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Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics
Accepted Manuscript Title: Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics Authors: Danli Wang, Xiaobin Ma, Lufeng Yan, Thunthacha Chantapakul, Wenjun Wang, Tian Ding, Xingqan Ye, Donghong Liu PII: DOI: Reference:
S0144-8617(17)30730-0 http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.093 CARP 12481
To appear in: Received date: Revised date: Accepted date:
25-4-2017 21-6-2017 22-6-2017
Please cite this article as: Wang, Danli., Ma, Xiaobin., Yan, Lufeng., Chantapakul, Thunthacha., Wang, Wenjun., Ding, Tian., Ye, Xingqan., & Liu, Donghong., Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.06.093 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics Danli Wanga, Xiaobin Maa, Lufeng Yana, Thunthacha Chantapakula, Wenjun Wanga, Tian Dingac, Xingqan Yeabc, Donghong Liu*abc a
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
b
Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China
c
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technology and
Equipment, Hangzhou 310058, China
Danli Wang: [email protected] Xiaobin Ma: [email protected] Lufeng Yan: [email protected] Thunthacha Chantapakul: [email protected] Wenjun Wang: [email protected] Tian Ding: [email protected] Xingqan Ye: [email protected]
Corresponding author at: College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China. E-mail address: [email protected]. Telephone number: +86-13858088582 Fax number: +86-571-88982169
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Highlight
Ultrasound assisted enzymatic process was influenced by temperature and ultrasonic intensity.
Ultrasound accelerated starch degradation and the enzymatic process catalyzed by glucoamylase.
Combination of ultrasound and glucoamylase resulted in the highest starch degradation extent.
Abstract The present work investigates the synergistic impact of glucoamylase and ultrasound on starch hydrolysis. The extent of starch hydrolysis at different reaction parameters (ultrasonic intensity, temperature, reaction time) was analyzed. The hydrolysis extent increased with the reaction time and reached a maximum value under ultrasonic intensity of 7.20 W/mL at 10 min. Ultrasound did not alter the optimum enzymatic temperature but speeded up the thermal inactivation of glucoamylase. The evaluation of enzymatic kinetics and starch degradation kinetics indicated a promotion of the reaction rate and enzyme-substrate affinity. According to the thermodynamic results, sonoenzymolysis reactions require less energy than enzymolysis reactions. The measurement of molecular weight, solubility, thermal properties, and structures of the substrates revealed that sonoenzymolysis reaction generated greater impacts on starch properties. The molecular weight and radii of gyration decreased by 80.19% and 90.05% respectively while the starch solubility improved by 136.50%. Keywords ultrasound; glucoamylase; starch hydrolysis; enzymatic kinetics; degradation kinetics
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1. Introduction Starch, as the main source of carbohydrate in our daily life, is widely utilized in the food industry as thickener, colloidal stabilizer, gelling agent, bulking agent, water retention agent, and adhesive (Singh, Singh, Kaur, Singh Sodhi & Singh Gill, 2003). They could be extracted from plants such as maize, wheat, potato, rice, tapioca, etc. where physicochemical and functional properties may vary according to their botanical sources. Potato is notable as a rich source of starch for industrial process, owing to its wide availability and attractive price. Potato starch is unique among other commercial starch, due to large granule sizes, relatively long amylose and amylopectin chain, presence of phosphate ester groups on amylopectin and capacity to form clear gels (Alvani, Qi, Tester & Snape, 2011). On the another hand, potato starch has limitations including low solubility, high viscosity, thermal decomposition, low shear and thermal resistance, while high affinity for retrogradation，which impede its uses in some applications (Atrous, Benbettaieb, Chouaibi, Attia & Ghorbel, 2016). To overcome these disadvantages, various methods have been used to modify starch in attempting to enhance its quality (Kaur, Ariffin, Bhat & Karim, 2012). Enzymatic modification is a popular and common method due to its high selectivity, substrate specificity and mild reaction conditions. However, the enzymatic hydrolysis is high-cost and has slow kinetics sometimes. Therefore, intensification of enzymatic reactions is crucial to enhance the efficiency. Recently, ultrasound assisted conventional enzymatic and chemical methods have been paid more attention (Singh, Agarwal, Bhatt, Goyal & Moholkar, 2015). Ultrasound is well known for its positive influence on various processes in the food industry including: sterilization, emulsification, extraction, depolymerization, and defoaming etc. (Chemat, Zill e & Khan, 2011) Ultrasound assisted reactions are more attractive than the conventional reactions because of the improved
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mass transfer and reaction efficiency (Adulkar & Rathod, 2014). The facilitation of mass transfer mainly results from the ultrasonic cavitation. The mechanical effect of cavitation induced by the collage of microbubbles would uniform the mixture, showing notable enhancement of mass transfer. Szabo and Csiszar (Szabo & Csiszar, 2017) used different agitation methods (i.e. magnetic stirring, horizontal and vertical mechanical agitation) and ultrasound in the enzymatic hydrolysis of cellulose. It claimed that cellulase was more effective on cellulose hydrolysis in the ultrasonic systems than agitation systems. The influence of ultrasound in the kinetic parameters and reduction of esterification reaction time by Novozym 435 were reported (Martins, Schein, Friedrich, Fernandez-Lafuente, Ayub & Rodrigues, 2013). Ultrasound assisted technology improved the process productivity about 7.5 fold, compared to the traditional mechanical agitation. The advantages of ultrasound assisted enzymatic reactions may be summarized as follows: 1) it doesn’t introduce extra chemicals or additives; 2) it is simple and efficient; 3) it minimizes the reaction time; 4) the process does not dramatically alter the chemical structure, the properties of starch in particular (Iida, Tuziuti, Yasui, Towata & Kozuka, 2008). Although some researches have reported that ultrasound could accelerate enzymatic reactions when combining with pectinase (Ma et al., 2016), cellulase (Szabo & Csiszar, 2017), lipase (Zhao et al., 2016), alpha amylase (Hu et al., 2013), and alcalase (Wang et al., 2016), there are only few reports available on ultrasound assisted glucoamylase catalyzed hydrolysis of starch. Furthermore, ultrasound could degrade and depolymerize starch directly, which was discovered since 1933 (Szent-gyorgyi, 1933). Several researches have studied the properties of ultrasound-treated starch from different botanical origins and the results confirmed that ultrasound affects the morphological, physicochemical, functional, and rheological properties of starch (Izidoro,
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Sierakowski, Haminiuk, de Souza & Scheer, 2011; Mohammad Amini, Razavi & Mortazavi, 2015; Zhu, Li, Chen & Li, 2012). R. Carmona-García et al. looked into the effect of ultrasonic treatment on starch of different granule sizes and found ultrasound generated profound cavities and fractures on the granule surface (Carmona-García, Bello-Pérez, Aguirre-Cruz, Aparicio-Saguilán, Hernández-Torres & Alvarez-Ramirez, 2016). Additionally, starch with larger granule size was more vulnerable to ultrasonic treatment. The ultrasonic effect on enzymatic process and starch provide the basic of ultrasound assisted enzymatic hydrolysis of starch. In the present work, ultrasound was introduced to act on potato starch enzymatic hydrolysis by glucoamylase under different ultrasonic conditions. Its effect on the enzymatic kinetics and starch degradation kinetics were studied. Some properties of starch and glucoamylase were further investigated, helping to explore the mechanism of ultrasound assisted enzymatic process.
2. Materials and Methods 2.1 Materials Glucoamylase from Aspergillus niger (EC3.2.1.3) was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China) and potato starch was purchased from Aladdin Reagent Company (Shanghai, China). Both the enzyme and the substrate were used without further purification while all other chemicals involved were of analytical grade. 2.2 Preparation of the Enzyme and Substrate Samples Glucoamylase was dissolved in 0.2 mol/L acetate buffer (pH 4.0) at a concentration of 1mg/mL when potato starch (5% (w/v)) was dispersed in 0.2 mol/L acetate buffer (pH 4.0) and heated in a water-bath at 100℃ for 30min.
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2.3 Enzymatic treatment The prepared starch solution (24 mL) was first preheated in a water bath for 5min to approach the reaction temperature, and glucoamylase solution (1 mL) was quickly added afterwards. The mixture was then incubated in the water bath at different temperatures (35-75 oC) for different period of time (5-40 min) with magnetic stirring. After enzymolysis, 1 mL 20% (w/v) NaOH was added into the mixture to inactivate the glucoamylase and later cooled down in icy water (see Supplementary Data). 2.4 Ultrasonic treatment The samples were treated in different conditions using a probe ultrasonic processor (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ningbo, China) with a 10 mm ultrasonic horn creating 22 kHz frequency. The instrument could deliver a maximum power of 900 W. 2.4.1 Sonoenzymolysis sample The prepared starch solution (24 mL, preheated in a water bath for 5min) and glucoamylase solution (1 mL) were quickly mixed in a cylindrical glass reactor and the ultrasound generator probe was immediately inserted (approximately half of the liquid height) to introduce ultrasonic field. The mixture was treated with different ultrasonic power (45–360 W), temperatures (35-75oC) and treatment times (5-40 min). After sonication, 1 mL 20% (w/v) NaOH was added to inactivate the glucoamylase and then the mixture was cooled down in icy water. During the ultrasonic process, the desired temperatures of subjected solution were controlled by a circulating water bath (DC−1006, Safe Corporation, Ningbo, China). 2.4.2 Sonolysis sample Instead of mixing 1 mL glucoamylase solution, 1mL acetate buffer (pH 4.0) was mixed with starch
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solution. Then the same ultrasonic treatment procedures as mentioned above were repeated. 2.5 Determination of hydrolysis degree The reducing sugar yield was measured using the 3, 5-dinitrosalicylic acid (DNS) method as described by Ma (Ma, Wang, Zou, Ding, Ye & Liu, 2015) with a little modification. The absorbance was detected at 540 nm with a spectrophotometer (UV-2550, SHIMADZU Co., Japan). The hydrolysis degree of starch was expressed by dextrose equivalent (DE) and calculated by the following equation: DE
reducing
sugar
expressed
dry weight
glucose/g
(1)
starch/g
2.6 Enzymatic Kinetics for the Enzymolysis and Sonoenzymolysis Reactions The Michaelis–Menten model was constructed to describe the enzymolysis and sonoenzymolysis reactions. Different concentrations of substrates (24 mL, 0.25-2.5 %(w/v)) and 1 mL glucoamylase were incubated at 35 oC for 10min with and without ultrasonic treatment to measure the reaction rate of each sample. The values of enzyme kinetics parameters, including the Michaelis–Menten constant (Km) and maximum rate of reaction (Vm), were attained from Lineweaver–Burk plots： 1
V
Km
1
Vm [S ]
1
(2)
Vm
where V is the enzymatic reaction rate and [S] is concentration of substrate. 2.7 Degradation Kinetics of the Sonolysis, Enzymolysis and Sonoenzymolysis Reactions The chemical kinetic model of starch hydrolysis established in the current work was based on the first-order kinetics:
ln (
C
) kt
(3)
C0
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where t is the reaction time (min), C0 is the initial concentration of potato starch (μg/mL), C is the concentration of starch at time t (µg/mL), and k is the effective rate constant of different degradation reactions (min−1). As it was hard to measure the decrement of starch concentration, the reaction rate was demonstrated by the escalating amount of reducing sugar released by starch instead with the following equation: ln (V V t ) kt ln V
(4)
where Vt is the concentration of reducing sugar at time t (μg/mL) and V∞ is the ultimate concentration of reducing sugar (μg/mL) obtained by the hydrolysis reaction in 2%(v/v) HCl solution under 100oC for 4h. The rate constant k (min−1) can then be determined from the slope by plotting ln(V∞− Vt) against t. 2.8 Thermodynamic parameters The activation energy (Ea) can be described by Arrhenius equation as follow: k Ae
- E a / RT
(5)
where A is the pre-exponential factor, Ea is the activation energy (J·mol−1) and R is the universal gas constant (8.314 J·mol−1K−1). The thermodynamic parameters were obtained by the Eyring transition state theory in the current study: k
k BT h
exp(
G RT
)
k BT h
exp(
H
RT
S
)
(6)
R
where T is the absolute temperature (K), kB is Boltzman constant (1.38×10-23 J·K−1) and h is Planck constant (6.6256×10-34 J·s−1). G, H and S are the parameters of changes in free energy, enthalpy and entropy of the reaction, respectively. 2.9 Determination of the starch properties
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The samples were dialyzed by dialysis bag MD44 (Mw: 8−14 kDa) through running water for 24h, then placed in distill water for 48h. After desalination, the samples were pre-frozen at -80oC then freeze-dried for later use. 2.9.1 SEC-MALLS-RI measurements The Mw of potato starch samples was measured by a SEC-MALLS-RI system (Wei et al., 2016). It consists of a pump(Waters 1525, US), a injector(Rheodyne, USA ) with a 50 μL sample loop, a SHODEX OHpak LB-806M column (300×8 mm, 13 μm; Shodex, Japan), a multi angle laser light scattering detector (Dawn HELEOS-II, Wyatt Technology, USA) and a refractive index detector (Waters 2414, US). The samples were suspended in 0.1 mol/ L NaNO3 (at a concentration about 2 mg/ mL) and heated in 100oC water bath for 1 h with agitation to complete dissolution. After cooling, samples were filtered through a 0.22μm membrane and then these filtered samples (50 μL) were injected into the column and eluted with 0.1 mol/ L NaNO3 at a flow rate of 0.5mL/min for 30 min at 40 ◦C. The eluent was then monitored with MALLS and RI at 40 oC. The Mw and Rg were calculated by the Astra 6.1 software (Wyatt Technology). 2.9.2 Solubility The solubility of starch was measured according to the procedure described by Sujka M and Jamroz J (Sujka & Jamroz, 2013). Samples (0.1 g) were suspended in 25 mL distilled water at 60 oC
with magnetic stirring. After 30 min, the mixture was centrifuged at 8000×g for 15 min. Then
the supernatant was collected and dried to constant weight (Ws). The solubility could be calculated by the equation:
S olubility
Ws
% the initial
weight
100 % of starch
2.9.3 Differential scanning calorimetry (DSC) measurements -9-
(7)
DSC measurements were performed using a Modulated Differential Scanning Calorimeter MDS1 instrument (Mettler, Toledo, Switzerland) equipped with a thermal analysis data station and data recording software. First of all, approximately 3.0 mg of a sample was weighed in an aluminum pan, distilled water (about 9.0 μL) was added to create a suspension of 25% (w/w).(Yan, Yayuan, Ling & Zhengbiao, 2011) Then the pan was hermetically sealed and rest at room temperature for 15h. After equilibration, the pan was heated from 30 to 180oC at a rate of 10oC/min where an empty pan was used as a reference. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and melting enthalpy (H) were validated. 2.9.4 Fourier transform infrared (FT-IR) spectroscopy The functional groups were verified by FT-IR spectra, collected with an IR spectrometer (Nicolet 5700; Thermo Fisher Scientific, MA, USA). The spectra were recorded in transmission mode from 4000 to 400 cm (mid-infrared region) at a resolution of 4 cm (Kim et al., 2008). Samples (approx. 2mg) were mixed with KBr (approx. 0.5g) and pressed into KBr pellets before acquisition. 2.10 Determination of enzyme structure 2.10.1 Intrinsic fluorescence analysis Fluorescence detections were performed at room temperature through a fluorescence spectrophotometer (Varian Inc., Palo Alto, USA; Model Cary Eclipse). The excitation wavelength was 280 nm (slit = 5 nm) while the emission wavelength was scanned from 300 to 500 nm (slit = 5 nm) at a scanning speed of 600 nm/min. The buffer for dissolving glucoamylase was chosen as blank solution for the sample. 2.10.2 Circular dichroism (CD) Circular dichroism (CD) spectra of glucoamylase samples with and without ultrasonic treatment
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were recorded at room temperature by a spectropolarimeter (JASCO Corporation, Japan, Model J-815) using a quartz cuvette of 1 mm optical path length. Scanning of CD spectra occurred in Far UV region (190 to 250 nm) at 200 nm/ min with a bandwidth of 5 nm. The CD data was expressed in terms of mean residue ellipticity, θ (deg·cm2·dmol−1). The secondary structures of glucoamylase with or without ultrasonic treatment were further analyzed using DICHROWEB. 2.11 Statistical analysis All experiments were conducted in triplicate, leading to achieve the evaluation by the mean ± standard deviation (SD). The figures were plotted by Origin Software Version 8.5 (OriginLab Corp.,MA, USA).
3. Results and discussions 3.1 Effect of ultrasound on the enzymatic hydrolysis of the potato starch. 3.1.1 Effect of ultrasonic intensity The enzymatic hydrolysis degree of starch solutions treated with different ultrasonic intensities is displayed in Fig. 1(a), indicating that DE was promoted by the increasing ultrasonic intensity until reaching the optimum level at 7.20 W/mL. However, DE decreased when intensity was further raised. This finding matches with the result from the study on ultrasound assisted guar gum hydrolysis (Prajapat, Subhedar & Gogate, 2016). Fundamentally, the main mechanisms for creating this phenomenon are associated with the generation of micro-jet, streaming, turbulence and sheer force by sonication. The promotion of enzymatic reactions by ultrasound could roughly be explained in three aspects: the enzyme, the substrate, or the combination of both enzyme and substrate. Firstly, ultrasonic cavitation affects
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enzyme conformation, expose the active site and unfold the polypeptide chains under appropriate conditions. These changes in the enzyme conformation promote the binding of enzyme with substrates, and thus increase the enzymatic activity (Jadhav & Gogate, 2014). Secondly, ultrasound can directly degrade starch and break the chains within starch granule, boosting the favorability for enzymatic hydrolysis (Kang, Zuo, Hilliou, Ashokkumar & Hemar, 2016). Ultrasonic treatment even resulted in better reduction effect on the starch molecular weight than glucoamylase treatment (as showed in 3.5.1). Lastly, ultrasound could overcome the mass transfer limitations and increase the mass transfer rate, which improve the affinity between enzymes and substrates. In addition, ultrasound could improve the binding of enzymes and substrates, as well as the removal of products from enzymes(Ma et al., 2016). Ultrasonic intensity is one of the most important parameters which affect ultrasonic cavitation. In general increasing ultrasonic intensity leads to the enhancement of cavitation effect. However, exorbitant intensity can disrupt bubble dynamics as it helps bubbles grow abnormally during expansion that may result in poor cavitation. (Pokhrel, Vabbina & Pala, 2016). Bubbles get bigger at higher ultrasonic intensity, causing weak explosion. Meanwhile, more bubbles are formed with further increase in ultrasonic intensity, impeding the propagation of ultrasonic waves so that DE decreased beyond 7.2 W/mL (Sutar & Rathod, 2015). Accordingly, an optimum ultrasonic intensity (7.20 W/mL) is applied to obtain the most beneficial effects. 3.1.2 Effect of temperature Enzymatic reactions can be easily influenced by temperature change due to the temperature sensitivity of enzyme. Fig.1(b) illustrates the hydrolysis degree of enzymolysis and sonoenzymolysis reactions at different temperatures (35oC, 45oC, 55oC, 65oC and 75oC). Overall,
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ultrasonic treatment did not change the optimum reaction temperature of glucoamylase. As the temperature increased from 35oC to 65oC, DE rose from 3.23% to 10.70% under enzymolysis and 3.73% to 13.49% under sonoenzymolysis reaction. However, when the temperature reached 75oC, DE decreased for both reactions, especially in the sonoenzymolysis reaction which indicated that the ultrasonic treatment reduces enzymatic hydrolysis efficiency at redundantly high temperature. This incident may have occurred because ultrasonic effect decreases thermal stability and accelerates thermal inactivation of glucoamylase. Furthermore, high temperature could also hinder the ultrasonic cavitation effect. Since ultrasonic cavitation is corresponded with the reaction temperature, the improvement of enzymatic reactions by ultrasound could vary. To be more specific, cavitation bubbles collapse with higher intensity and at lower temperature, which enhance the mass transfer (Sutar & Rathod, 2015). According to J. Raso et al., the actual output cavitation power is also temperature dependent where the cavitation between 20oC and 70oC is hardly affected but decreases dramatically when the temperature is beyond 70oC (Raso, Manas, Pagan & Sala, 1999). For this reason, the amount of reducing sugar yields fell. 3.1.3 Effect of reaction time The starch hydrolysis degree of the enzymolysis and sonoenzymolysis reactions (35oC, 7.20W/mL) conducted for 40 min are shown in Fig. 1(c). Within this period, the experiment with sonoenzymolysis reaction resulted higher DE than the one with enzymolysis reaction. Ma et al. (Ma et al., 2016) studied the synergetic effect of ultrasound and pectinase on pectin hydrolysis. The hydrolysis reaction was conducted for 30 min at the ultrasonic intensity of 4.5 W/mL. It was found that the enzymatic reaction rate increased by ultrasound within 12 min and later declined
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with the increasing time. This does not agree with our research finding but indicates that glucoamylase possesses better ultrasonic tolerance than pectinase. In general, the combination of ultrasound with glucoamylase obviously accelerates starch hydrolysis. 3.2 Enzymatic Kinetics The enzymatic kinetics experiment was conducted using different concentrations of starch solutions as reaction substrates. The results of Lineweaver-Burk analysis are given in Fig. 2 while the values of Km, Vm and R2 from the data fitting are listed in Table 1. To summarize, ultrasonic treatment increased Vm by 25.87% and decreased Km by 45.00%. In other words, ultrasound may increase the reaction velocity and enzyme-substrate affinity, promoting reactions by enhancing the binding of enzymes and substrates. This phenomenon is a consequence to the mechanical effects caused by ultrasonic cavitation, supported by the reported ability of ultrasound to improve the mass transfer rate during the enzymatic reaction (Sajjadi, Asgharzadehahmadi, Asaithambi, Raman & Parthasarathy, 2017). The mechanical effects of micro-jet, streaming, and turbulence are positive motivations for the mass transfer, which accelerate the formation of enzyme-substrate complexes as well as the diffusion rate of products into solution. Meanwhile, ultrasonic cavitation also eliminates the associated mass transfer resistance leading to a more rapid hydrolysis (Prajapat, Subhedar & Gogate, 2016).
3.3 Degradation Kinetics of enzymolysis and sonoenzymolysis starch The first-order kinetic model was used to describe starch hydrolysis. Fig.3(a) & (b) exhibits relationships between ln (V ∞ − Vt) and reaction time of enzymolysis and sonoenzymolysis
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reactions. Moreover, the degradation rate constants were calculated and listed in Table 2. The rate constants of both reactions increased as the temperature rose from 25oC to 65oC. However, the growth in rate constants of sonoenzymolysis reaction between 55 oC and 65 oC was minor in comparison to the growth in the rate constants between 55oC and lower temperatures. At 65 oC, the rate constant of sonoenzymolysis reaction even was lower than that of enzymolysis reaction. As mentioned above, the intensification of enzymatic hydrolysis by ultrasound would weaken at redundantly high temperature, owing to the reduction of cavitation effects and development in inactivation of glucoamylase with the prolongation of reaction time. Nevertheless, sonoenzymolysis reactions could provide greater rate constants than enzymolysis reactions at the same temperatures below 55oC. It indicates that ultrasound assisted methods work better at lower temperature. Since low operation temperature is preferred in industry for energy saving, cost saving and products protection.
3.4 Thermodynamic parameters Since the rate constant of sonoenzymolysis reaction at 65oC reduced appreciably, the thermodynamic parameters were calculated using the data from 25oC to 55oC. The Arrhenius plot of lnk against 1/T was depicted in Fig. 3(c) where activation energy (Ea) could be obtained from slopes of the Arrhenius plots. It appeared that Ea of enzymolysis and sonoenzymolysis were 46.71 kJ/ mol and 40.92 kJ/ mol respectively. The decrease of activation energy after ultrasonic treatment could be
explained that ultrasound has lessened the energy barrier necessary for the enzymatic reaction. The
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relationship between ln (k/T) and 1/T was displayed in Fig. 3(d). Correspondingly, H, S and G
were calculated according to Eyring transition state theory, as listed in Table 3. The difference in G of the two reactions was insignificant. The positive values of H illustrate that the processes are endothermic. The lower H value of sonoenzymolysis reaction signifies the requirement for less energy during starch hydrolysis as compared to enzymolysis. Furthermore, the change in entropy (S) reflects the change of disorderness over the process. The decrease in the S can be resulted from the oxidative alteration of amino acid residues and beginning of enzyme cross-linking and aggregation (Prajapat, Subhedar & Gogate, 2016). The decreased S value indicates a more complete reaction after ultrasonic treatment. Changes of thermodynamic parameters ascertain the acceleration of enzymatic reaction by ultrasound. 3.5 Substrate properties 3.5.1 Mw and Solubility The molecular weights (Mw), radii of gyration (Rg) and solubility of starch samples with different treatment were determined and established in Table 4. As expected, the starch samples treated by enzymolysis, sonolysis and sonoenzymolysis had lower Mw and Rg as well as higher solubility. This indicates that both enzymatic and ultrasonic treatment could effectively decrease the Mw of starch and therefore, improve starch solubility in water. It has been proved that ultrasound could depolymerize starch polymer molecules, as a result of radical and mechanical effects induced by ultrasonic cavitation (BeMiller & Huber, 2015). Accordingly, ultrasonic treatment leads to further reduction of the starch molecular weight and enhances starch degrading operation by glucoamylase. The solubility of starch depends strongly on the molecular structures. Referring to the current
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research, the glucoamylase treatment alone increased the solubility of starch approximately by 20%, while treatments by sonolysis and sonoenzymolysis overly doubled the solubility. Obviously, ultrasound assisted enzymatic treatment is more effective in boosting starch solubility, which is consistent with the results of molecular weights. The mechanism could be described that ultrasound has degraded the starch granules by loosening their structures, allowing water to reach into the net structures more easily. 3.5.2 Thermal properties of starch samples DSC analysis was applied to assess thermal properties of starch and results are presented in Table S1. The onset gelatinization temperatures of all starch samples showed little differences. The peak temperatures of sonolysis and sonoenzymolysis starch decreased slightly compared with those of untreated or enzymolysis starch. Besides, the conclusive temperatures of treated starch were lower than that of untreated starch (sonoenzymolysis< sonolysis< enzymolysis< untreated). Consequently, the gelatinization temperature range (Tc -To) also decreased respectively after the different treatments, among which the sonoenzymolysis starch reached the minimum (Tc -To) value. It suggests that both enzymatic and ultrasonic treatments could affect the degree of heterogeneity of crystallites in starch (Luo, Fu, He, Luo, Gao & Yu, 2008). Moreover, ultrasound promotes the breakage of the crystallites caused by enzymolysis. The gelatinization enthalpy (H) reflects the loss of double helical order rather than loss of crystalline register (Thirumdas, Trimukhe, Deshmukh & Annapure, 2017). The shrinkage in H of enzymolysis, sonolysis and sonoenzymolysis starch compared with untreated control indicates that some of the double helical of the granules may have been disrupted. 3.5.3 FT-IR analysis of starch samples
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Fig. S3 shows the FT-IR spectra of starch samples from different treatments. From the acquired spectra, several discernible absorption peaks was detected at 1154, 1081, and 1021cm−1, which were attributed to C–O bond stretching. Additional characteristic absorption bands at 929, 856, 763, and 576 cm−1 were due to the skeletal vibration of glucose pyranose ring. Furthermore, an extremely broad band due to O−H stretching appeared around 3384cm−1 and a weak absorption derived from C−H stretching appeared at 2927 cm−1 (Zhang, Xie, Zhao, Liu & Gao, 2009). The bands at 1154 cm−1 and 1081 cm−1 were related to the ordered structures of starch, whereas, the band at 1021 cm−1 was associated with the amorphous structures of starch (Khatoon, Sreerama, Raghavendra, Bhattacharya & Bhat, 2009). The results illustrated that starch crystallinity has barely been altered, as the spectra of four samples are similar with only minor difference between the absorption peak intensities. There is no new formation or loss of chemical bonds and functional groups during the treatments. Generally, little difference was seen among untreated, enzymolysis, sonolysis and sonoenzymolysis starch in our FT-IR study. 3.6 Enzyme structures 3.6.1 Intrinsic fluorescence analysis In order to reveal the effect of ultrasonic treatment on the tertiary structures of glucoamylase, intrinsic fluorescence of the enzyme was explored. Intrinsic fluorescence is mainly originated from tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) presented in the enzyme, especially the Trp residue (Galban, Andreu, Sierra, de Marcos & Castillo, 2001). In this research, we studied the conformational changes of glucoamylase by Trp fluorescence. The Trp residue is the dominant source
of
intrinsic
protein
fluorescence
which
is
highly
sensitive
to
the
local
microenvironment(Ghisaidoobe & Chung, 2014). As indicated in Fig. S4 (a), the fluorescence
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intensity was weakened after ultrasonic treatment with no changes on the maximum emission (λmax=336 nm). It means that ultrasonic treatment has altered the microenvironment of impressible Trp residues, decreasing the amount of Trp on glucoamylase’s surface (Jafari-Aghdam, Khajeh, Ranjbar & Nemat-Gorgani, 2005). This phenomenon is relevant to the unfolding of enzyme protein, induced by the ultrasonic cavitation (Wang, Zhu, Xiao, Liu & Wang, 2011). The external Trp residues were buried partially, while other internal residues were exposed to the surface. This reduction of external Trp residues leads to the reduction of the fluorescence intensity. 3.6.2 Circular Dichroism CD spectroscopy can provide information on the secondary structures of proteins, including α-helix, β-sheet, β-turn, and random coil. The CD spectra of glucoamylase treated with and without ultrasound are shown in Fig. S4 (b), and the contents of α-helix, β-sheet, turn and random coil in the secondary structures are summarized in Table 5. It can be seen that the content of random coil in the glucoamylase increased after undergoing ultrasonic treatment, while the other fractions of the structures revealed an opposite trend. This structural transformation caused by ultrasonic treatment could be traced back to the effects from cavitation pressure and turbulence (Dai, Li & Jiang, 1999). The increase in random coil conformation implies a more flexible glucoamylase structure, enhancing the catalytic efficiency (Subhedar & Gogate, 2014). This transformation might have slightly loosened glucoamylase structure as well as exposing the catalytic center and binding sites, thus accelerating the enzymatic processes.
4. Conclusion A significant intensification of enzymatic hydrolysis extent by ultrasound assisted method was
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established in the current study. Glucoamylase worked effectively upon reducing sugar production, but barely affected the starch molecular degradation when comparing to sonication treatment. Favorably with ultrasound assisted treatment, sonoenzymolysis showed even greater effects both on reducing sugar yield and starch molecular degradation than the individual enzymolysis and sonolysis reactions. Redundantly high temperature generates an adverse effect to the ultrasound assisted enzymatic treatments due to enzyme’s resistance to heat and ultrasound. The rate constants of enzymatic reactions had an increasing trend after ultrasonic treatment within 55oC. Meanwhile sonication raised the Vm while it decreased the Km of the enzymatic reactions, confirming that ultrasound could enhance the enzymatic hydrolysis rate and the enzyme-substrate affinity. Furthermore, the molecular weight of starch deceased sharply after undergoing sonoenzymolysis treatment while the solubility of hydrolyzed starch improved. All of the above claims that the modification of starch by sonication and enzymatic reactions was a synergistic effect. Additionally, starch properties were analyzed by DSC and FT-IR spectra and the results demonstrated that the starch structures were hardly changed. Fluorescence and circular dichroism spectra reflected that ultrasound partially eliminated the external tryptophan residues of glucoamylase and slightly changed the secondary structure. In either case, the remaining effect of ultrasonic modification on glucoamylase requires further study.
Acknowledgements This work was financially supported by the National Key Research and Development Program of China (grant 2016YFD0400301), the Key Research and Development Program of Zhejiang Province (grant 2017C02015) and National Science Foundation of China (Project 31371872).
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Fig. 1. Effect of (a) ultrasonic intensity, (b) temperature, (c) reaction time on the hydrolysis of starch
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Fig. 2. Plots of the initial velocity values obtained as a function of the correspondent substrate concentration values using Lineweaver– linearization
Fig. 3. (a) Relationship between ln(V∞− Vt)and reaction time for enzymolysis starch. (b) Relationship between ln(V∞− Vt) and reaction time for sonoenzymolysis starch. (c) Relationship between lnk and 1/T. (d) Relationship between ln(k/T) and 1/T
Table 1 Enzymatic kinetics parameters of the enzymolysis and sonoenzymolysis reactions Samples Enzymolysis
Vm (mg/mL·min−1)
Km (mg/mL)
R2
0.0545±0.0007
0.6511±0.0515
0.9986
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sonoenzymolysis
0.0686±0.0004
0.3581±0.529
0.9948
Table 2 Rate constants of the enzymolysis and sonoenzymolysis reactions Temperature (oC)
25
35
45
55
65
kenzymolysis (10-3min-1)
2.36±0.11
3.74±0.17
7.61±0.09
12.66±0.11
17.73±0.43
ksonoenzymolysis (10-3min-1)
3.20±0.07
4.98±0.02
8.44±0.39
14.40±0.15
15.11±0.75
+35.59
+33.16
+10.91
+13.74
−14.78
Change rate (%)
Table 3 Thermodynamic parameters of the enzymolysis and sonoenzymolysis reactions Ea (kJ/mol)
H (kJ/mol)
S (kJ/mol)
G (kJ/mol)
Enzymolysis
46.71±0.37
44.12±0.37
-147.67±3.82
89.62±1.40
Sonoenzymolysis
40.92±0.25
38.32±0.24
-164.47±3.16
89.00±0.77
Table 4
The average molecular weights (Mw), radii of gyration (Rg) and solubility of starch samples. Untreated
Enzymolysis
Sonolysis
Sonoenzymolysis
Mw (10^5 g/mol)
266.80±19.26
71.80±2.36
8.21±0.09
5.28±0.07
Rg (nm)
267.40±2.67
92.90±1.76
30.90±1.29
26.60±2.02
Solubility (%)
17.13±0.53
20.75±0.71
34.38±0.88
40.50±0.71
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Table 5 Secondary structures of glucoamylase treated with and without ultrasound. Treatment
α-Helix (%)
β-Sheet (%)
Turn (%)
Random coil (%)
Untreated
61.1±11.4
8.6±1.6
13.3±2.6
17.1±3.2
Ultrasound-treated
55.9±7.1
3.8±0.5
11.4±1.5
28.9±3.7
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S0144-8617(17)30730-0 http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.093 CARP 12481
To appear in: Received date: Revised date: Accepted date:
25-4-2017 21-6-2017 22-6-2017
Please cite this article as: Wang, Danli., Ma, Xiaobin., Yan, Lufeng., Chantapakul, Thunthacha., Wang, Wenjun., Ding, Tian., Ye, Xingqan., & Liu, Donghong., Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.06.093 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasound assisted enzymatic hydrolysis of starch catalyzed by glucoamylase: Investigation on starch properties and degradation kinetics Danli Wanga, Xiaobin Maa, Lufeng Yana, Thunthacha Chantapakula, Wenjun Wanga, Tian Dingac, Xingqan Yeabc, Donghong Liu*abc a
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
b
Fuli Institute of Food Science, Zhejiang University, Hangzhou 310058, China
c
Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R&D Center for Food Technology and
Equipment, Hangzhou 310058, China
Danli Wang: [email protected] Xiaobin Ma: [email protected] Lufeng Yan: [email protected] Thunthacha Chantapakul: [email protected] Wenjun Wang: [email protected] Tian Ding: [email protected] Xingqan Ye: [email protected]
Corresponding author at: College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China. E-mail address: [email protected]. Telephone number: +86-13858088582 Fax number: +86-571-88982169
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Highlight
Ultrasound assisted enzymatic process was influenced by temperature and ultrasonic intensity.
Ultrasound accelerated starch degradation and the enzymatic process catalyzed by glucoamylase.
Combination of ultrasound and glucoamylase resulted in the highest starch degradation extent.
Abstract The present work investigates the synergistic impact of glucoamylase and ultrasound on starch hydrolysis. The extent of starch hydrolysis at different reaction parameters (ultrasonic intensity, temperature, reaction time) was analyzed. The hydrolysis extent increased with the reaction time and reached a maximum value under ultrasonic intensity of 7.20 W/mL at 10 min. Ultrasound did not alter the optimum enzymatic temperature but speeded up the thermal inactivation of glucoamylase. The evaluation of enzymatic kinetics and starch degradation kinetics indicated a promotion of the reaction rate and enzyme-substrate affinity. According to the thermodynamic results, sonoenzymolysis reactions require less energy than enzymolysis reactions. The measurement of molecular weight, solubility, thermal properties, and structures of the substrates revealed that sonoenzymolysis reaction generated greater impacts on starch properties. The molecular weight and radii of gyration decreased by 80.19% and 90.05% respectively while the starch solubility improved by 136.50%. Keywords ultrasound; glucoamylase; starch hydrolysis; enzymatic kinetics; degradation kinetics
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1. Introduction Starch, as the main source of carbohydrate in our daily life, is widely utilized in the food industry as thickener, colloidal stabilizer, gelling agent, bulking agent, water retention agent, and adhesive (Singh, Singh, Kaur, Singh Sodhi & Singh Gill, 2003). They could be extracted from plants such as maize, wheat, potato, rice, tapioca, etc. where physicochemical and functional properties may vary according to their botanical sources. Potato is notable as a rich source of starch for industrial process, owing to its wide availability and attractive price. Potato starch is unique among other commercial starch, due to large granule sizes, relatively long amylose and amylopectin chain, presence of phosphate ester groups on amylopectin and capacity to form clear gels (Alvani, Qi, Tester & Snape, 2011). On the another hand, potato starch has limitations including low solubility, high viscosity, thermal decomposition, low shear and thermal resistance, while high affinity for retrogradation，which impede its uses in some applications (Atrous, Benbettaieb, Chouaibi, Attia & Ghorbel, 2016). To overcome these disadvantages, various methods have been used to modify starch in attempting to enhance its quality (Kaur, Ariffin, Bhat & Karim, 2012). Enzymatic modification is a popular and common method due to its high selectivity, substrate specificity and mild reaction conditions. However, the enzymatic hydrolysis is high-cost and has slow kinetics sometimes. Therefore, intensification of enzymatic reactions is crucial to enhance the efficiency. Recently, ultrasound assisted conventional enzymatic and chemical methods have been paid more attention (Singh, Agarwal, Bhatt, Goyal & Moholkar, 2015). Ultrasound is well known for its positive influence on various processes in the food industry including: sterilization, emulsification, extraction, depolymerization, and defoaming etc. (Chemat, Zill e & Khan, 2011) Ultrasound assisted reactions are more attractive than the conventional reactions because of the improved
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mass transfer and reaction efficiency (Adulkar & Rathod, 2014). The facilitation of mass transfer mainly results from the ultrasonic cavitation. The mechanical effect of cavitation induced by the collage of microbubbles would uniform the mixture, showing notable enhancement of mass transfer. Szabo and Csiszar (Szabo & Csiszar, 2017) used different agitation methods (i.e. magnetic stirring, horizontal and vertical mechanical agitation) and ultrasound in the enzymatic hydrolysis of cellulose. It claimed that cellulase was more effective on cellulose hydrolysis in the ultrasonic systems than agitation systems. The influence of ultrasound in the kinetic parameters and reduction of esterification reaction time by Novozym 435 were reported (Martins, Schein, Friedrich, Fernandez-Lafuente, Ayub & Rodrigues, 2013). Ultrasound assisted technology improved the process productivity about 7.5 fold, compared to the traditional mechanical agitation. The advantages of ultrasound assisted enzymatic reactions may be summarized as follows: 1) it doesn’t introduce extra chemicals or additives; 2) it is simple and efficient; 3) it minimizes the reaction time; 4) the process does not dramatically alter the chemical structure, the properties of starch in particular (Iida, Tuziuti, Yasui, Towata & Kozuka, 2008). Although some researches have reported that ultrasound could accelerate enzymatic reactions when combining with pectinase (Ma et al., 2016), cellulase (Szabo & Csiszar, 2017), lipase (Zhao et al., 2016), alpha amylase (Hu et al., 2013), and alcalase (Wang et al., 2016), there are only few reports available on ultrasound assisted glucoamylase catalyzed hydrolysis of starch. Furthermore, ultrasound could degrade and depolymerize starch directly, which was discovered since 1933 (Szent-gyorgyi, 1933). Several researches have studied the properties of ultrasound-treated starch from different botanical origins and the results confirmed that ultrasound affects the morphological, physicochemical, functional, and rheological properties of starch (Izidoro,
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Sierakowski, Haminiuk, de Souza & Scheer, 2011; Mohammad Amini, Razavi & Mortazavi, 2015; Zhu, Li, Chen & Li, 2012). R. Carmona-García et al. looked into the effect of ultrasonic treatment on starch of different granule sizes and found ultrasound generated profound cavities and fractures on the granule surface (Carmona-García, Bello-Pérez, Aguirre-Cruz, Aparicio-Saguilán, Hernández-Torres & Alvarez-Ramirez, 2016). Additionally, starch with larger granule size was more vulnerable to ultrasonic treatment. The ultrasonic effect on enzymatic process and starch provide the basic of ultrasound assisted enzymatic hydrolysis of starch. In the present work, ultrasound was introduced to act on potato starch enzymatic hydrolysis by glucoamylase under different ultrasonic conditions. Its effect on the enzymatic kinetics and starch degradation kinetics were studied. Some properties of starch and glucoamylase were further investigated, helping to explore the mechanism of ultrasound assisted enzymatic process.
2. Materials and Methods 2.1 Materials Glucoamylase from Aspergillus niger (EC3.2.1.3) was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China) and potato starch was purchased from Aladdin Reagent Company (Shanghai, China). Both the enzyme and the substrate were used without further purification while all other chemicals involved were of analytical grade. 2.2 Preparation of the Enzyme and Substrate Samples Glucoamylase was dissolved in 0.2 mol/L acetate buffer (pH 4.0) at a concentration of 1mg/mL when potato starch (5% (w/v)) was dispersed in 0.2 mol/L acetate buffer (pH 4.0) and heated in a water-bath at 100℃ for 30min.
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2.3 Enzymatic treatment The prepared starch solution (24 mL) was first preheated in a water bath for 5min to approach the reaction temperature, and glucoamylase solution (1 mL) was quickly added afterwards. The mixture was then incubated in the water bath at different temperatures (35-75 oC) for different period of time (5-40 min) with magnetic stirring. After enzymolysis, 1 mL 20% (w/v) NaOH was added into the mixture to inactivate the glucoamylase and later cooled down in icy water (see Supplementary Data). 2.4 Ultrasonic treatment The samples were treated in different conditions using a probe ultrasonic processor (JY92-IIDN, Ningbo Scientz Biotechnology Co., Ningbo, China) with a 10 mm ultrasonic horn creating 22 kHz frequency. The instrument could deliver a maximum power of 900 W. 2.4.1 Sonoenzymolysis sample The prepared starch solution (24 mL, preheated in a water bath for 5min) and glucoamylase solution (1 mL) were quickly mixed in a cylindrical glass reactor and the ultrasound generator probe was immediately inserted (approximately half of the liquid height) to introduce ultrasonic field. The mixture was treated with different ultrasonic power (45–360 W), temperatures (35-75oC) and treatment times (5-40 min). After sonication, 1 mL 20% (w/v) NaOH was added to inactivate the glucoamylase and then the mixture was cooled down in icy water. During the ultrasonic process, the desired temperatures of subjected solution were controlled by a circulating water bath (DC−1006, Safe Corporation, Ningbo, China). 2.4.2 Sonolysis sample Instead of mixing 1 mL glucoamylase solution, 1mL acetate buffer (pH 4.0) was mixed with starch
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solution. Then the same ultrasonic treatment procedures as mentioned above were repeated. 2.5 Determination of hydrolysis degree The reducing sugar yield was measured using the 3, 5-dinitrosalicylic acid (DNS) method as described by Ma (Ma, Wang, Zou, Ding, Ye & Liu, 2015) with a little modification. The absorbance was detected at 540 nm with a spectrophotometer (UV-2550, SHIMADZU Co., Japan). The hydrolysis degree of starch was expressed by dextrose equivalent (DE) and calculated by the following equation: DE
reducing
sugar
expressed
dry weight
glucose/g
(1)
starch/g
2.6 Enzymatic Kinetics for the Enzymolysis and Sonoenzymolysis Reactions The Michaelis–Menten model was constructed to describe the enzymolysis and sonoenzymolysis reactions. Different concentrations of substrates (24 mL, 0.25-2.5 %(w/v)) and 1 mL glucoamylase were incubated at 35 oC for 10min with and without ultrasonic treatment to measure the reaction rate of each sample. The values of enzyme kinetics parameters, including the Michaelis–Menten constant (Km) and maximum rate of reaction (Vm), were attained from Lineweaver–Burk plots： 1
V
Km
1
Vm [S ]
1
(2)
Vm
where V is the enzymatic reaction rate and [S] is concentration of substrate. 2.7 Degradation Kinetics of the Sonolysis, Enzymolysis and Sonoenzymolysis Reactions The chemical kinetic model of starch hydrolysis established in the current work was based on the first-order kinetics:
ln (
C
) kt
(3)
C0
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where t is the reaction time (min), C0 is the initial concentration of potato starch (μg/mL), C is the concentration of starch at time t (µg/mL), and k is the effective rate constant of different degradation reactions (min−1). As it was hard to measure the decrement of starch concentration, the reaction rate was demonstrated by the escalating amount of reducing sugar released by starch instead with the following equation: ln (V V t ) kt ln V
(4)
where Vt is the concentration of reducing sugar at time t (μg/mL) and V∞ is the ultimate concentration of reducing sugar (μg/mL) obtained by the hydrolysis reaction in 2%(v/v) HCl solution under 100oC for 4h. The rate constant k (min−1) can then be determined from the slope by plotting ln(V∞− Vt) against t. 2.8 Thermodynamic parameters The activation energy (Ea) can be described by Arrhenius equation as follow: k Ae
- E a / RT
(5)
where A is the pre-exponential factor, Ea is the activation energy (J·mol−1) and R is the universal gas constant (8.314 J·mol−1K−1). The thermodynamic parameters were obtained by the Eyring transition state theory in the current study: k
k BT h
exp(
G RT
)
k BT h
exp(
H
RT
S
)
(6)
R
where T is the absolute temperature (K), kB is Boltzman constant (1.38×10-23 J·K−1) and h is Planck constant (6.6256×10-34 J·s−1). G, H and S are the parameters of changes in free energy, enthalpy and entropy of the reaction, respectively. 2.9 Determination of the starch properties
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The samples were dialyzed by dialysis bag MD44 (Mw: 8−14 kDa) through running water for 24h, then placed in distill water for 48h. After desalination, the samples were pre-frozen at -80oC then freeze-dried for later use. 2.9.1 SEC-MALLS-RI measurements The Mw of potato starch samples was measured by a SEC-MALLS-RI system (Wei et al., 2016). It consists of a pump(Waters 1525, US), a injector(Rheodyne, USA ) with a 50 μL sample loop, a SHODEX OHpak LB-806M column (300×8 mm, 13 μm; Shodex, Japan), a multi angle laser light scattering detector (Dawn HELEOS-II, Wyatt Technology, USA) and a refractive index detector (Waters 2414, US). The samples were suspended in 0.1 mol/ L NaNO3 (at a concentration about 2 mg/ mL) and heated in 100oC water bath for 1 h with agitation to complete dissolution. After cooling, samples were filtered through a 0.22μm membrane and then these filtered samples (50 μL) were injected into the column and eluted with 0.1 mol/ L NaNO3 at a flow rate of 0.5mL/min for 30 min at 40 ◦C. The eluent was then monitored with MALLS and RI at 40 oC. The Mw and Rg were calculated by the Astra 6.1 software (Wyatt Technology). 2.9.2 Solubility The solubility of starch was measured according to the procedure described by Sujka M and Jamroz J (Sujka & Jamroz, 2013). Samples (0.1 g) were suspended in 25 mL distilled water at 60 oC
with magnetic stirring. After 30 min, the mixture was centrifuged at 8000×g for 15 min. Then
the supernatant was collected and dried to constant weight (Ws). The solubility could be calculated by the equation:
S olubility
Ws
% the initial
weight
100 % of starch
2.9.3 Differential scanning calorimetry (DSC) measurements -9-
(7)
DSC measurements were performed using a Modulated Differential Scanning Calorimeter MDS1 instrument (Mettler, Toledo, Switzerland) equipped with a thermal analysis data station and data recording software. First of all, approximately 3.0 mg of a sample was weighed in an aluminum pan, distilled water (about 9.0 μL) was added to create a suspension of 25% (w/w).(Yan, Yayuan, Ling & Zhengbiao, 2011) Then the pan was hermetically sealed and rest at room temperature for 15h. After equilibration, the pan was heated from 30 to 180oC at a rate of 10oC/min where an empty pan was used as a reference. The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and melting enthalpy (H) were validated. 2.9.4 Fourier transform infrared (FT-IR) spectroscopy The functional groups were verified by FT-IR spectra, collected with an IR spectrometer (Nicolet 5700; Thermo Fisher Scientific, MA, USA). The spectra were recorded in transmission mode from 4000 to 400 cm (mid-infrared region) at a resolution of 4 cm (Kim et al., 2008). Samples (approx. 2mg) were mixed with KBr (approx. 0.5g) and pressed into KBr pellets before acquisition. 2.10 Determination of enzyme structure 2.10.1 Intrinsic fluorescence analysis Fluorescence detections were performed at room temperature through a fluorescence spectrophotometer (Varian Inc., Palo Alto, USA; Model Cary Eclipse). The excitation wavelength was 280 nm (slit = 5 nm) while the emission wavelength was scanned from 300 to 500 nm (slit = 5 nm) at a scanning speed of 600 nm/min. The buffer for dissolving glucoamylase was chosen as blank solution for the sample. 2.10.2 Circular dichroism (CD) Circular dichroism (CD) spectra of glucoamylase samples with and without ultrasonic treatment
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were recorded at room temperature by a spectropolarimeter (JASCO Corporation, Japan, Model J-815) using a quartz cuvette of 1 mm optical path length. Scanning of CD spectra occurred in Far UV region (190 to 250 nm) at 200 nm/ min with a bandwidth of 5 nm. The CD data was expressed in terms of mean residue ellipticity, θ (deg·cm2·dmol−1). The secondary structures of glucoamylase with or without ultrasonic treatment were further analyzed using DICHROWEB. 2.11 Statistical analysis All experiments were conducted in triplicate, leading to achieve the evaluation by the mean ± standard deviation (SD). The figures were plotted by Origin Software Version 8.5 (OriginLab Corp.,MA, USA).
3. Results and discussions 3.1 Effect of ultrasound on the enzymatic hydrolysis of the potato starch. 3.1.1 Effect of ultrasonic intensity The enzymatic hydrolysis degree of starch solutions treated with different ultrasonic intensities is displayed in Fig. 1(a), indicating that DE was promoted by the increasing ultrasonic intensity until reaching the optimum level at 7.20 W/mL. However, DE decreased when intensity was further raised. This finding matches with the result from the study on ultrasound assisted guar gum hydrolysis (Prajapat, Subhedar & Gogate, 2016). Fundamentally, the main mechanisms for creating this phenomenon are associated with the generation of micro-jet, streaming, turbulence and sheer force by sonication. The promotion of enzymatic reactions by ultrasound could roughly be explained in three aspects: the enzyme, the substrate, or the combination of both enzyme and substrate. Firstly, ultrasonic cavitation affects
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enzyme conformation, expose the active site and unfold the polypeptide chains under appropriate conditions. These changes in the enzyme conformation promote the binding of enzyme with substrates, and thus increase the enzymatic activity (Jadhav & Gogate, 2014). Secondly, ultrasound can directly degrade starch and break the chains within starch granule, boosting the favorability for enzymatic hydrolysis (Kang, Zuo, Hilliou, Ashokkumar & Hemar, 2016). Ultrasonic treatment even resulted in better reduction effect on the starch molecular weight than glucoamylase treatment (as showed in 3.5.1). Lastly, ultrasound could overcome the mass transfer limitations and increase the mass transfer rate, which improve the affinity between enzymes and substrates. In addition, ultrasound could improve the binding of enzymes and substrates, as well as the removal of products from enzymes(Ma et al., 2016). Ultrasonic intensity is one of the most important parameters which affect ultrasonic cavitation. In general increasing ultrasonic intensity leads to the enhancement of cavitation effect. However, exorbitant intensity can disrupt bubble dynamics as it helps bubbles grow abnormally during expansion that may result in poor cavitation. (Pokhrel, Vabbina & Pala, 2016). Bubbles get bigger at higher ultrasonic intensity, causing weak explosion. Meanwhile, more bubbles are formed with further increase in ultrasonic intensity, impeding the propagation of ultrasonic waves so that DE decreased beyond 7.2 W/mL (Sutar & Rathod, 2015). Accordingly, an optimum ultrasonic intensity (7.20 W/mL) is applied to obtain the most beneficial effects. 3.1.2 Effect of temperature Enzymatic reactions can be easily influenced by temperature change due to the temperature sensitivity of enzyme. Fig.1(b) illustrates the hydrolysis degree of enzymolysis and sonoenzymolysis reactions at different temperatures (35oC, 45oC, 55oC, 65oC and 75oC). Overall,
- 12 -
ultrasonic treatment did not change the optimum reaction temperature of glucoamylase. As the temperature increased from 35oC to 65oC, DE rose from 3.23% to 10.70% under enzymolysis and 3.73% to 13.49% under sonoenzymolysis reaction. However, when the temperature reached 75oC, DE decreased for both reactions, especially in the sonoenzymolysis reaction which indicated that the ultrasonic treatment reduces enzymatic hydrolysis efficiency at redundantly high temperature. This incident may have occurred because ultrasonic effect decreases thermal stability and accelerates thermal inactivation of glucoamylase. Furthermore, high temperature could also hinder the ultrasonic cavitation effect. Since ultrasonic cavitation is corresponded with the reaction temperature, the improvement of enzymatic reactions by ultrasound could vary. To be more specific, cavitation bubbles collapse with higher intensity and at lower temperature, which enhance the mass transfer (Sutar & Rathod, 2015). According to J. Raso et al., the actual output cavitation power is also temperature dependent where the cavitation between 20oC and 70oC is hardly affected but decreases dramatically when the temperature is beyond 70oC (Raso, Manas, Pagan & Sala, 1999). For this reason, the amount of reducing sugar yields fell. 3.1.3 Effect of reaction time The starch hydrolysis degree of the enzymolysis and sonoenzymolysis reactions (35oC, 7.20W/mL) conducted for 40 min are shown in Fig. 1(c). Within this period, the experiment with sonoenzymolysis reaction resulted higher DE than the one with enzymolysis reaction. Ma et al. (Ma et al., 2016) studied the synergetic effect of ultrasound and pectinase on pectin hydrolysis. The hydrolysis reaction was conducted for 30 min at the ultrasonic intensity of 4.5 W/mL. It was found that the enzymatic reaction rate increased by ultrasound within 12 min and later declined
- 13 -
with the increasing time. This does not agree with our research finding but indicates that glucoamylase possesses better ultrasonic tolerance than pectinase. In general, the combination of ultrasound with glucoamylase obviously accelerates starch hydrolysis. 3.2 Enzymatic Kinetics The enzymatic kinetics experiment was conducted using different concentrations of starch solutions as reaction substrates. The results of Lineweaver-Burk analysis are given in Fig. 2 while the values of Km, Vm and R2 from the data fitting are listed in Table 1. To summarize, ultrasonic treatment increased Vm by 25.87% and decreased Km by 45.00%. In other words, ultrasound may increase the reaction velocity and enzyme-substrate affinity, promoting reactions by enhancing the binding of enzymes and substrates. This phenomenon is a consequence to the mechanical effects caused by ultrasonic cavitation, supported by the reported ability of ultrasound to improve the mass transfer rate during the enzymatic reaction (Sajjadi, Asgharzadehahmadi, Asaithambi, Raman & Parthasarathy, 2017). The mechanical effects of micro-jet, streaming, and turbulence are positive motivations for the mass transfer, which accelerate the formation of enzyme-substrate complexes as well as the diffusion rate of products into solution. Meanwhile, ultrasonic cavitation also eliminates the associated mass transfer resistance leading to a more rapid hydrolysis (Prajapat, Subhedar & Gogate, 2016).
3.3 Degradation Kinetics of enzymolysis and sonoenzymolysis starch The first-order kinetic model was used to describe starch hydrolysis. Fig.3(a) & (b) exhibits relationships between ln (V ∞ − Vt) and reaction time of enzymolysis and sonoenzymolysis
- 14 -
reactions. Moreover, the degradation rate constants were calculated and listed in Table 2. The rate constants of both reactions increased as the temperature rose from 25oC to 65oC. However, the growth in rate constants of sonoenzymolysis reaction between 55 oC and 65 oC was minor in comparison to the growth in the rate constants between 55oC and lower temperatures. At 65 oC, the rate constant of sonoenzymolysis reaction even was lower than that of enzymolysis reaction. As mentioned above, the intensification of enzymatic hydrolysis by ultrasound would weaken at redundantly high temperature, owing to the reduction of cavitation effects and development in inactivation of glucoamylase with the prolongation of reaction time. Nevertheless, sonoenzymolysis reactions could provide greater rate constants than enzymolysis reactions at the same temperatures below 55oC. It indicates that ultrasound assisted methods work better at lower temperature. Since low operation temperature is preferred in industry for energy saving, cost saving and products protection.
3.4 Thermodynamic parameters Since the rate constant of sonoenzymolysis reaction at 65oC reduced appreciably, the thermodynamic parameters were calculated using the data from 25oC to 55oC. The Arrhenius plot of lnk against 1/T was depicted in Fig. 3(c) where activation energy (Ea) could be obtained from slopes of the Arrhenius plots. It appeared that Ea of enzymolysis and sonoenzymolysis were 46.71 kJ/ mol and 40.92 kJ/ mol respectively. The decrease of activation energy after ultrasonic treatment could be
explained that ultrasound has lessened the energy barrier necessary for the enzymatic reaction. The
- 15 -
relationship between ln (k/T) and 1/T was displayed in Fig. 3(d). Correspondingly, H, S and G
were calculated according to Eyring transition state theory, as listed in Table 3. The difference in G of the two reactions was insignificant. The positive values of H illustrate that the processes are endothermic. The lower H value of sonoenzymolysis reaction signifies the requirement for less energy during starch hydrolysis as compared to enzymolysis. Furthermore, the change in entropy (S) reflects the change of disorderness over the process. The decrease in the S can be resulted from the oxidative alteration of amino acid residues and beginning of enzyme cross-linking and aggregation (Prajapat, Subhedar & Gogate, 2016). The decreased S value indicates a more complete reaction after ultrasonic treatment. Changes of thermodynamic parameters ascertain the acceleration of enzymatic reaction by ultrasound. 3.5 Substrate properties 3.5.1 Mw and Solubility The molecular weights (Mw), radii of gyration (Rg) and solubility of starch samples with different treatment were determined and established in Table 4. As expected, the starch samples treated by enzymolysis, sonolysis and sonoenzymolysis had lower Mw and Rg as well as higher solubility. This indicates that both enzymatic and ultrasonic treatment could effectively decrease the Mw of starch and therefore, improve starch solubility in water. It has been proved that ultrasound could depolymerize starch polymer molecules, as a result of radical and mechanical effects induced by ultrasonic cavitation (BeMiller & Huber, 2015). Accordingly, ultrasonic treatment leads to further reduction of the starch molecular weight and enhances starch degrading operation by glucoamylase. The solubility of starch depends strongly on the molecular structures. Referring to the current
- 16 -
research, the glucoamylase treatment alone increased the solubility of starch approximately by 20%, while treatments by sonolysis and sonoenzymolysis overly doubled the solubility. Obviously, ultrasound assisted enzymatic treatment is more effective in boosting starch solubility, which is consistent with the results of molecular weights. The mechanism could be described that ultrasound has degraded the starch granules by loosening their structures, allowing water to reach into the net structures more easily. 3.5.2 Thermal properties of starch samples DSC analysis was applied to assess thermal properties of starch and results are presented in Table S1. The onset gelatinization temperatures of all starch samples showed little differences. The peak temperatures of sonolysis and sonoenzymolysis starch decreased slightly compared with those of untreated or enzymolysis starch. Besides, the conclusive temperatures of treated starch were lower than that of untreated starch (sonoenzymolysis< sonolysis< enzymolysis< untreated). Consequently, the gelatinization temperature range (Tc -To) also decreased respectively after the different treatments, among which the sonoenzymolysis starch reached the minimum (Tc -To) value. It suggests that both enzymatic and ultrasonic treatments could affect the degree of heterogeneity of crystallites in starch (Luo, Fu, He, Luo, Gao & Yu, 2008). Moreover, ultrasound promotes the breakage of the crystallites caused by enzymolysis. The gelatinization enthalpy (H) reflects the loss of double helical order rather than loss of crystalline register (Thirumdas, Trimukhe, Deshmukh & Annapure, 2017). The shrinkage in H of enzymolysis, sonolysis and sonoenzymolysis starch compared with untreated control indicates that some of the double helical of the granules may have been disrupted. 3.5.3 FT-IR analysis of starch samples
- 17 -
Fig. S3 shows the FT-IR spectra of starch samples from different treatments. From the acquired spectra, several discernible absorption peaks was detected at 1154, 1081, and 1021cm−1, which were attributed to C–O bond stretching. Additional characteristic absorption bands at 929, 856, 763, and 576 cm−1 were due to the skeletal vibration of glucose pyranose ring. Furthermore, an extremely broad band due to O−H stretching appeared around 3384cm−1 and a weak absorption derived from C−H stretching appeared at 2927 cm−1 (Zhang, Xie, Zhao, Liu & Gao, 2009). The bands at 1154 cm−1 and 1081 cm−1 were related to the ordered structures of starch, whereas, the band at 1021 cm−1 was associated with the amorphous structures of starch (Khatoon, Sreerama, Raghavendra, Bhattacharya & Bhat, 2009). The results illustrated that starch crystallinity has barely been altered, as the spectra of four samples are similar with only minor difference between the absorption peak intensities. There is no new formation or loss of chemical bonds and functional groups during the treatments. Generally, little difference was seen among untreated, enzymolysis, sonolysis and sonoenzymolysis starch in our FT-IR study. 3.6 Enzyme structures 3.6.1 Intrinsic fluorescence analysis In order to reveal the effect of ultrasonic treatment on the tertiary structures of glucoamylase, intrinsic fluorescence of the enzyme was explored. Intrinsic fluorescence is mainly originated from tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) presented in the enzyme, especially the Trp residue (Galban, Andreu, Sierra, de Marcos & Castillo, 2001). In this research, we studied the conformational changes of glucoamylase by Trp fluorescence. The Trp residue is the dominant source
of
intrinsic
protein
fluorescence
which
is
highly
sensitive
to
the
local
microenvironment(Ghisaidoobe & Chung, 2014). As indicated in Fig. S4 (a), the fluorescence
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intensity was weakened after ultrasonic treatment with no changes on the maximum emission (λmax=336 nm). It means that ultrasonic treatment has altered the microenvironment of impressible Trp residues, decreasing the amount of Trp on glucoamylase’s surface (Jafari-Aghdam, Khajeh, Ranjbar & Nemat-Gorgani, 2005). This phenomenon is relevant to the unfolding of enzyme protein, induced by the ultrasonic cavitation (Wang, Zhu, Xiao, Liu & Wang, 2011). The external Trp residues were buried partially, while other internal residues were exposed to the surface. This reduction of external Trp residues leads to the reduction of the fluorescence intensity. 3.6.2 Circular Dichroism CD spectroscopy can provide information on the secondary structures of proteins, including α-helix, β-sheet, β-turn, and random coil. The CD spectra of glucoamylase treated with and without ultrasound are shown in Fig. S4 (b), and the contents of α-helix, β-sheet, turn and random coil in the secondary structures are summarized in Table 5. It can be seen that the content of random coil in the glucoamylase increased after undergoing ultrasonic treatment, while the other fractions of the structures revealed an opposite trend. This structural transformation caused by ultrasonic treatment could be traced back to the effects from cavitation pressure and turbulence (Dai, Li & Jiang, 1999). The increase in random coil conformation implies a more flexible glucoamylase structure, enhancing the catalytic efficiency (Subhedar & Gogate, 2014). This transformation might have slightly loosened glucoamylase structure as well as exposing the catalytic center and binding sites, thus accelerating the enzymatic processes.
4. Conclusion A significant intensification of enzymatic hydrolysis extent by ultrasound assisted method was
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established in the current study. Glucoamylase worked effectively upon reducing sugar production, but barely affected the starch molecular degradation when comparing to sonication treatment. Favorably with ultrasound assisted treatment, sonoenzymolysis showed even greater effects both on reducing sugar yield and starch molecular degradation than the individual enzymolysis and sonolysis reactions. Redundantly high temperature generates an adverse effect to the ultrasound assisted enzymatic treatments due to enzyme’s resistance to heat and ultrasound. The rate constants of enzymatic reactions had an increasing trend after ultrasonic treatment within 55oC. Meanwhile sonication raised the Vm while it decreased the Km of the enzymatic reactions, confirming that ultrasound could enhance the enzymatic hydrolysis rate and the enzyme-substrate affinity. Furthermore, the molecular weight of starch deceased sharply after undergoing sonoenzymolysis treatment while the solubility of hydrolyzed starch improved. All of the above claims that the modification of starch by sonication and enzymatic reactions was a synergistic effect. Additionally, starch properties were analyzed by DSC and FT-IR spectra and the results demonstrated that the starch structures were hardly changed. Fluorescence and circular dichroism spectra reflected that ultrasound partially eliminated the external tryptophan residues of glucoamylase and slightly changed the secondary structure. In either case, the remaining effect of ultrasonic modification on glucoamylase requires further study.
Acknowledgements This work was financially supported by the National Key Research and Development Program of China (grant 2016YFD0400301), the Key Research and Development Program of Zhejiang Province (grant 2017C02015) and National Science Foundation of China (Project 31371872).
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Fig. 1. Effect of (a) ultrasonic intensity, (b) temperature, (c) reaction time on the hydrolysis of starch
- 24 -
Fig. 2. Plots of the initial velocity values obtained as a function of the correspondent substrate concentration values using Lineweaver– linearization
Fig. 3. (a) Relationship between ln(V∞− Vt)and reaction time for enzymolysis starch. (b) Relationship between ln(V∞− Vt) and reaction time for sonoenzymolysis starch. (c) Relationship between lnk and 1/T. (d) Relationship between ln(k/T) and 1/T
Table 1 Enzymatic kinetics parameters of the enzymolysis and sonoenzymolysis reactions Samples Enzymolysis
Vm (mg/mL·min−1)
Km (mg/mL)
R2
0.0545±0.0007
0.6511±0.0515
0.9986
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sonoenzymolysis
0.0686±0.0004
0.3581±0.529
0.9948
Table 2 Rate constants of the enzymolysis and sonoenzymolysis reactions Temperature (oC)
25
35
45
55
65
kenzymolysis (10-3min-1)
2.36±0.11
3.74±0.17
7.61±0.09
12.66±0.11
17.73±0.43
ksonoenzymolysis (10-3min-1)
3.20±0.07
4.98±0.02
8.44±0.39
14.40±0.15
15.11±0.75
+35.59
+33.16
+10.91
+13.74
−14.78
Change rate (%)
Table 3 Thermodynamic parameters of the enzymolysis and sonoenzymolysis reactions Ea (kJ/mol)
H (kJ/mol)
S (kJ/mol)
G (kJ/mol)
Enzymolysis
46.71±0.37
44.12±0.37
-147.67±3.82
89.62±1.40
Sonoenzymolysis
40.92±0.25
38.32±0.24
-164.47±3.16
89.00±0.77
Table 4
The average molecular weights (Mw), radii of gyration (Rg) and solubility of starch samples. Untreated
Enzymolysis
Sonolysis
Sonoenzymolysis
Mw (10^5 g/mol)
266.80±19.26
71.80±2.36
8.21±0.09
5.28±0.07
Rg (nm)
267.40±2.67
92.90±1.76
30.90±1.29
26.60±2.02
Solubility (%)
17.13±0.53
20.75±0.71
34.38±0.88
40.50±0.71
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Table 5 Secondary structures of glucoamylase treated with and without ultrasound. Treatment
α-Helix (%)
β-Sheet (%)
Turn (%)
Random coil (%)
Untreated
61.1±11.4
8.6±1.6
13.3±2.6
17.1±3.2
Ultrasound-treated
55.9±7.1
3.8±0.5
11.4±1.5
28.9±3.7
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