Effects of power ultrasound on the activity and structure of β‐D‐glucosidase with potentially aroma‐enhancing capability

Abstract β‐d‐glucosidase can release aroma precursors to improve the flavor of plant food, but the hydrolysis efficiency of the enzyme is low; the purpose of this study was to improve the enzyme activity using ultrasound. The effects of ultrasound parameters on β‐d‐glucosidase activity were investigated, and the respective structures of enzyme activated and enzyme inhibited were further analyzed. Low temperature (20–45°C), low ultrasonic intensity (<181.53 W/cm2), and short treatment time (<15 min) led to the activation of β‐d‐glucosidase, whereas high temperature (45–60°C), high ultrasonic intensity (>181.53 W/cm2), and long treatment time (>15 min) led to its inhibition. Application of ultrasound lowered the optimum temperature for β‐d‐glucosidase activity from 50 to 40°C. Ultrasound did not change the primary structures of the enzyme, but changed the secondary structures. When ultrasound activated β‐d‐glucosidase, the α‐helix contents were increased, the β‐fold and irregular coil content were reduced. When ultrasound inhibited β‐d‐glucosidase, the contents of β‐folds were increased, the α‐helix and irregular coil contents were reduced.. In summary, activation or inhibition of β‐d‐glucosidase under ultrasound was determined by the ultrasound conditions. This study suggests that ultrasound combined with β‐D‐glucosidase can be used in aroma‐enhancing.

sonic intensity (<181.53 W/cm 2 ), and short treatment time (<15 min) led to the activation of β-d-glucosidase, whereas high temperature (45-60°C), high ultrasonic intensity (>181.53 W/cm 2 ), and long treatment time (>15 min) led to its inhibition. Application of ultrasound lowered the optimum temperature for β-d-glucosidase activity from 50 to 40°C. Ultrasound did not change the primary structures of the enzyme, but changed the secondary structures. When ultrasound activated β-d-glucosidase, the α-helix contents were increased, the β-fold and irregular coil content were reduced. When ultrasound inhibited β-d-glucosidase, the contents of β-folds were increased, the α-helix and irregular coil contents were reduced.. In summary, activation or inhibition of β-d-glucosidase under ultrasound was determined by the ultrasound conditions. This study suggests that ultrasound combined with β-D-glucosidase can be used in aroma-enhancing.
However, the method of β-d-glucosidase hydrolysis of glycosidically bound volatiles has a major drawback in that the hydrolysis time is too long which has limited its application in food industry. Thus, it is essential to improve the enzyme activity. Ultrasound is a potential method for modifying enzyme activity (Delgado-Povedano & Castro, 2015;Huang, Chen, et al., 2017;Nadar & Rathod, 2017), and many researchers have used it to improve food enzymes. For example, Dalagnol, Silveira, Silva, Manfroi, and Rodrigues (2017) found that ultrasound improved the activities of pectinase, xylanase, and cellulase. Ma et al. (2011) found that energy-gathered ultrasound improved the activity of alcalase by 5.8%. Barton, Bullock, and Weir (1996) found that ultrasound improved the activities of some glycosidase enzymes. Conversely, some researchers have used ultrasound to inhibit enzyme activity. Terefe et al. (2009) investigated the ultrasonic inactivation of polygalacturonase and pectin methylesterase in tomato juice at a frequency of 20 kHz and temperatures between 50 and 75°C. Rodrigues, Fernandes, García-Pérez José, and Cárcel (2016) found that ultrasound-assisted drying increased the activities of polyphenoloxidase, peroxidase, and peroxidase ascorbate in apples, but led to partial inactivation of pectin methylesterase. To the best of our knowledge, there have been no reports on the effects of ultrasound on β-d-glucosidase.
The objectives of this study were to evaluate the effects of ultrasonic power, time, temperature, medium pH, and duty cycle on β-d-glucosidase activity, to analyze the structures of the enzyme under its activation and inhibition conditions conducive to, and to explore the mechanism whereby ultrasound affects enzyme activity. The results could expedite the application of ultrasound in enhancing aroma in fruit juice.
Enzyme solution (0.1 ml), pH 5.0 citric acid-trisodium citrate buffer (0.3 ml), and 5 mM pNPG (0.2 ml) were combined in a 10-ml stopper tube, and the mixture was incubated for 10 min at 50°C. Thereafter, 1 M Na 2 CO 3 solution (2 ml) was added to terminate the reaction, and the mixture was diluted to a fixed volume of 5 ml with distilled water.
The absorbance of the yellow mixture was measured at 400 nm to determine the amount of p-nitrophenol. β-d-Glucosidase activity was calculated according to the following formula: where X is the β-d-glucosidase activity (U/ml), c is the concentration of p-nitrophenol (mM), V is the final volume of reaction solution (ml), V′ is the volume of β-d-glucosidase solution (ml), and t is the reaction time (min).

| Ultrasound treatment
Ultrasound treatment was carried out with a probe ultrasonic processor (Scientz-IID; Ningbo Scientz Biotechnology Co.).
Salient parameters of the probe ultrasonic processor were maximum power 950 W, frequency 25 kHz, diameter of horn microtip 2 mm. Aliquots (2 ml) of enzyme solution (prepared as described in Section 2.2) were added to glass tubes (1 cm diameter, 7 cm height), and then, these tubes were immersed in a low-temperature water bath (DC-1006; Safe Corporation) at a preset constant temperature and subjected to ultrasound treatment.
Apart from the specific ultrasound conditions, as mentioned in the Results Section, the general ultrasound conditions were kept constant. The probe was placed 0.5 cm from the top surface of the extraction cell. The liquid height, measured as the distance from the horn microtip to the bottom of the tube, was 1.5 cm. The temperature was 40°C, pulsed mode was applied (1s on and 2s off), the treatment time was 10 min, the pH was 5, and the ultrasonic intensity was 60.51 W/cm 2 .

| Calculation of electrical acoustic intensity
The electrical acoustic intensity dissipated from the probe microtip was calculated according to the following formula (Li, Pordesimo, & Weiss, 2004): where r is the radius of the probe microtip and P is the input power.

| Circular dichroism (CD)
β-d-Glucosidase solution was sonicated, and its circular dichroism spectrum was measured with respect to buffer solution as the blank in a quartz cuvette of 1 mm optical path length at room temperature (20 ± 1°C). Scanning was conducted in the far-UV range of 185-290 nm at 200 nm/min, with 1 nm as spectral spacing (Subhedar & Gogate, 2014). CD data are expressed in the form of mean residue ellipticity [θ] deg cm 2 /dmol. The secondary structures (α-helices, βturns, and β-sheets) of β-d-glucosidase, with or without ultrasound treatment, were analyzed using Dichroweb (Ma et al., 2015).   Figure 1 shows the effect of ultrasonic intensity on β-d-glucosidase

| Effect of ultrasonic intensity on the activity of β-d-glucosidase
activity. The β-d-glucosidase activity was improved by ultrasonic intensity in the range from 0 to 181.53 W/cm 2 (p < 0.05) compared with the untreated enzyme. However, its activity was inhibited by ultrasonic intensity in the range from 181.53 to 484.08 W/cm 2 (p < 0.05).
The highest enzyme activity was achieved at 12.10 W/cm 2 .
The results indicated that ultrasonic intensity is an important factor with regard to enhancing or inhibitory effects of ultrasound on enzyme activity. Under the condition of low intensity, ultrasound generates cavitation, magnetostrictive effects, and mechanical oscillation, which affect the conformation of the enzyme and increase its contact with the substrate. Conversely, high-intensity ultrasound inhibited the enzyme activity, which may be due to the reaction of generated hydroxyl or hydrogen radicals with the protein backbone. This could in turn lead to enzyme aggregation, thereby obstructing the active sites.

| Effect of ultrasonication time on the activity of β-d-glucosidase
The effect of ultrasonication time on the activity of β-d-glucosidase is shown in Figure 2. The β-d-glucosidase activity was improved compared with the untreated enzyme at treatment time ranging from 0 to 15 min (p < 0.05), but was inhibited at treatment times ranging from 15 to 25 min (p < 0.05). The results may be explained that short-term ultrasound treatment at low power intensity changes the conformation and causes more active sites to appear on the surface of the enzyme, whereas long-term ultrasound treatment, even at the appropriate power intensity, can still destroy the conformation.

| Effect of ultrasonication temperature on the activity of β-d-glucosidase
The optimum temperature for β-d-glucosidase activity under untreated conditions was found to be 50°C, but this was lowered to 40°C following ultrasonic treatment (Figure 3). Compared to the untreated enzyme,  (2011) found that ultrasound improved alliinase activity within the tested temperature range and did not affect the optimal temperature for enzyme activity. Liu et al. (2008) reported that ultrasonic treatment raised the optimal temperature for lipase activity by about 5-10°C. These differences may be attributed to the different characteristics of the enzymes.

| Effect of pH on the activity of β-d-glucosidase under ultrasonication
pH is another key factor affecting enzyme activity. Higher or lower pH can lead to partial or complete inactivation of an enzyme. The effect of ultrasound on optimal pH is plotted in Figure 4. For the enzyme under ultrasound irradiation, pH 5 was identified as optimal, consistent with that without ultrasound treatment. This result suggested that ultrasound did not change the optimum pH of β-d-glucosidase and improved its activity under the studied pH conditions. It is well known that pH can influence the ionization state of an enzyme, modifying its active site. The optimal pH conditions in the presence and absence of ultrasound in this study were similar, implying that ultrasound caused little change to the ionization state of β-d-glucosidase. Figure 5 shows the effect of ultrasound duty cycle on β-d-glucosidase

| Circular dichroism(CD) analysis
CD spectroscopy is the most widely applied technique for obtaining information about enzyme secondary structure. The contents of α-helices, β-sheets, β-turns, and random coils in β-d-glucosidase were determined to describe the relationship between the enzyme activity and its secondary structure. Figure 6 shows the CD spectra of control and ultrasound-treated β-d-glucosidase at 60.51 W/ suggesting that ultrasound did not affect the β-turn and random coil contents of the enzyme.
The results suggest that the active site of β-d-glucosidase may lie within an α-helix. The changes of enzyme secondary structure under ultrasound may be due to the high temperature and high pressure of cavitation, and the mechanism needs future studies.

| Fluorescence spectrometric analysis
Fluorescence spectroscopy is a useful technique for following tertiary structural transitions in proteins, because the intrinsic fluorescence of aromatic amino acid residues (such as Trp, Tyr, and Phe residues, particularly Trp) is sensitive to the polarity of microenvironments along the transition (Zhao & Yang, 2008). As shown in Figure 7, the emission fluorescence spectra of untreated and ultrasonically treated β-d-glucosidase each featured a peak at 330 nm.
After treatment with 484.08 W/cm 2 ultrasound, the fluorescence intensity decreased significantly (from 275.0 to 227.5) and the peak was red-shifted (from 330 to 338 nm). After treatment with 60.51 W/cm 2 ultrasound, the intensity showed a weak decrease (275.0-267.0) and the peak was slightly red-shifted (from 330 to 334 nm

| CON CLUS IONS
In summary, the effects of ultrasound on β-d-glucosidase activity were studied, and the mechanism whereby ultrasound affects enzyme activity was further explored. It was found that temperature, ultrasonic intensity, and treatment time determined the activation or inhibition of β-d-glucosidase, whereas duty cycle and pH only impacted the effects. The activation or inhibition of β-d-glucosidase under ultrasound may be attributed to changes in secondary structure. For activation, ultrasound increased the content of α-helices, decreased the content of β-folds and irregular coils of β-d-glucosidase. For inhibition, ultrasound increased the content of β-folds and reduced the content of α-helices and irregular coils. This study gives very useful information concerning the application of the ultrasound technique for enhancing aroma in fruit juice.

ACK N OWLED G M ENTS
This work was supported by National Natural Science Foundation of China (31771982) and China Scholarship (201708330203).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

O RCI D
Yujing Sun https://orcid.org/0000-0002-0449-2917 F I G U R E 7 Fluorescence spectra of β-D-glucosidase treated by ultrasound F I G U R E 8 UV absorption spectroscopy of β-D-glucosidase treated by ultrasound