝”品种）果肉在两种酶抑制剂组合溶液（A：5 g/L柠檬酸+2.5 g/L L-抗坏血酸+5 g/L氯化钙；B：10 g/L柠檬酸+5 g/L L-抗坏血酸+10 g/L氯化钙）中分别浸泡10 min，并在100~400 MPa压力、10 ℃温度条件下处理30 min，采用分光光度法测定果肉中POD、PME的活性。结果表明：A、B两种组合处理能够明显钝化POD，但却显著激活了PME；超高压与A组合联合处理不能使POD、PME活性下降；超高压与B组合联合处理对POD、PME的影响与压力值有关系，100~300 MPa的超高压与B组合联合处理使POD活性下降，200~400 MPa的超高压与B组合联合处理则使PME活性升高。因此，超高压与酶抑制剂联合处理对荔枝果肉中POD的钝化存在一定的协同效应，且浓度越高，协同抑制效应越明显；而超高压与酶抑制剂联合处理对荔枝果肉中PME的钝化却表现出一定的拮抗性。     

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.     

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.     

Crystallization of potash alum: effect of power ultrasound.

The influence of power ultrasound on the crystallization of potash alum was investigated. Experiments have been carried out in a batch stirred vessel. It was found that ultrasonic waves decrease the supersaturation limits and modify the morphology of the crystals produced. The average crystal size decreases with an increase of ultrasonic power. To investigate also the action of ultrasound on already existing crystals, crystals produced in silent conditions were suspended in saturated potash alum solution at various ultrasonic powers. The results show that ultrasound has also an abrasive effect on potash alum crystals for high power inputs.     

Synergistic effects of combining ultrasound with the Fenton process in the degradation of Reactive Blue 19

The decoloration of reactive dye C.I. Reactive Blue 19 (RB 19) using combined ultrasound with the Fenton process has been investigated. The effect of varying the concentrations of hydrogen peroxide and iron sulfate, initial pH, ultrasonic power, initial dye concentration and dissolved gas on the decoloration and degradation efficiencies was measured. Calibration of the ultrasound systems was performed using calorimetric measurements and oxidative species monitoring using the Fricke dosimeter and degradations were carried out with a 20 kHz probe type transducer at 2, 4, 6 and 8 W cm⁻² of acoustic intensity at 15, 25, 50 and 75 mg L⁻¹ initial dye concentrations. First order rate kinetics was observed. It was found that while the degradation rate due to ultrasound alone was slow, sonication significantly accelerated the Fenton reaction. While the results were similar to those reported for other dyes, the effects occurred at lower concentrations. The rate and extent of decoloration of RB 19 increased with rising hydrogen peroxide concentration, ultrasonic powers and iron sulfate concentration but decreased with increasing dye concentration. An optimum pH value of pH = 3.5 was found. The rate of decoloration was higher when dissolved oxygen was present as compared with nitrogen and argon confirming the solution phase mechanism of the degradation.     

~(12)C~(6+)离子辐照对苜蓿M1代低温胁迫存活率、过氧化氢酶及过氧化物酶活性的影响

研究了不同剂量12C6+离子辐照对中兰1号、BC-04-477、塔城3种苜蓿M1代个体在低温胁迫下存活率、过氧化氢酶(CAT)及过氧化物酶(POD)活性的影响。在辐照剂量为400 Gy时,中兰1号低温胁迫组存活率、CAT活性比未辐照的对照分别提高了33.3%,56.3%,POD活性与未辐照的对照无差异;在辐照剂量为400 Gy时,BC-04-477低温胁迫组存活率、CAT及POD活性比未辐照的对照组分别提高了33.3%,69.2%,5.1%;塔城在辐照剂量为800 Gy时,低温胁迫组的存活率、CAT及POD活性比未辐照的对照组分别提高了25%,26%,22.8%。以上结果表明,12C6+离子辐照可以提高中兰1号、BC-04-477、塔城的低温环境的存活能力,提高苜蓿抗寒性能。     

Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution

The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60 W of ultrasonic rated power and ultrasonic treatment time of 30 min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (E_a), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products.     

Ultrasonic inactivation of Bacillus alpha-amylase. I. effect of gas content and emitting face of probe

Sonoinactivation of alpha-amylase from Bacillus amyloliquefacience was studied at a constant frequency of 30 kHz. The effect of sonotrode emitting face and gas content of medium on the efficiency of enzyme inactivation were investigated at different time-temperature combinations and generation of OH free radicals was also monitored. The results showed that ultrasound effectively inactivated alpha-amylase with a minimum overall inactivation rate at 50 degrees C. The tip diameter of the sonotrode and the gas content of the medium both significantly affected the rate of enzyme inactivation. The increase of tip diameter increased the effect of ultrasound on the enzyme, while the removal of dissolved gas adversely influenced the cavitational events and reduced the rate of enzyme inactivation. Calculation of the kinetic and activation parameters revealed that ultrasound decreased the activation energy, E(a), activation enthalpy, DeltaH(#), and the activation Gibbs free energy, DeltaG(#), and strongly reduced the activation entropy, DeltaS(#), down to negative values. This huge reduction in activation entropy was attributed to the different mechanisms of inactivation induced by heat and ultrasound. It is proved in this study that ultrasonically generated OH free radicals and shear forces, which arise from pulsation- or collapse of bubbles, both can destabilize the enzyme, although their contribution to the overall inactivation varies depending on the temperature and the tip diameter of the sonotrode.     

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.     

Effect of ultrasonic pretreatment on kinetics of gelatin hydrolysis by collagenase and its mechanism

Gelatin is a mixture of soluble proteins prepared by partial hydrolysis of native collagen. Gelatin can be enzymatically hydrolyzed to produce bioactive hydrolysates. However, the preparation of gelatin peptide with expected activity is usually a time-consuming process. The production efficiency of gelatin hydrolysates needs to be improved. In present work, effect of ultrasonic pretreatment on kinetic parameters of gelatin hydrolysis by collagenase was investigated based on an established kinetic model. With ultrasonic pretreatment, reaction rate constant and enzyme inactivation constant were increased by 27.5% and 27.8%, respectively. Meanwhile, hydrolysis activation energy and enzyme inactivation energy were reduced by 36.3% and 43.0%, respectively. In order to explore its possible mechanism, influence of sonication on structural properties of gelatin was determined using atomic force microscopy, particle size analyzer, fluorescence spectroscopy, protein solubility test and Fourier transform infrared spectroscopy. Moreover, hydrogen peroxide was used as a positive control for potential sonochemical effect. It was found that reduction of gelatin particle size was mainly caused by physical effect of ultrasound. Increased solubility and variation in β-sheet and random coil elements of gelatin were due to sonochemical effect. Both physical and chemical effects of sonication contributed to the change in α-helix and β-turn structures. The current results suggest that ultrasound can be potentially applied to stimulate the production efficiency of gelatin peptides, mainly due to its effects on modification of protein structures.     

Effect of focused ultrasound stimulation at different ultrasonic power levels on the local field potential power spectrum

Local field potential(LFP) signals of the rat hippocampus were recorded under noninvasive focused ultrasound stimulation(FUS) with different ultrasonic powers. The LFP mean absolute power was calculated with the Welch algorithm at the delta, theta, alpha, beta, and gamma frequency bands. The experimental results demonstrate that the LFP mean absolute power at different frequency bands increases as the ultrasound power increases.     

Effect of ultrasound in enantioselective hydrogenation of 1-phenyl-1,2-propanedione: comparison of catalyst activation, solvents and supports

The enantioselective hydrogenation of 1-phenyl-1,2-propanedione was carried out over Pt/Al2O3, Pt/SiO2, Pt/SF (silica fiber), Pt/C catalysts modified with cinchonidine under ultrasonic irradiation. The initial rate, regioselectivity and enantioselectivity were investigated for different catalyst pretreatments, solvents and ultrasonic powers. The ultrasound effects were very catalyst dependent. The sonication significantly enhanced enantioselectivity and activity of the Pt/SF (silica fiber) catalyst. For the other Pt supported catalysts the reaction rate, enantioselectivity and regioselectivity increased moderately. The choice of solvent influenced the impact of ultrasound effect, namely in mesitylene, which has the lowest vapor pressure, the highest ultrasound enhancement was observed. The effect of sonication on catalysts surface was studied by transmission electron microscopy and scanning electron microscopy (SEM). No significant change in the metal particle size distribution due to sonication was observed. However, in the case of the Pt/SF catalyst, acoustic irradiation induced morphological changes on the catalyst particle surface (SEM), which might be the cause for enhancement of the initial reaction rate and enantioselectivity.