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Effects of low intensity ultrasound on cellulase pretreatment
Bioresource Technology 117 (2012) 222–227
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Effects of low intensity ultrasound on cellulase pretreatment Zhenbin Wang a,b,⇑, Xiaoming Lin a, Pingping Li b, Jie Zhang a,c, Shiqing Wang a, Haile Ma a a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education (Jiangsu University), Jiangsu Provincial Key Laboratory of Modern Agricultural Equipment and Technology, Jiangsu University, Zhenjiang 212013, China c College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China b
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 3 April 2012 Accepted 4 April 2012 Available online 13 April 2012 Keywords: Ultrasound Cellulase Enzyme activity Immobilized enzyme Pretreatment
a b s t r a c t This research was to explore the mechanism of ultrasonic impact on free cellulase activity and immobilize cellulase activities. The highest free cellulase activity was achieved when the sample was treated with low intensity ultrasound at 15 W, 24 kHz for 10 min, under which the enzyme activity was increased by 18.17% over the control. Fluorescence and CD spectra revealed that the ultrasonic treatment had increased the number of tryptophan on cellulase surface slightly, with the deformation of certain number of a-helix structure and increase of random coil content in cellulase protein. The highest immobilized cellulase activity was achieved when the sample was treated with low intensity ultrasound at 60 W, 24 kHz for 10 min, under which the enzyme activity was increased by 24.67% over the control. Scanning electron microscopy revealed that the ultrasonic treatment had increased the surface area of immobilized cellulase. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Bioethanol produced from lignocellulosic biomass is an interesting alternative source of energy since lignocellulosic raw materials do not compete with food crops and they are also less expensive than conventional agricultural feedstocks (Alviraa et al., 2010). To achieve an efficient conversion of waste cellulose to soluble sugars, the enzymatic hydrolysis of cellulose is suggested to be preferred to the various acid catalyzed processes using inorganic acid, subcritical or supercritical waters, etc. since the former not only offers a bioconversion process under the simpler and milder operating conditions but also produces no by-products detrimental to fermentative microorganisms (Li et al., 2005). Although the enzymatic route has the highest current cost, it has long-term potential for cost reduction (Zheng et al., 2009). Therefore, the present cellulosic ethanol research is driven by the need to reduce the production cost. The bioethanol process based on the enzymatic hydrolysis of cellulose primarily includes three steps such as biomass pretreatment, enzymatic hydrolysis and fermentation, in which the hydrolysis step is one of the major contributors to the total production cost. Typically, it accounts for over 20% of the total production cost (Goh et al., 2010). A reduction in cellulase production cost, an ⇑ Corresponding author at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China. Tel.: +86 159 0528 8309; fax: +86 511 8878 0201. E-mail address: [email protected] (Z. Wang).
improvement in cellulase performance, and an increase in sugar yield are all vital to reduce the processing costs of bioethanol. Cellulases are relatively costly enzymes, and a significant reduction in cost will be important for their commercial use in bioethanol processing. Cellulase-based strategies that will make the bioethanol processing more economical include: increasing commercial enzyme volumetric productivity, producing enzymes using cheaper substrates, producing enzyme preparations with greater stability for specific processes, and producing cellulases with higher specific activity on solid substrates. It is also an important way to enhance the commercial cellulase activity by molecular modification (Singh et al., 2010). Recently, application of ultrasonic technology in biological processing has widely attracted attention. Ultrasound has been used in the laboratory or processing plant to effect novel changes in the physicochemical properties of biological material in various areas, such as nanoemulsion preparation (Kentish et al., 2008), ultrasound-assisted extraction (Vilkhu et al., 2008), and reduction of viscosity (Iida et al., 2008). More recently the interest of food technologists has turned to the use of power ultrasound in altering enzyme activities. Prolonged exposure to high-intensity ultrasound has been shown to inhibit the catalytic activity of a number of enzymes (Kadkhodaee and Povey, 2008). However, in some cases, enzyme activities have been found to have increased activity following short exposures to ultrasound (Duan et al., 2011; Lee et al., 2008). Some authors have recently reported that ultrasound can accelerate the enzymatic hydrolysis of solid leather waste (Jian et al., 2008). Little is known about the effects of ultrasonic
0960-8524/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.04.015
Z. Wang et al. / Bioresource Technology 117 (2012) 222–227
treatment on the hydrolysis pattern of cellulose with free or immobilized cellulase. The objective of this work was to evaluate the impacts of low intensity ultrasound pretreatment, which ultrasonic power lower than 60 W, on catalytic activity of free enzymatic solution and immobilized cellulase. The fluorescence spectrum and the CD spectra of free cellulase, and the scanning electron micrographs on the microstructure of immobilized cellulase were also analyzed for exploring the mechanism of the ultrasound pretreatment. 2. Methods 2.1. Material and chemicals Cellulase (300 mg/ml) was purchased from Novozymes (China) Biotechnology Co. Chitosan (DAC >95%) was purchased from Nantong Okinari Biochemical Factory (China). Carboxymethyl cellulose (CMC–Na), citric acid, 3,5-dinitrosalicylic acid (DNS), phenol, NaOH, anhydrous sodium sulfite and potassium sodium tartrate, were all of analytical grade. 2.2. Test of effect of low intensity ultrasound on the cellulase Ultrasonic pretreatment experiment was performed in Ultrasonic biological promote growth apparatus (CY-5D, Xinzhi Biological Technology Co., Ltd., Ningbo, China). The instrument can promote growth of biological samples, having five different 2.0 cm flat tip probes at five different frequencies (18, 20, 24, 26, 29 kHz); all can deliver a maximum power of 200 W. Fifty milliliter cellulase sample solution was put into a 100 ml beaker, and the beaker was placed in a water bath at initial temperature of 60 °C. The surface of the cellulase solution in the beaker was 2 cm lower than that of the water bath. Each treatment was replicated three times. 2.2.1. The ultrasonic treatment of free cellulase Cellulase solution was prepared by diluting the purchased cellulase (300 mg/ml) in pH 4.8 citric acid buffer with magnetic stirring for 5 min and the final concentration obtained was 1.5 g/l. The first set of experiments was used to investigate the effect of ultrasound at different ultrasonic time (5, 10, 15, 20, 25, 30 min) at 15 W and 24 kHz. The second set of experiments was used to investigate the effect of ultrasound at different power (5, 10, 15, 20, 25, 30, 35, 40 W) for 10 min at 24 kHz. The third set of experiments was used to investigate the effect of ultrasound at different frequency (18, 20, 24, 26, 29 kHz) for 10 min at 15 W. After being treated by ultrasound for 10 min at 15 W, 24 kHz, the cellulase activities were investigated at different initial temperature (20, 30, 40, 50, 60, 70, 80, 90 °C). 2.2.2. The ultrasonic treatment of immobilized cellulase Chitosan transparent gel solution was prepared by dissolving 1.0 g chitosan flakes into 100 ml of 2% (v/v) acetic acid solution. Seventy-five milliliters of 5% NaOH solution was added to the transparent gel solution and flocculent precipitate came out. The precipitate was obtained by centrifuging at 8000 rpm and then washing with distilled water until its pH value was adjusted to 7.0. It was mixed with 50 ml of 5% glutaraldehyde by stirring in a water bath at room temperature for 2 h and allowed to stand overnight. Cross-linked chitosan were separated from the mixture by centrifuging at 8000 rpm for 15 min, and was washed several times with distilled water to wipe out the residual glutaraldehyde, after which 50 ml of 3.0 g/l cellulase solution was introduced before incubation at 4 °C with gently shaking for 24 h. The immobilized cellulase was filtered and washed with distilled water until the free
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cellulase in the washing could not be detected. It was then transferred into a 50 ml distilled water in 100 ml beaker and explored in different ultrasound condition. After ultrasound treatment, the immobilized cellulase was collected by filtration and then freezedried, after which its catalytic activity was measured and its scanning electron microscopy was analyzed immediately. The first set of experiments was used to investigate the cellulase activities at different initial temperature (30, 40, 50, 60, 70, 80 °C) after being treated by ultrasound for 10 min at 15 W, 24 kHz and pH 3.0. The second set of experiments was used to investigate the cellulase activities at different pH value (2.0, 3.0, 4.0, 4.8, 6.0, 7.0) after being treated by ultrasound at 15 W, 24 kHz, 60 °C for 10 min. The third set of experiments was used to investigate the effect of ultrasound at different frequency (18, 20, 24, 26, 29 kHz) for 10 min at 15 W. The fourth group was used to investigate the effect of ultrasound at different ultrasonic time (5, 10, 15, 20, 25, 30 min) at 15 W, 24 kHz. The fourth set of experiments was used to investigate the effect of ultrasound at different power (10, 20, 30, 40, 50, 60, 70, 80 W) for 10 min at 24 kHz. 2.2.3. Determination of cellulase activity The hydrolytic activity of cellulase was measured by using a 0.51% (w/v) CMC–Na as the substrate. Reaction mixture was incubated in a water bath at 50 °C for 3 min, the amount of generated glucose was measured by the spectrophotometer at 540 nm with DNS agent as a color indicator (Dinçer and Telefoncu, 2007). One unit of cellulase activity was defined by the amount of enzyme, which produced 1.0 lg of reducing sugar from the substrate per minute. 2.3. Test of effect of low intensity ultrasound on the structure of cellulase 2.3.1. Measurement of intrinsic fluorescence and circular dichroism of free cellulase Five series samples were prepared. Series 1 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (18 kHz) at 5 W for 5 min. Series 2 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (24 kHz) at 15 W for 10 min. Series 3 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (26 kHz) at 20 W for 10 min. Series 4 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (29 kHz) at 50 W for 30 min. Series 5 is free cellulase solution (1.5 g/l) without ultrasonic treatment. Intrinsic fluorescence spectra of untreated (control) and ultrasound-treated samples in water were measured at room temperature (25 ± 1 °C) with fluorescence spectrophotometer (Varian Inc., Palo Alto, USA; Model Cary Eclipse) at 280 nm (excitation wavelength, slit = 5 nm), 300–500 nm (emission wavelength, slit = 5 nm) and 10 nm/s of scanning speed. Water used to dissolve cellulase was used as blank solution for the sample. Circular dichroism (CD) spectra were recorded with a spectropolarimeter (French Biologic Company, Grenoble, French; Model MOS-450), using a quartz cuvette of 1 mm optical path length at room temperature (25 ± 1 °C). CD spectra were scanned in the far UV range (250–190 nm) with three replicates at 100 nm/min and with 0.1 nm as bandwidth. The CD data were expressed in terms of mean residue ellipticity, [h], in deg cm2 dmol 1. The a-helix content of cellulase was calculated from the [h] value at 208 nm (Ma et al., 2011). 2.3.2. Scanning electron microscopy (SEM) of immobilized cellulase Four series samples were prepared. Immobilization of cellulase was described as in Section 2.2.2. Series 1 is 50 ml immobilized cellulase solution treated with ultrasound probe (26 kHz) at 27 W for 17 min. Series 2 is 50 ml immobilized cellulase solution treated with ultrasound probe (29 kHz) at 47 W for 30 min. Series 3 is
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3.1. Effect of low intensity ultrasonic pretreatment on the cellulase activity
activity within the first 15 min. When the treatment duration exceeded 15 min, cellulase activity was lower than the control. The ultrasonic power is an important factor to be considered during cellulase pretreatment. Free cellulase activity was enhanced with increasing ultrasonic power up to 15 W than that of the control (without exposure to ultrasound). Further increase in ultrasonic power did not improve the value of cellulase activity, but decreased the catalytic activity at 35 and 40 W. The effect of the ultrasound pretreatment frequency on cellulase activity was not significant. This is probably because the power ultrasound cannot affect the cellulase molecular bonds directly. Its frequency ranges from 20 to 50 kHz, with corresponding ultrasonic wavelength range from 5 to 0.03 mm, which is much greater than the size of cellulase molecule. The effect of temperature on catalytic activity was also investigated. It was observed that the ultrasonic pretreated cellulase activity was not significantly different compared to the control (0 min) (p < 0.05), and the optimum catalytic temperature was 50 °C.
3.1.1. The ultrasonic treatment of free cellulase The effect of low intensity ultrasonic pretreatment on free cellulase activity is shown in Fig. 1. The results show that an increase in pretreatment time significantly increased the cellulase
3.1.2. The ultrasonic treatment of immobilized cellulase The results on the effects of enzymatic condition on the immobilized cellulase activity are shown in Fig. 2. It can be seen that the thermal and pH stability of the ultrasound treated immobilized
immobilized cellulase without ultrasonic treatment. Series 4 is chitosan flakes. A thin layer of the sample granules was mounted on the copper sample-holder, using a double sided carbon tape and coated with gold of 10 nm thicknesses to make the samples conductive. SEM studies were carried out using a scanning electron microscope (JSM-7001F, JEOL, Tokyo, Japan) at acceleration voltage of 15 kV. 2.4. Statistical analysis Analysis of variance (ANOVA) was performed to compare the effects of ultrasound under the significance level of p < 0.05. All graphs and calculations were performed with OriginPro 8.0 and Microsoft Office Excel 2007, respectively.
Enzyme activity, U
Enzyme activity, U
3. Results and discussion
Ultrasonic time, min
Ultrasonic power, W
(B)
Enzyme activity, U
Enzyme activity, U
(A)
Ultrasonic frequencye, kHz
(C)
Temperature, oC
(D)
Fig. 1. Effect of low intensity ultrasound pretreatment factors of ultrasonic time (A), ultrasonic power (B), ultrasonic frequency (C) and temperature (D) on the free cellulase activity. Conditions: (A) ultrasound was at 24 kHz and 15 W; (B) ultrasonic frequency was at 24 kHz for 10 min; (C) ultrasonic power was at 15 W for 10 min; (D) ultrasound was at 24 kHz and 15 W for 10 min.
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Enzyme activity, U
Enzyme activity, U
Z. Wang et al. / Bioresource Technology 117 (2012) 222–227
Temperature,o C
pH
(A)
(B)
Enzyme activity, U
Enzyme activity, U
Fig. 2. Effect of enzymatic condition of temperature (A) and pH (B) on the immobilized cellulase activity. Conditions: (A) ultrasound was at 24 kHz, 15 W for 10 min, and the pH 3.0; (B) ultrasound was at 24 kHz and 15 W for 10 min, and temperature 60 °C.
Ultrasonic frequency, kHz
(B)
Enzyme activity, U
(A)
Ultrasonic time, min
Ultrasonic power, W
(C) Fig. 3. Effect of low intensity ultrasonic pretreatment factors of ultrasonic frequency (A), ultrasonic time (B) and ultrasonic power (C) on the immobilized cellulase activity. Conditions: (A) ultrasonic power was at 15 W for 10 min, the pH 3.0 and temperature 60 °C; (B) ultrasound was at 15 W and 24 kHz, the pH 3.0 and temperature 60 °C; (C) Ultrasonic frequency at 24 kHz for 10 min.
Z. Wang et al. / Bioresource Technology 117 (2012) 222–227
cellulase are enhanced respectively. The optimum temperature of the native and the immobilized enzymes were 50 and 60 °C, respectively. This variation may be caused by the covalent bond formation leading to the reduction of the conformational flexibility. The shifts of optimum pH were observed as well: 4.8 for the native enzyme and 3.0 for immobilized enzymes. It was known that the secondary interaction such as ionic and polar interactions between the enzymes and ionic matrices was the major factor for the shift of optimum pH. It seems that cation carriers of chitosan play an important role in changing the charge that caused the optimum pH to shift. The results on the effects of low intensity ultrasonic pretreatment on the immobilized cellulase activity are shown in Fig. 3. The effect of ultrasonic frequency on the immobilized cellulase catalytic activity can be seen from Fig. 3A. The catalytic activity of immobilized cellulase were increased by 6.56%, 14.79%, 17.85%, and 10.45%, respectively at 18, 20, 24 and 26 kHz, respectively, and decreased by 1.02% at 29 kHz than the control. This was probably due to the ability of ultrasound to increase the surface area of immobilization cellulase particles. But the surface was destroyed and the catalytic activity was decreased when too much ultrasound energy was absorbed by the immobilization cellulase particles. The percentage of ultrasound energy absorbed by immobilization cellulase particles varied with the difference between wavelength of ultrasound and size of immobilization cellulase particles, the little the difference, the more the ultrasound energy absorbed because of the resonance effect. The effect of ultrasonic pretreatment time and power on the immobilized cellulase activity is shown in Fig. 3B and C. The trends are similar to the effect of ultrasonic pretreatment time and power on the free cellulase, but the due pretreatment time and power were prolonged to 20 min and 60 W, the cellulase activity were 6.34 U and 6.36 U, increased by 21.16% and 24.67%, respectively than the control 5.23 U. It is probably because immobilization protected the cellulase and lessened the ultrasound energy exerted, in other words it improved the stability of cellulase. 3.2. Effect of ultrasonic treatment on the structure of cellulase 3.2.1. Fluorescence spectra analysis of free cellulase The fluorescence spectrum is mainly attributed to the Trp, Tyr and Phe residues, particularly the Trp residue (Jia et al., 2010). In this work, changes of enzyme conformation were investigated by Trp fluorescence spectrum (the maximum fluorescence emission wavelength is 348 nm). As seen in Fig. 4, the fluorescence intensity of the enzyme molecule decreased gradually with the increase of ultrasonic power, ultrasonic frequency and ultrasonic time. However, the optimum fluorescence emission wavelength (kmax = 348 nm) changed a little. That is to say, the optimum fluorescence emission wavelength did not show a red or blue shift. The results suggest that enzyme conformation changed regularly with ultrasonic pretreatment The reason could be that ultrasonic pretreatment induced molecular unfolding of protein, destroyed hydrophobic interactions of protein molecules, caused more hydrophobic groups and regions inside the molecules to expose to the outside (Gülseren et al., 2007; Jambrak et al., 2008). 3.2.2. Circular dichroism analysis of free cellulase The enzyme protein secondary structure change can be analyzed by circular dichroism (CD) spectroscopy. The content of ahelix, b-sheet and random coil of cellulase was calculated to clarify the connection between enzyme activity and secondary structure. The data in Table 1 show that the activity of cellulase increased to 52.41 U/ml, 59.58 U/ml and 54.77 U/ml after ultrasonic treatment at 18 kHz, 5 W for 5 min, at 24 kHz, 15 W for 10 min and at 26 kHz, 20 W for 10 min, respectively, Meanwhile, ultrasound
1000
Fluorescence intensity (a.u.)
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800
600
400
200
0 300
320
340
360
380
400
Wavelength (nm)
Control Ultrasound at 18KHz, 5W for 5 min. Ultrasound at 24KHz, 15W for 10 min. Ultrasound at 26KHz, 20W for 10 min. Ultrasound at 29KHz, 50W for 30 min.
Fig. 4. Intrinsic fluorescence spectra of control and ultrasonic-treated cellulase.
Table 1 The contents of secondary structure of cellulase after treatment with ultrasound. (Enzyme activity given is expressed as mean ± standard deviation.) Treatment
Control Ultrasound at 18 kHz, 5 W for 5 min. Ultrasound at 24 kHz, 15 W for 10 min. Ultrasound at 26 kHz, 20 W for 10 min. Ultrasound at 29 kHz, 50 W for 30 min.
aHelix (%)
bSheet (%)
bTurn (%)
Random coil (%)
Enzyme activity (U/ml)
26. 2 23.9
26.6 25.0
21.9 23.7
24.8 30.4
50.42 ± 1.75 52.21 ± 1.38
23.4
25.0
23.7
32.1
59.58 ± 1.44
24.8
24.6
22.8
26.9
54.77 ± 1.71
27.6
26.4
23.5
22.2
31.62 ± 4.23
caused conformational changes of the protein with the deformation of a certain number of a-helix structure and increase in random coil content of cellulase protein. These changes made cellulase exhibit softness and more flexibility, which made the substance more accessible to the cellulase activity center and was helpful for the improvement of cellulase activity. However, with the increase in a-helix structure content and a decrease in random coil content of cellulase protein the activity of cellulase decreased to 31.62 U/ml after excessive ultrasonic treatment at 29 kHz, 50 W for 30 min, which indicated that the tighter structure may be covered by the enzyme activity center thereby hindering the substance from reacting with the enzyme. 3.2.3. Scanning electron microscopy analysis of immobilized cellulase The microstructure of chitosan, immobilized cellulase without pretreatment and the immobilized cellulase pretreated by ultrasound were examined using scanning electron microscopy. Immobilized cellulase after ultrasonic pretreatment became looser than that without ultrasonic pretreatment. It suggests that appropriate ultrasonic pretreatment may increase the surface area of immobilized cellulase, and help enzyme to attack substrate easily during enzymatic hydrolysis, which resulted in a higher enzyme activity than that without ultrasonic pretreatment. However, higher ultrasound power for a long time may destroy the cross linking of immobilized cellulase, reducing the attachment probability of the
Z. Wang et al. / Bioresource Technology 117 (2012) 222–227
free cellulase on the enzyme loading, and cellulase making it easy for the cellulase to fall off. Hence the usage repeatability was reduced, and may result in inactivation of the enzyme. The effect of ultrasonic treatment on enzyme can be attributed to the formation of localized hot spots upon collapse of bubbles, mechanical stresses or shear forces created by microstreaming and shock waves, and generation of free radicals through sonolysis of water. Of these it has been reported that the role of free radicals in denaturation and inactivation of enzymes is the most important mechanism (Jambrak et al., 2008; Kadkhodaee and Povey, 2008; Ma et al., 2008). Essentially any amino acid radical formed within a peptide chain could cross-link with an amino acid radical in another protein. Alternatively, some radicals may cross-link with amino acid molecules. The selectivity and rate constants for such reactions are not well understood (Van Tilbeurgh and Claeyssens, 1985). The authors think that the increase in catalytic activity was due to the cellulase molecular structure which was modestly modified by the ultrasound free radicals, making the enzymes more readily accessible for reaction and thus resulting in the increase of cellulase activity, but it does not mean that the more the modifier, the higher the catalytic activity, on the contrary, when the molecular structure is excessively modified than the modest, the obstacle of cellulase attacking the substrate is augmented and catalytic activity declined. Ma et al. think the increase of catalytic activity was probably due to the ability of ultrasound to break down molecular aggregates (Ma et al., 2008), the decrease of catalytic activity was probably due to excessive pressure, temperature or shear force generated by the ultrasonic treatment, and generation of free radicals through sonolysis of water. 4. Conclusion In this study, low intensity ultrasound was demonstrated to have a positive effect on free cellulase activity and immobilize cellulase activities. The molecular structure of free cellulase was changed showed by Fluorescence and CD spectra when ultrasound was at 15 W, 24 kHz for 10 min, and the enzyme activity was increased by 18.17% over the control. To the immobilize cellulase, its surface area was increased while the ultrasound was at 60 W, 24 kHz for 10 min, and its activity was increased by 24.67% over the control. Acknowledgements This research was supported by grants from the 863 Research Program of China (No. 2007AA10Z321), the Natural Science
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Foundation of Jiangsu Province (No. BK2008241) and China Postdoctoral Science Foundation (Nos. 20090461072, 0902108C). References Alviraa, P., Ballesterosa, M., Tomás-Pejóa, E., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861. Dinçer, A., Telefoncu, A., 2007. Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads. J. Mol. Catal. B: Enzym. 45, 10–14. Duan, X., Zhou, J., Qiao, S., Wei, H., 2011. Application of low intensity ultrasound to enhance the activity of anammox microbial consortium for nitrogen removal. Bioresour. Technol. 102, 4290–4293. _ Güzey, D., Bruce, B.D., Weiss, J., 2007. Structural and functional changes Gülseren, I., in ultrasonicated bovine serum albumin solutions. Ultrason. Sonochem. 14, 173–183. Goh, C.S., Tan, K.T., Lee, K.T., Bhatia, S., 2010. Bio-ethanol from lignocellulose: status, perspectives and challenges in Malaysia. Bioresour. Technol. 101, 4834–4841. Iida, Y., Toru, T., Yasui, K., Towata, A., Kozuka, T., 2008. Control of viscosity in starch and polysaccharide solutions with ultrasound after gelatinization. Innov. Food Sci. Emerg. 9, 140–146. Jambrak, A.R., Mason, T.J., Lelas, V., Herceg, Z., Herceg, I.L., 2008. Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions. J. Food Eng. 86, 281–287. Jia, J.Q., Ma, H.L., Zhao, W.R., Wang, Z.B., Tian, W.M., Luo, L., He, R.H., 2010. The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein. Food Chem. 119, 336–342. Jian, S., Wenyi, T., Wuyong, C., 2008. Ultrasound-accelerated enzymatic hydrolysis of solid leather waste. J. Clean Prod. 16, 591–597. Kadkhodaee, R., Povey, M.J.W., 2008. Ultrasonic inactivation of Bacillus [alpha]amylase. I. Effect of gas content and emitting face of probe. Ultrason. Sonochem. 15, 133–142. Kentish, S., Wooster, T.J., Ashokkumar, M., Balachandran, S., Mawson, R., Simons, L., 2008. The use of ultrasonics for nanoemulsion preparation. Innov. Food Sci. Emerg. 9, 170–175. Lee, S.H., Nguyen, H.M., Koo, Y.-M., Ha, S.H., 2008. Ultrasound-enhanced lipase activity in the synthesis of sugar ester using ionic liquids. Process Biochem. 43, 1009–1012. Li, C.Z., Yoshimoto, M., Ogata, H., Tsukuda, N., Fukunaga, K., Nakao, K., 2005. Effects of ultrasonic intensity and reactor scale on kinetics of enzymatic saccharification of various waste papers in continuously irradiated stirred tanks. Ultrason. Sonochem. 12, 373–384. Ma, H., Huang, L., Jia, J., He, R., Luo, L., Zhu, W., 2011. Effect of energy-gathered ultrasound on Alcalase. Ultrason. Sonochem. 18, 419–424. Ma, Y.Q., Ye, X.Q., Hao, Y.B., Xu, G.N., Xu, G.H., Liu, D.H., 2008. Ultrasound-assisted extraction of hesperidin from Penggan (Citrus reticulata) peel. Ultrason. Sonochem. 15, 227–232. Singh, A., Pant, D., Korres, N.E., Nizami, A.-S., Prasad, S., Murphy, J.D., 2010. Key issues in life cycle assessment of ethanol production from lignocellulosic biomass: challenges and perspectives. Bioresour. Technol. 101, 5003–5012. Van Tilbeurgh, H., Claeyssens, M., 1985. Detection and differentiation of cellulase components using low molecular mass fluorogenic substrates. FEBS Lett. 187, 283–288. Vilkhu, K., Mawson, R., Simons, L., Bates, D., 2008. Applications and opportunities for ultrasound assisted extraction in the food industry – A review. Innov. Food Sci. Emerg. 9, 161–169. Zheng, Y., Pan, Z.L., Zhang, R.H., Wang, D.H., 2009. Enzymatic saccharification of dilute acid pretreated saline crops for fermentable sugar production. Appl. Energ. 86, 2459–2465.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effects of low intensity ultrasound on cellulase pretreatment Zhenbin Wang a,b,⇑, Xiaoming Lin a, Pingping Li b, Jie Zhang a,c, Shiqing Wang a, Haile Ma a a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education (Jiangsu University), Jiangsu Provincial Key Laboratory of Modern Agricultural Equipment and Technology, Jiangsu University, Zhenjiang 212013, China c College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China b
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 3 April 2012 Accepted 4 April 2012 Available online 13 April 2012 Keywords: Ultrasound Cellulase Enzyme activity Immobilized enzyme Pretreatment
a b s t r a c t This research was to explore the mechanism of ultrasonic impact on free cellulase activity and immobilize cellulase activities. The highest free cellulase activity was achieved when the sample was treated with low intensity ultrasound at 15 W, 24 kHz for 10 min, under which the enzyme activity was increased by 18.17% over the control. Fluorescence and CD spectra revealed that the ultrasonic treatment had increased the number of tryptophan on cellulase surface slightly, with the deformation of certain number of a-helix structure and increase of random coil content in cellulase protein. The highest immobilized cellulase activity was achieved when the sample was treated with low intensity ultrasound at 60 W, 24 kHz for 10 min, under which the enzyme activity was increased by 24.67% over the control. Scanning electron microscopy revealed that the ultrasonic treatment had increased the surface area of immobilized cellulase. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction Bioethanol produced from lignocellulosic biomass is an interesting alternative source of energy since lignocellulosic raw materials do not compete with food crops and they are also less expensive than conventional agricultural feedstocks (Alviraa et al., 2010). To achieve an efficient conversion of waste cellulose to soluble sugars, the enzymatic hydrolysis of cellulose is suggested to be preferred to the various acid catalyzed processes using inorganic acid, subcritical or supercritical waters, etc. since the former not only offers a bioconversion process under the simpler and milder operating conditions but also produces no by-products detrimental to fermentative microorganisms (Li et al., 2005). Although the enzymatic route has the highest current cost, it has long-term potential for cost reduction (Zheng et al., 2009). Therefore, the present cellulosic ethanol research is driven by the need to reduce the production cost. The bioethanol process based on the enzymatic hydrolysis of cellulose primarily includes three steps such as biomass pretreatment, enzymatic hydrolysis and fermentation, in which the hydrolysis step is one of the major contributors to the total production cost. Typically, it accounts for over 20% of the total production cost (Goh et al., 2010). A reduction in cellulase production cost, an ⇑ Corresponding author at: School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China. Tel.: +86 159 0528 8309; fax: +86 511 8878 0201. E-mail address: [email protected] (Z. Wang).
improvement in cellulase performance, and an increase in sugar yield are all vital to reduce the processing costs of bioethanol. Cellulases are relatively costly enzymes, and a significant reduction in cost will be important for their commercial use in bioethanol processing. Cellulase-based strategies that will make the bioethanol processing more economical include: increasing commercial enzyme volumetric productivity, producing enzymes using cheaper substrates, producing enzyme preparations with greater stability for specific processes, and producing cellulases with higher specific activity on solid substrates. It is also an important way to enhance the commercial cellulase activity by molecular modification (Singh et al., 2010). Recently, application of ultrasonic technology in biological processing has widely attracted attention. Ultrasound has been used in the laboratory or processing plant to effect novel changes in the physicochemical properties of biological material in various areas, such as nanoemulsion preparation (Kentish et al., 2008), ultrasound-assisted extraction (Vilkhu et al., 2008), and reduction of viscosity (Iida et al., 2008). More recently the interest of food technologists has turned to the use of power ultrasound in altering enzyme activities. Prolonged exposure to high-intensity ultrasound has been shown to inhibit the catalytic activity of a number of enzymes (Kadkhodaee and Povey, 2008). However, in some cases, enzyme activities have been found to have increased activity following short exposures to ultrasound (Duan et al., 2011; Lee et al., 2008). Some authors have recently reported that ultrasound can accelerate the enzymatic hydrolysis of solid leather waste (Jian et al., 2008). Little is known about the effects of ultrasonic
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treatment on the hydrolysis pattern of cellulose with free or immobilized cellulase. The objective of this work was to evaluate the impacts of low intensity ultrasound pretreatment, which ultrasonic power lower than 60 W, on catalytic activity of free enzymatic solution and immobilized cellulase. The fluorescence spectrum and the CD spectra of free cellulase, and the scanning electron micrographs on the microstructure of immobilized cellulase were also analyzed for exploring the mechanism of the ultrasound pretreatment. 2. Methods 2.1. Material and chemicals Cellulase (300 mg/ml) was purchased from Novozymes (China) Biotechnology Co. Chitosan (DAC >95%) was purchased from Nantong Okinari Biochemical Factory (China). Carboxymethyl cellulose (CMC–Na), citric acid, 3,5-dinitrosalicylic acid (DNS), phenol, NaOH, anhydrous sodium sulfite and potassium sodium tartrate, were all of analytical grade. 2.2. Test of effect of low intensity ultrasound on the cellulase Ultrasonic pretreatment experiment was performed in Ultrasonic biological promote growth apparatus (CY-5D, Xinzhi Biological Technology Co., Ltd., Ningbo, China). The instrument can promote growth of biological samples, having five different 2.0 cm flat tip probes at five different frequencies (18, 20, 24, 26, 29 kHz); all can deliver a maximum power of 200 W. Fifty milliliter cellulase sample solution was put into a 100 ml beaker, and the beaker was placed in a water bath at initial temperature of 60 °C. The surface of the cellulase solution in the beaker was 2 cm lower than that of the water bath. Each treatment was replicated three times. 2.2.1. The ultrasonic treatment of free cellulase Cellulase solution was prepared by diluting the purchased cellulase (300 mg/ml) in pH 4.8 citric acid buffer with magnetic stirring for 5 min and the final concentration obtained was 1.5 g/l. The first set of experiments was used to investigate the effect of ultrasound at different ultrasonic time (5, 10, 15, 20, 25, 30 min) at 15 W and 24 kHz. The second set of experiments was used to investigate the effect of ultrasound at different power (5, 10, 15, 20, 25, 30, 35, 40 W) for 10 min at 24 kHz. The third set of experiments was used to investigate the effect of ultrasound at different frequency (18, 20, 24, 26, 29 kHz) for 10 min at 15 W. After being treated by ultrasound for 10 min at 15 W, 24 kHz, the cellulase activities were investigated at different initial temperature (20, 30, 40, 50, 60, 70, 80, 90 °C). 2.2.2. The ultrasonic treatment of immobilized cellulase Chitosan transparent gel solution was prepared by dissolving 1.0 g chitosan flakes into 100 ml of 2% (v/v) acetic acid solution. Seventy-five milliliters of 5% NaOH solution was added to the transparent gel solution and flocculent precipitate came out. The precipitate was obtained by centrifuging at 8000 rpm and then washing with distilled water until its pH value was adjusted to 7.0. It was mixed with 50 ml of 5% glutaraldehyde by stirring in a water bath at room temperature for 2 h and allowed to stand overnight. Cross-linked chitosan were separated from the mixture by centrifuging at 8000 rpm for 15 min, and was washed several times with distilled water to wipe out the residual glutaraldehyde, after which 50 ml of 3.0 g/l cellulase solution was introduced before incubation at 4 °C with gently shaking for 24 h. The immobilized cellulase was filtered and washed with distilled water until the free
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cellulase in the washing could not be detected. It was then transferred into a 50 ml distilled water in 100 ml beaker and explored in different ultrasound condition. After ultrasound treatment, the immobilized cellulase was collected by filtration and then freezedried, after which its catalytic activity was measured and its scanning electron microscopy was analyzed immediately. The first set of experiments was used to investigate the cellulase activities at different initial temperature (30, 40, 50, 60, 70, 80 °C) after being treated by ultrasound for 10 min at 15 W, 24 kHz and pH 3.0. The second set of experiments was used to investigate the cellulase activities at different pH value (2.0, 3.0, 4.0, 4.8, 6.0, 7.0) after being treated by ultrasound at 15 W, 24 kHz, 60 °C for 10 min. The third set of experiments was used to investigate the effect of ultrasound at different frequency (18, 20, 24, 26, 29 kHz) for 10 min at 15 W. The fourth group was used to investigate the effect of ultrasound at different ultrasonic time (5, 10, 15, 20, 25, 30 min) at 15 W, 24 kHz. The fourth set of experiments was used to investigate the effect of ultrasound at different power (10, 20, 30, 40, 50, 60, 70, 80 W) for 10 min at 24 kHz. 2.2.3. Determination of cellulase activity The hydrolytic activity of cellulase was measured by using a 0.51% (w/v) CMC–Na as the substrate. Reaction mixture was incubated in a water bath at 50 °C for 3 min, the amount of generated glucose was measured by the spectrophotometer at 540 nm with DNS agent as a color indicator (Dinçer and Telefoncu, 2007). One unit of cellulase activity was defined by the amount of enzyme, which produced 1.0 lg of reducing sugar from the substrate per minute. 2.3. Test of effect of low intensity ultrasound on the structure of cellulase 2.3.1. Measurement of intrinsic fluorescence and circular dichroism of free cellulase Five series samples were prepared. Series 1 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (18 kHz) at 5 W for 5 min. Series 2 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (24 kHz) at 15 W for 10 min. Series 3 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (26 kHz) at 20 W for 10 min. Series 4 is 50 ml cellulase solution (1.5 g/l) treated with ultrasound probe (29 kHz) at 50 W for 30 min. Series 5 is free cellulase solution (1.5 g/l) without ultrasonic treatment. Intrinsic fluorescence spectra of untreated (control) and ultrasound-treated samples in water were measured at room temperature (25 ± 1 °C) with fluorescence spectrophotometer (Varian Inc., Palo Alto, USA; Model Cary Eclipse) at 280 nm (excitation wavelength, slit = 5 nm), 300–500 nm (emission wavelength, slit = 5 nm) and 10 nm/s of scanning speed. Water used to dissolve cellulase was used as blank solution for the sample. Circular dichroism (CD) spectra were recorded with a spectropolarimeter (French Biologic Company, Grenoble, French; Model MOS-450), using a quartz cuvette of 1 mm optical path length at room temperature (25 ± 1 °C). CD spectra were scanned in the far UV range (250–190 nm) with three replicates at 100 nm/min and with 0.1 nm as bandwidth. The CD data were expressed in terms of mean residue ellipticity, [h], in deg cm2 dmol 1. The a-helix content of cellulase was calculated from the [h] value at 208 nm (Ma et al., 2011). 2.3.2. Scanning electron microscopy (SEM) of immobilized cellulase Four series samples were prepared. Immobilization of cellulase was described as in Section 2.2.2. Series 1 is 50 ml immobilized cellulase solution treated with ultrasound probe (26 kHz) at 27 W for 17 min. Series 2 is 50 ml immobilized cellulase solution treated with ultrasound probe (29 kHz) at 47 W for 30 min. Series 3 is
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3.1. Effect of low intensity ultrasonic pretreatment on the cellulase activity
activity within the first 15 min. When the treatment duration exceeded 15 min, cellulase activity was lower than the control. The ultrasonic power is an important factor to be considered during cellulase pretreatment. Free cellulase activity was enhanced with increasing ultrasonic power up to 15 W than that of the control (without exposure to ultrasound). Further increase in ultrasonic power did not improve the value of cellulase activity, but decreased the catalytic activity at 35 and 40 W. The effect of the ultrasound pretreatment frequency on cellulase activity was not significant. This is probably because the power ultrasound cannot affect the cellulase molecular bonds directly. Its frequency ranges from 20 to 50 kHz, with corresponding ultrasonic wavelength range from 5 to 0.03 mm, which is much greater than the size of cellulase molecule. The effect of temperature on catalytic activity was also investigated. It was observed that the ultrasonic pretreated cellulase activity was not significantly different compared to the control (0 min) (p < 0.05), and the optimum catalytic temperature was 50 °C.
3.1.1. The ultrasonic treatment of free cellulase The effect of low intensity ultrasonic pretreatment on free cellulase activity is shown in Fig. 1. The results show that an increase in pretreatment time significantly increased the cellulase
3.1.2. The ultrasonic treatment of immobilized cellulase The results on the effects of enzymatic condition on the immobilized cellulase activity are shown in Fig. 2. It can be seen that the thermal and pH stability of the ultrasound treated immobilized
immobilized cellulase without ultrasonic treatment. Series 4 is chitosan flakes. A thin layer of the sample granules was mounted on the copper sample-holder, using a double sided carbon tape and coated with gold of 10 nm thicknesses to make the samples conductive. SEM studies were carried out using a scanning electron microscope (JSM-7001F, JEOL, Tokyo, Japan) at acceleration voltage of 15 kV. 2.4. Statistical analysis Analysis of variance (ANOVA) was performed to compare the effects of ultrasound under the significance level of p < 0.05. All graphs and calculations were performed with OriginPro 8.0 and Microsoft Office Excel 2007, respectively.
Enzyme activity, U
Enzyme activity, U
3. Results and discussion
Ultrasonic time, min
Ultrasonic power, W
(B)
Enzyme activity, U
Enzyme activity, U
(A)
Ultrasonic frequencye, kHz
(C)
Temperature, oC
(D)
Fig. 1. Effect of low intensity ultrasound pretreatment factors of ultrasonic time (A), ultrasonic power (B), ultrasonic frequency (C) and temperature (D) on the free cellulase activity. Conditions: (A) ultrasound was at 24 kHz and 15 W; (B) ultrasonic frequency was at 24 kHz for 10 min; (C) ultrasonic power was at 15 W for 10 min; (D) ultrasound was at 24 kHz and 15 W for 10 min.
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Enzyme activity, U
Enzyme activity, U
Z. Wang et al. / Bioresource Technology 117 (2012) 222–227
Temperature,o C
pH
(A)
(B)
Enzyme activity, U
Enzyme activity, U
Fig. 2. Effect of enzymatic condition of temperature (A) and pH (B) on the immobilized cellulase activity. Conditions: (A) ultrasound was at 24 kHz, 15 W for 10 min, and the pH 3.0; (B) ultrasound was at 24 kHz and 15 W for 10 min, and temperature 60 °C.
Ultrasonic frequency, kHz
(B)
Enzyme activity, U
(A)
Ultrasonic time, min
Ultrasonic power, W
(C) Fig. 3. Effect of low intensity ultrasonic pretreatment factors of ultrasonic frequency (A), ultrasonic time (B) and ultrasonic power (C) on the immobilized cellulase activity. Conditions: (A) ultrasonic power was at 15 W for 10 min, the pH 3.0 and temperature 60 °C; (B) ultrasound was at 15 W and 24 kHz, the pH 3.0 and temperature 60 °C; (C) Ultrasonic frequency at 24 kHz for 10 min.
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cellulase are enhanced respectively. The optimum temperature of the native and the immobilized enzymes were 50 and 60 °C, respectively. This variation may be caused by the covalent bond formation leading to the reduction of the conformational flexibility. The shifts of optimum pH were observed as well: 4.8 for the native enzyme and 3.0 for immobilized enzymes. It was known that the secondary interaction such as ionic and polar interactions between the enzymes and ionic matrices was the major factor for the shift of optimum pH. It seems that cation carriers of chitosan play an important role in changing the charge that caused the optimum pH to shift. The results on the effects of low intensity ultrasonic pretreatment on the immobilized cellulase activity are shown in Fig. 3. The effect of ultrasonic frequency on the immobilized cellulase catalytic activity can be seen from Fig. 3A. The catalytic activity of immobilized cellulase were increased by 6.56%, 14.79%, 17.85%, and 10.45%, respectively at 18, 20, 24 and 26 kHz, respectively, and decreased by 1.02% at 29 kHz than the control. This was probably due to the ability of ultrasound to increase the surface area of immobilization cellulase particles. But the surface was destroyed and the catalytic activity was decreased when too much ultrasound energy was absorbed by the immobilization cellulase particles. The percentage of ultrasound energy absorbed by immobilization cellulase particles varied with the difference between wavelength of ultrasound and size of immobilization cellulase particles, the little the difference, the more the ultrasound energy absorbed because of the resonance effect. The effect of ultrasonic pretreatment time and power on the immobilized cellulase activity is shown in Fig. 3B and C. The trends are similar to the effect of ultrasonic pretreatment time and power on the free cellulase, but the due pretreatment time and power were prolonged to 20 min and 60 W, the cellulase activity were 6.34 U and 6.36 U, increased by 21.16% and 24.67%, respectively than the control 5.23 U. It is probably because immobilization protected the cellulase and lessened the ultrasound energy exerted, in other words it improved the stability of cellulase. 3.2. Effect of ultrasonic treatment on the structure of cellulase 3.2.1. Fluorescence spectra analysis of free cellulase The fluorescence spectrum is mainly attributed to the Trp, Tyr and Phe residues, particularly the Trp residue (Jia et al., 2010). In this work, changes of enzyme conformation were investigated by Trp fluorescence spectrum (the maximum fluorescence emission wavelength is 348 nm). As seen in Fig. 4, the fluorescence intensity of the enzyme molecule decreased gradually with the increase of ultrasonic power, ultrasonic frequency and ultrasonic time. However, the optimum fluorescence emission wavelength (kmax = 348 nm) changed a little. That is to say, the optimum fluorescence emission wavelength did not show a red or blue shift. The results suggest that enzyme conformation changed regularly with ultrasonic pretreatment The reason could be that ultrasonic pretreatment induced molecular unfolding of protein, destroyed hydrophobic interactions of protein molecules, caused more hydrophobic groups and regions inside the molecules to expose to the outside (Gülseren et al., 2007; Jambrak et al., 2008). 3.2.2. Circular dichroism analysis of free cellulase The enzyme protein secondary structure change can be analyzed by circular dichroism (CD) spectroscopy. The content of ahelix, b-sheet and random coil of cellulase was calculated to clarify the connection between enzyme activity and secondary structure. The data in Table 1 show that the activity of cellulase increased to 52.41 U/ml, 59.58 U/ml and 54.77 U/ml after ultrasonic treatment at 18 kHz, 5 W for 5 min, at 24 kHz, 15 W for 10 min and at 26 kHz, 20 W for 10 min, respectively, Meanwhile, ultrasound
1000
Fluorescence intensity (a.u.)
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800
600
400
200
0 300
320
340
360
380
400
Wavelength (nm)
Control Ultrasound at 18KHz, 5W for 5 min. Ultrasound at 24KHz, 15W for 10 min. Ultrasound at 26KHz, 20W for 10 min. Ultrasound at 29KHz, 50W for 30 min.
Fig. 4. Intrinsic fluorescence spectra of control and ultrasonic-treated cellulase.
Table 1 The contents of secondary structure of cellulase after treatment with ultrasound. (Enzyme activity given is expressed as mean ± standard deviation.) Treatment
Control Ultrasound at 18 kHz, 5 W for 5 min. Ultrasound at 24 kHz, 15 W for 10 min. Ultrasound at 26 kHz, 20 W for 10 min. Ultrasound at 29 kHz, 50 W for 30 min.
aHelix (%)
bSheet (%)
bTurn (%)
Random coil (%)
Enzyme activity (U/ml)
26. 2 23.9
26.6 25.0
21.9 23.7
24.8 30.4
50.42 ± 1.75 52.21 ± 1.38
23.4
25.0
23.7
32.1
59.58 ± 1.44
24.8
24.6
22.8
26.9
54.77 ± 1.71
27.6
26.4
23.5
22.2
31.62 ± 4.23
caused conformational changes of the protein with the deformation of a certain number of a-helix structure and increase in random coil content of cellulase protein. These changes made cellulase exhibit softness and more flexibility, which made the substance more accessible to the cellulase activity center and was helpful for the improvement of cellulase activity. However, with the increase in a-helix structure content and a decrease in random coil content of cellulase protein the activity of cellulase decreased to 31.62 U/ml after excessive ultrasonic treatment at 29 kHz, 50 W for 30 min, which indicated that the tighter structure may be covered by the enzyme activity center thereby hindering the substance from reacting with the enzyme. 3.2.3. Scanning electron microscopy analysis of immobilized cellulase The microstructure of chitosan, immobilized cellulase without pretreatment and the immobilized cellulase pretreated by ultrasound were examined using scanning electron microscopy. Immobilized cellulase after ultrasonic pretreatment became looser than that without ultrasonic pretreatment. It suggests that appropriate ultrasonic pretreatment may increase the surface area of immobilized cellulase, and help enzyme to attack substrate easily during enzymatic hydrolysis, which resulted in a higher enzyme activity than that without ultrasonic pretreatment. However, higher ultrasound power for a long time may destroy the cross linking of immobilized cellulase, reducing the attachment probability of the
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free cellulase on the enzyme loading, and cellulase making it easy for the cellulase to fall off. Hence the usage repeatability was reduced, and may result in inactivation of the enzyme. The effect of ultrasonic treatment on enzyme can be attributed to the formation of localized hot spots upon collapse of bubbles, mechanical stresses or shear forces created by microstreaming and shock waves, and generation of free radicals through sonolysis of water. Of these it has been reported that the role of free radicals in denaturation and inactivation of enzymes is the most important mechanism (Jambrak et al., 2008; Kadkhodaee and Povey, 2008; Ma et al., 2008). Essentially any amino acid radical formed within a peptide chain could cross-link with an amino acid radical in another protein. Alternatively, some radicals may cross-link with amino acid molecules. The selectivity and rate constants for such reactions are not well understood (Van Tilbeurgh and Claeyssens, 1985). The authors think that the increase in catalytic activity was due to the cellulase molecular structure which was modestly modified by the ultrasound free radicals, making the enzymes more readily accessible for reaction and thus resulting in the increase of cellulase activity, but it does not mean that the more the modifier, the higher the catalytic activity, on the contrary, when the molecular structure is excessively modified than the modest, the obstacle of cellulase attacking the substrate is augmented and catalytic activity declined. Ma et al. think the increase of catalytic activity was probably due to the ability of ultrasound to break down molecular aggregates (Ma et al., 2008), the decrease of catalytic activity was probably due to excessive pressure, temperature or shear force generated by the ultrasonic treatment, and generation of free radicals through sonolysis of water. 4. Conclusion In this study, low intensity ultrasound was demonstrated to have a positive effect on free cellulase activity and immobilize cellulase activities. The molecular structure of free cellulase was changed showed by Fluorescence and CD spectra when ultrasound was at 15 W, 24 kHz for 10 min, and the enzyme activity was increased by 18.17% over the control. To the immobilize cellulase, its surface area was increased while the ultrasound was at 60 W, 24 kHz for 10 min, and its activity was increased by 24.67% over the control. Acknowledgements This research was supported by grants from the 863 Research Program of China (No. 2007AA10Z321), the Natural Science
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