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Investigation of enzymatic activity, stability and structure changes of pectinase treated in supercritical carbon dioxide
Journal of Cleaner Production 125 (2016) 331e340
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Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Investigation of enzymatic activity, stability and structure changes of pectinase treated in supercritical carbon dioxide Yi-Kang Peng, Long-Long Sun, Wei Shi, Jia-Jie Long* College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2016 Received in revised form 20 March 2016 Accepted 22 March 2016 Available online 7 April 2016
The activity and stability of enzymes in supercritical carbon dioxide fluid are the crucial points and basis for developing and applying green, environmentally friendly processes and/or reactions in this water free media in different industries, which has attracted increasing interesting recently. The objective of the present work is to investigate the activity and stability of pectinase in supercritical carbon dioxide media, as well as for its structure and conformation changes. The results show that the activity and stability of pectinase were significantly improved under appropriate conditions. Significant increases in activity and stability of treated pectinase could be available with pressure lower than 15.0 MPa, whereas, temperature tends to reduce enzymatic activity and stability. An excellent stability of pectinase with improved activity was observed with duration from 0.5 h to 4.0 h. Fourier transfer infrared spectra, ultraviolet spectra, fluorescence spectra and scanning electron microscopy analyses indicate that alterations in the secondary and tertiary structures, and morphology of treated pectinase, were occurred without conspicuous changes in its primary structure. An exposure of the residual side aromatic groups of tryptophan on polypeptide to outer surface of the enzyme in solution was also detected. Therefore, all the investigations further demonstrate that the supercritical treatment is an efficient method to improve the activity and stability of the enzyme due to conformational changes, and there is also a feasible to perform cleaner and sustainable production processes or reactions with pectinase in supercritical carbon dioxide media. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Supercritical carbon dioxide Pectinase Enzymatic activity Stability Structure Conformation
1. Introduction As a biological catalyst, enzyme has received a growing demand in the last decades as an alternative to most of conventional chemical catalysts in different industries, due to its high catalytic efficiency, specificity and selectivity, minimum side reactions, mild reaction conditions and readily available, as well as industrially economically feasible and more environmentally friendly, etc (Wohlgemuth, 2010; Ciftci and Saldana, 2012; Mukhopadhyay et al., 2013). Up to date, there are many available reports about various enzymes and their applications in various industries, such as aamylase, cellulase, b-galactosidase, protease, lipases, pectinase, Amyloglucosidasee pullanase, glucose oxidase, xylanase, which frequently employed in chemical engineering, organic synthesis, food engineering, textiles, environmental treatments, etc (Choi et al., 2015; Kalantzi et al., 2008; Chand et al., 2012). Among all
* Corresponding author. Tel.: þ86 0512 67164993; fax: þ86 0512 67246786. E-mail address: [email protected] (J.-J. Long). http://dx.doi.org/10.1016/j.jclepro.2016.03.058 0959-6526/© 2016 Elsevier Ltd. All rights reserved.
the employed enzymes, pectinase or pectinolytic enzyme refers to all the species that can catalyze the hydrolysis of pectin substances, and can be divided into protopectinase, pectin esterase and depolymerizing enzymes according to their reaction mechanisms, methods, action patterns, as well as substrates of hydrolysis (Kalantzi et al., 2008; Mukhopadhyay et al., 2013). Pectinase is widely utilized in food industry for extraction and clarification of fruit and vegetable juice, fermentation of red wine and tea, extraction of oil (Kashyap et al., 2001), as well as for scouring of cotton fabric and degumming of ramie fiber in textile industry, etc (Mukhopadhyay et al., 2013). However, all the traditional enzymatic processes for biocatalysis reactions in aqueous bath involve a large amount of water and energy consumptions, and readily cause some concerns of environment pollution by discharging wastewater with high biologic oxygen demand (BOD) and high chemical oxygen demand (COD) values, such as in textile industry. In fact, the conventional enzymatic processes employed for textile treatments usually still require a large bath ratio and long cultivation duration for a
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satisfactory efficiency of enzymatic reactions and commercial production, especially for the biodesizing, biodegumming and bioscouring of natural fiber based substrates by employing various enzymes. Thus, a lot of water resource, energy and effluents with high concentration of pollutants still can't be avoided in the conventional enzymatic processes, although they possess more advantages than traditionally harsh chemical processes. Therefore, the effective, useful and Ecofriendly methodologies or processes with non-aqueous media or in which water is not the predominant content for enzyme applications, are very popular in textile and/or other industries. Fortunately, supercritical carbon dioxide fluid, a green and hydrophobic medium, has received increasing attentions in recent years as an alternative to conventional water and organic solvents in industries, due to its numerous advantages such as nontoxic, environmentally friendly, high diffusion rates, less side reactions, readily downstream treatments, absence of gaseliquid mass transfer limitations (Gremos et al., 2012; Long et al., 2011; Long et al., 2015). Consequently, it is very meaningful and necessary to investigate the activity and stability of enzymes in supercritical carbon dioxide media in order to develop different biocatalytical processes by integrating or combining enzymes with non- or less content aqueous media of supercritical carbon dioxide fluid for cleaner and sustainable production in textile and other industries, with more advantages than conventional enzymatic processes in elimination and/or reduction of the consumption of water resource and the discharge of effluents inherent as well as decreasing total production costs, etc. Theoretically, the activity and stability of enzymes in supercritical carbon dioxide media are the crucial points and basis prior to a development and/or an application of any bio-catalytical methodologies or processes in this water free media. Up to date, part of research for some enzymes is available in literature. Melgosa et al. (2015) investigated the effect of supercritical carbon dioxide treatment on the activity and conformational, morphology of four commercial lipases, and revealed that all the employed enzymes remained activities in the supercritical medium and the activity of some enzymes could be improved, whereas some other enzymes reduced their activity by means of conformational changes and structural alterations according to the treatment conditions. Leitgeb et al. (2013) investigated the activity of cellulase and aamylase from Hortaea werneckii after cell treatment with supercritical carbon dioxide, and found that sufficient residual activity of both the examined enzymes were detected and could be still used as biocatalysts in this medium. Natalia et al. (2012) explored the stability, activity, and selectivity of benzaldehyde lyase in supercritical fluids including carbon dioxide, and revealed that supercritical fluids could be an alternative media to organic solvents. Senyay-Oncel and Yesil-Celiktas (2013) treated immobilized aamylase in supercritical carbon dioxide in order to enhance enzyme stability and activity, and found that the activity of immobilized aamylase after consecutive enzymatic reactions could be enhanced by the retreatment with supercritical carbon dioxide. In addition, an enhancement in activity and stability for free a-amylase was also achieved after being treated in sub- and supercritical carbon dioxide by Senyay-Oncel and Yesil-Celiktas (2011). Moreover, SenyayOncel et al. (2014) investigated the activity and stability of protease in sub- and supercritical carbon dioxide, and demonstrated that enhanced activity and stability were observed with the raise of pressure, and the potential mechanism was also explored. Andrade et al. (2008) assessed the influence of compressed CO2 and propane treatment on the specific activity of partially purified D-hydantoinase from adzuki bean (Vigna angularis), then found that good stability of this enzyme in the two solvents was observed although some activity losses were occurred for resolubilized enzyme extract with compressed CO2. Manera et al. (2011) explored the influences
of pressure, exposure time and depressurization rate on the bgalactosidase activity of permeabilized cells of Kluyveromyces marxianus CCT 7082 submitted to treatment with compressed carbon dioxide, propane and n-butane, and showed that the activities of this biocatalyst were always higher than those of the nontreated ones. Furthermore, Rezaei et al. (2007) reviewed effects of pressure and temperature on enzymatic reactions in supercritical fluids including carbon dioxide, and demonstrated that most of the enzymes were active in supercritical fluids, and their activity could be improved with temperature within limits. However, some negative effects from treatments with supercritical carbon dioxide on the activity and stability of some enzymes were also reported. Santos et al. (2016) investigated the activity of immobilized lipase from Candida antarctica (Lipozyme 435) and its performance on the esterification of oleic acid in supercritical carbon dioxide, and showed that the activity of Lipozyme 435 decreased with the increase of pressure, temperature, exposure time and the number of pressurization/depressurization cycles. Oliveira et al. (2006) also investigated the influence of temperature, pressure, exposure time and depressurization rate on the activity of an immobilized lipase from Yarrowia lipolytica in compressed carbon dioxide, propane and n-butane, and showed that significant activity losses were obtained in carbon dioxide in comparison with other organic solvents. Moreover, the effects of treatment parameters on enzyme activity depended on its nature and source, as well as its forms. However, Dhake et al. (2011) investigated the activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide, and revealed that the immobilization method by using the blended polymer of hydroxylpropyl methyl cellulose and polyvinyl alcohol could greatly overcome the drawback by enhancing the catalytic activity of R. oryzae lipase for synthesis in supercritical carbon dioxide. It is obvious that most of the reported research works about the activity and stability of enzymes in supercritical carbon dioxide are mainly concentrated on a-amylase, cellulase, b-galactosidase, protease, free and/or immobilized lipases, etc. Very few works have been reported about the activity and stability of pectinase in supercritical carbon dioxide; especially, it is very lack of investigations about the alterations of pectinase in primary, secondary and tertiary structures, as well as its conformational and morphology changes induced by the supercritical media, although it is very important as a crucial basis for developing or performing green and environmentally friendly processes, reactions by combination of the two ecological technologies in different industries. The objective of the present work is to investigate the activity and stability of pectinase in supercritical carbon dioxide media, and also to explore some information about its structural, conformational and morphology alterations during treatment. The effects of system pressure, temperature and treatment duration on the activity and stability of pectinase were evaluated. Moreover, Fourier transfer infrared spectra (FT-IR), ultraviolet (UV) spectra, fluorescence spectra and scanning electron microscopy (SEM) analyses were performed for investigating the structural, conformational and morphology information of treated pectinase. 2. Material and methods 2.1. Materials A pectinase (from aspergillus Niger) in an analytical pure grade was purchased from Beijing Solarbio Technology Co., Ltd. (Beijing of China), and was used without further purification. A pectin (Galacturonic acid > 74.0%, dried basis) from citrus peel was obtained from SigmaeAldrich Co., Ltd. (Shanghai of China), and used as a
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1.2
(a) 0.25 /g L 0.30 0.35 0.40 0.60 0.80 1.20 1.60 2.00
1.0
Absorbance (A)
substrate for enzymatic hydrolysis reaction. Glucose anhydrouse was purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai of China). Dinitrosalicylic acid (DNS; 3, 5-dinitro-2-hydroxybenzoic acid) in a chemical pure grade was supplied by Chinasun specialty products Co., Ltd. (Changsu of China), and employed as a reagent for measuring the content of reducing sugar. Other chemicals utilized in enzymatic activity determination, such as sodium hydroxide, citric acid monohydrate, potassium sodium tartrate tetrahydrate, etc., were commercially available products in an analytical pure grade. A pure carbon dioxide gas (99.6 vol.%) was employed for supercritical carbon dioxide treatment of pectinase samples.
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0.8 0.6
-1
0.4 0.2
2.2. Treatment of pectinase in supercritical carbon dioxide system
2.3. Evaluation of pectinase activity and stability The evaluation of pectinase activity was carried out on the basis of the determination of reducing sugars produced from pectin hydrolyzation. An aliquot of 1.0 mL pectinase sample solution at a concentration of 0.5 g L1 was added into substrate solution involving 10.0 g L1 pectin and 0.05 mol L1 citrate buffers at a pH value of 5.5. Then the enzymatic reaction was incubated at 50 C for 0.5 h, and terminated by boiling the mixed solution for 6.0 min. The produced reducing sugars were measured with the dinitrosalicylic acid (DNS) method declared by Miller (1959) employing galacturonic acid as a reference. Moreover, a construction of a calibration curve for determination of reducing sugars was carried out by employing standard addition method, in which glucose was used as a reference of reducing sugar with concentration ranging from 0.0 to 2.0 g L1 in a citrate buffer solution at a pH value of 5.5. After a complete reducing reaction between the glucose and the DNS indicator at boiling temperature for 5.0 min, the mixture solution was dilute with citrate buffer and its absorbance was monitored on an ultravioletevisible spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd, Beijing of China) at wavelength from 450.0 nm to 700.0 nm. Simultaneously, a control experiment was also carried out. Then, the calibration curve was constructed and regressed in a linear tendency with characteristic absorbance at maximum wavenumber against reducing sugar concentrations of glucose, as shown in Fig. 1(a, b) and Eq. (1):
C ¼ k DA þ b
(1)
where C refers to the concentration of reducing sugar of glucose; k is for the slope value of the calibration curve for activity determination; DA represents the difference of the absorbance at maximum wavenumber between the sample solution and the control; b is for the constant value from curve regression.
0.0 450
500
550
600
650
700
Wavelength (nm) 2.1
Concentration of glucose (g L-1)
The pectinase sample with a weight about 0.5 g were treated in the same batch system and the reactor as described elsewhere (Long et al., 2011; Long et al., 2015) at system temperature ranging from 30.0 C to 90.0 C, system pressure from 6.0 MPa to 23.0 MPa and treatment duration from 0.5 h to 4.0 h in supercritical carbon dioxide fluid, respectively. The solid sample of pectinase was packed with a metal mesh, and set at the up section of the reactor as described in detail elsewhere (Long et al., 2015). Then pure CO2 gas was pumped into the system after being preheated. As the system pressure and temperature were attained a request condition, the treatment process was begun by circulating the supercritical fluid with a syringe pump. After request treatment duration, the enzyme was took out and stored in refrigerator for further analysis.
(b)
1.8 1.5 1.2 0.9 0.6 0.3 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Absorbance (A) Fig. 1. The visible spectra (a) and calibration curve (b) for pectinase determination with DNS method from the standard solution with different concentration of glucose.
Accordingly, the activity of pectinase was described by Unit (U) against per gram of pectinase, and one unit of its activity was defined as the amount of pectinase that catalyze the degradation of pectin to release 1.0 mg galacturonic acid within the duration of per hour at the incubation conditions above. Consequently, the activity of pectinase (AP) could be evaluated according to the following Eq. (2):
AP ¼
ðk DA þ bÞ V ð194=180Þ m t
(2)
where AP refers to the activity of pectinase with a unit of U g1; k is for the slope value of the calibration curve for activity determination; DA represents the difference of the absorbance between the sample solution and the control; V is for the volume of the enzymatic reaction solution after a centrifugation, 10.0 mL was employed in this work; 194/180 refer to the equivalent conversion of the reducing sugar of glucose to galacturonic acid in terms of their individually relative molecular mass; m is for the diluted times of the reaction solution; t is for the enzymatic reaction time with a unit of hour in this work; b is for the constant value from curve regression. Furthermore, the stability of pectinase subjected to various conditions in supercritical carbon dioxide media, was also characterized by the residual activity of pectinase (APRe.) in comparison
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with its enzymatic activity before and after the treatment, and assessed according to Eq. (3):
APRe: ¼
AP1 100% AP0
(3)
where APRe. refers to the residual activity of pectinase; AP0, AP1 are for the activity of pectinase before and after a treatment in supercritical carbon dioxide media, respectively. 2.4. FT-IR analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions, were also analyzed on an FT-IR instrument (Nicolet 5700, Thermo electron Co., Ltd., US) with traditional KBr pellet sampling method under an identical measurement condition with a same quantity at 0.58 mg of the samples. The transmittance of the infrared in individual powder sample of pectinase was recorded from 400.0 cm1 to 3800.0 cm1 at a resolution of 2.0 cm1 for infrared spectra. 2.5. Ultraviolet spectroscopy analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were dissolved, respectively, in citrate buffer solution at a pH value of 5.5 with a concentration of 2.50 g L1. Then the prepared solutions were measured by employing an ultravioletevisible spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd, Beijing of China) at wavelength ranging from 250.0 nm to 350.0 nm at room temperature for scanning three times, in order to explore some information about the tertiary structure variations of pectinase samples after being subjected to supercritical carbon dioxide media. 2.6. Fluorescence spectroscopy analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were dissolved, respectively, in citrate buffer solution at a pH value of 5.5 with a concentration of 2.50 g L1. Then the prepared solutions were measured on a spectrofluorometer (FM4P-TCSPC, Horiba Jobin Yvon Co., Ltd, USA) with an excitation wavelength at 280.0 nm and an emission wavelength ranging from 300.0 nm to 540.0 nm at room temperature for scanning three times, in order to further explore some information about the tertiary structure variations of pectinase samples after being subjected to supercritical carbon dioxide media. 2.7. Investigation of surface morphology for pectinase samples The surface morphological investigations of pectinase samples before and after a supercritical treatment were carried out by employing a scanning electronic microscopy (SEM) (TM3030, Hitachi, Japan) with an acceleration voltage of 15.0 kV at 200 and 5000 magnifications, respectively. Before investigation, samples were sputtered with gold on an ion sputter (E-1045 Hitachi, Japan) at 10.0 mA for 60.0 s. 3. Results and discussion 3.1. Calibration curve for determination of the activity of pectinase samples By employing the standard addition method and utilizing DNS as an indicator, a calibration curve for determination of reducing
sugars was constructed by employing glucose as a reference in a citrate buffer solution at a pH value of 5.5. The obtained results are depicted in Fig. 1(a, b). Fig. 1(a) shows that good absorption curves with a main and single characteristic peak at a maximum wavenumber (lmax) of 490.0 nm for the reduced colorful product of DNS were obtained in a citrate buffer solution with a pH value of 5.5, by accompanying with a proportional increase of the peak height as glucose concentration ranging from 0.25 g L1 to 2.00 g L1. Furthermore, Fig. 1(b) indicates that a good linear relationship (r2 ¼ 0.9995) was obtained between the glucose concentration (C) and the peak absorbance (DA), as expressed in Eq. (1). The linear dynamic range for reducing sugar determination in the predetermined conditions was 0.25e2.00 g L1 with a satisfactory sensitivity (the slop of k is 1.679 g L1. DA1 and the intercept b is 0.142 g L1). The relative standard deviation was 2.65% (n ¼ 2) at 0.25 g L1 of glucose in the employed determination conditions. These notably indicate that the proposed standard addition method and the obtained calibration curve with the DNS indicator are feasible and adequate to quantitatively determine reducing sugars in the employed system and conditions, as well as proved by our previous experiments. In principle, the reducing sugars which are added or produced from the hydrolyzation of pectins by pectinase, can react with the indicator of DNS in an appropriate condition to reduce the DNS to 3amino-5-nitrosalicylic acid, which is a stable brownish red color and proportionally increased with the concentration of reducing sugars in a linear dynamic range in a favorable buffer solution. Consequently, according to this calibration curve and a further equivalent conversion of the reducing sugars of glucoses to galacturonic acids, the activities of all the pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were evaluated, as well as for the enzymatic stability in the following experiments. 3.2. Effect of system pressure on the supercritical treatment of pectinase An effect of system pressure on the activity and stability of pectinase in supercritical carbon dioxide media was investigated at a system temperature of 40.0 C for treatment duration of 1.0 h as system pressure ranging from 6.0 MPa to 23.0 MPa. The obtained results are shown in Fig. 2. Fig. 2 shows that a notable enhancement in the enzymatic activity and residual activity of pectinase was obtained with system pressure increased from 6.0 MPa to 15.0 MPa after a treatment in supercritical carbon dioxide media, and then a sharply decrease was also observed as system pressure further increased from 15.0 MPa to 23.0 MPa. Moreover, Fig. 2 further declare that the residual activities higher than 100% were achieved with the system pressure from 6.0 MPa to 20.0 MPa with a maximum value of 135% at 15.0 MPa in comparison with the one before treatment in supercritical carbon dioxide. These clearly indicate that the pectinase showed highly catalytical activity and stability in supercritical carbon dioxide media, and the supercritical treatment is benefit to improve its enzymatic activity during a favorable pressure range over 6.0 MPae20.0 MPa. Theoretically, supercritical carbon dioxide is a hydrophobic fluid medium and its density increases with system pressure at a fixed system temperature, which leads to enhanced interactions, or influences on the solutes. Therefore, an appropriate higher pressure probably shows more benefits to modify and/or rearrange the conformations of the protein structures such as the secondary and tertiary structures, via the interactions among the polypeptides, side chains and groups, and the solvent molecules, as well as by extracting some bound water and impurities, etc. It is important
Y.-K. Peng et al. / Journal of Cleaner Production 125 (2016) 331e340
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60
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6
9
12
15
18
21
24
Residual activity (APRe., %)
Enzymatic activity (×104, U g-1)
2.8
335
0
Pressure (MPa) Fig. 2. Effect of system pressure on enzymatic activity (B) and residual activity (D) of pectinase treated at a system temperature of 40.0 C for 1.0 h in supercritical carbon dioxide fluid by employing DNS determination method.
Fig. 3. Effect of system temperature on enzymatic activity (B) and residual activity (D) of pectinase treated at a system pressure of 15.0 MPa for 1.0 h in supercritical carbon dioxide fluid by employing DNS determination method.
that these interactions present a positive effect on the enzymatic activity and stability. However, the decrease tendencies of the enzymatic activity and stability as the system pressure higher than 15.0 MPa are probably due to some irreversible and/or destructive changes in the secondary and tertiary structures at an overhigh pressure of supercritical fluid. Accordingly, by consideration of the effects of system pressures on the activity and stability of pectinase, a system pressure of 15.0 MPa was recommended in further experiments for the supercritical treatment of pectinase. Moreover, an overhigh system pressure such as higher than 23.0 MPa should be avoided during a design and control of the enzymatic processes and/or reactions in supercritical carbon dioxide media, in order to maintain an adequate activity and enough lifetime of the pectinase.
and stability of pectinase. Consequently, a relative lower treatment temperature is favorable for pectinase applications in supercritical carbon dioxide media, and a temperature higher than 80.0 C should be avoided.
3.3. Effect of temperature on the supercritical treatment of pectinase An effect of system temperature on the enzymatic activity and residual activity of pectinase in supercritical carbon dioxide was explored at a system pressure of 15.0 MPa for treatment duration of 1.0 h with temperature ranging from 30.0 C to 90.0 C. The results are depicted in Fig. 3. Fig. 3 shows that evident enhancements in the enzymatic activity and residual activity of pectinase were achieved after being treated in supercritical carbon dioxide media as system temperature over 30.0 Ce80.0 C in comparison with the activity of untreated free pectinase at 1.88 104 U g1, although a gradual decrease tendency was also observed by accompanying with the whole tested temperatures. Moreover, serious reduces were also obtained in enzymatic activity and residual activity with lower values than the untreated samples as the system temperature higher than 80.0 C. These results demonstrate that there is feasible for pectinase to remain its enhanced catalytic activity and stability at a system temperature lower than 80.0 C for substrates in comparison with its untreated one. However, it is also obvious that the system temperature imposed a negative effect on the enzymatic activity and stability, especially for the system temperature higher than 80.0 C. Theoretically, system temperature usually presents notable affects on the polypeptide chain arrangements and irreversible variations in conformation of protein in secondary and tertiary structures, and readily makes it denaturation, restriction and even deactivation in catalytic activities. Therefore, the increase of the system temperature in supercritical carbon dioxide media reasonably resulted in the decrease of the enzymatic activity
3.4. Effect of treatment duration on the supercritical treatment of pectinase An effect of treatment duration on the enzymatic activity and residual activity of pectinase in supercritical carbon dioxide was investigated at a system pressure of 15.0 MPa and a temperature of 40.0 C with treatment duration from 0.5 h to 4.0 h. The results are depicted in Fig. 4. Fig. 4 shows that an evident enhancement in the enzymatic activity and residual activity was achieved, respectively, as the pectinase sample treated in supercritical carbon dioxide media only for 0.5 h, and then a platform was observed in the both curves of enzymatic activity and residual activity with prolonged treatment duration up to 4.0 h. In addition, for all the supercritically treated samples, the enzymatic activities were notably improved in comparison with the untreated one, and the residual activities were all higher than 100% for all the treatment durations. These clearly disclose a fact that the treatment of pectinase in supercritical carbon dioxide media is helpful and highly efficient to improve its enzymatic activity and stability by employing an appropriate
Fig. 4. Effect of treatment duration on enzymatic activity (B) and residual activity (D) of pectinase treated at a system pressure of 15.0 MPa and a temperature of 40 C in supercritical carbon dioxide fluid by employing DNS determination method.
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system temperature and pressure. Probably, the treatment of pectinase in the hydrophobic media at a favorable condition is benefit to present positive modifications in the secondary and tertiary structures of the protein via the rearrangements, interactions of its polypeptides, side chains and the solvent molecules. Therefore, it makes the pectinase become more suitable and efficient to combine with the substrate at its active centers, as well as to stimulate a higher catalytic performance of the enzyme than the untreated one. Consequently, treatment duration over 0.5 he4.0 h, and even longer durations are applicable for catalytic reactions and/ or processes with pectinase in supercritical carbon dioxide media by employing appropriate system pressures and temperatures. 3.5. FT-IR analysis of pectinase treated by supercritical carbon dioxide According to the above investigations, the treatment of pectinase with supercritical carbon dioxide media brought a significant influence on the enzymatic activity and stability. In order to further explore the influences of the supercritical treatment on the primary structure and conformation of the enzyme, the treated pectinase samples with highest and lowest enzymatic activity in supercritical carbon dioxide media were investigated by employing an FT-IR instrument. The results are shown in Fig. 5. Fig. 5 indicates that some significant changes in the infrared spectra of treated pectinase in supercritical carbon dioxide media were observed in comparison with the control sample, especially for the absorption intensities of the main and characteristic peaks. The infrared spectrum of pectinase sample with highest enzymatic activity and stability after a treatment in supercritical carbon dioxide media at conditions of 40.0 C 15.0 MPa 1.0 h shows the highest absorption intensities for all the characteristic peaks among all the samples. These indicate a structural change occurred during the treatment, which was responsible for the enhanced enzymatic activity and stability. Moreover, the characteristically strong peak at 3394.7 cm1 was attributed to the combination of the stretching vibrations of NeH and OeH (nNeH, nOeH), which was shifted to the lower location from 3410.7 cm1 for the control sample, accompanying with more evident peak splits at 3368.4 cm1 and
3293.1 cm1. These obviously demonstrate that more and stronger hydrogen bonds were formed between eOH, eNHe and eNH2 groups among inter- and/or intra-macrochains of the enzyme after the treatment in supercritical carbon dioxide fluid under the favorable conditions. The absorption bands at 2930.6 cm1 was assigned to the symmetrical stretching vibration of CeH in eCH3 and/or eCH2e groups, which was a slight shift from 2928.4 cm1 for the control sample, indicating a change occurred in the chemical environment of the aliphatic groups or chains. Furthermore, the characteristically strong and increased peak at 1647.1 cm1 was due to the stretching vibration of C]O from amide І (nC]O), which was closely relative and more sensitive than other amide bands to the change of the secondary structure of the main chains, such as the ahelix and b-sheet conformations in the treated pectinase (SenyayOncel and Yesil-Celiktas, 2013; Senyay-Oncel et al., 2014). It is obvious that the increased absorption intensity of the amide І indicates a significant change and/or a conformation transformation occurred in the secondary structure of the main chains, which is probably responsible for improving the enzymatic activity and stability. The medium intensity peaks at 1542.4 cm1 and 1451.9 cm1 were assigned to the bending vibration of NeH (dNeH) from amide ІІ and the stretching vibration of CeN (nCeN) from amide ІІІ, respectively (Dhake et al., 2013). In addition, the absorption band at 1405.7 cm1 was assigned to the bending vibration within plane of NeH (dNeH), and CeC (nCeC), CeN (nCeN) stretching vibrations (Dhake et al., 2013). Moreover, the absorption bands at 1154.9 cm1, 1079.9 cm1 and 1021.8 cm1 were due to the stretching vibrations of CeO (nCeO) and NeO (nNeO), etc. The weak absorption bands at 576.5 cm1 is attributed to CeS stretching vibrations of sulfides and disulfides (Senyay-Oncel and Yesil-Celiktas, 2013). However, the infrared spectrum of treated pectinase sample with lowest activity and stability at higher temperature condition of 90.0 C 15.0 MPa 1.0 h, exhibits notably reduced absorption intensities for all the corresponding peaks in comparison with the sample possessing highest enzymatic activity and stability, although their peak intensity are higher than those of the control sample. Moreover, the predominant peak for the combination of the stretching vibrations of NeH and OeH (nNeH, nOeH) was also
100
Transmittance (%)
95 90
576.5
85
1
80
1451.9 1542.4
2930.6
1405.7 1154.9
3
1079.9
1647.1
75
2
1021.8
3293.1 3368.4 3394.7
70 65 60 3800
3600
3400
3200
3000
1800
1600
1400
1200
1000
800
600
400
ν (cm-1) Fig. 5. FT-IR spectra of pectinase samples for (1) before treatment, and treated at (2) 40.0 C 15.0 MPa 1.0 h, (3) 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide fluid.
Y.-K. Peng et al. / Journal of Cleaner Production 125 (2016) 331e340
8.0 Fluorescence intensity (cps.E6)
shifted to a higher wavenumber at 3410.1 cm1 compared with the highest activity sample. These indicate that a change and/or a weakening of the hydrogen bonds or interactions were occurred among inter- and/or intra-macrochains of the enzyme. Moreover, the symmetrical stretching vibration of CeH in eCH3 and/or eCH2e groups was slightly shifted to a lower wavenumber at 2929.5 cm1, as well as for the amide ІІ band. Probably, it is due to irreversible variations occurred in secondary structure of the main chains of pectinase, such as unfolding actions, rearrangements of the chains, which was induced by the increased and overhigh system temperature for the enzyme. In addition, there were no new characteristic peaks observed for all the treated enzyme samples in the FT-IR spectra, which indicates that no conspicuous change in its primary structure was occurred during the supercritical treatment.
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3
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2
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5.0 4.0 3.0 2.0 1.0 0.0
300 330 360 390 420 450 480 510 540 Wavelength (nm)
3.6. Ultraviolet and fluorescence spectroscopy analysis of pectinase treated by supercritical carbon dioxide In order to further explore the effect of supercritical treatment on the tertiary structure of pectinase samples, the treated pectinase samples with highest and lowest enzymatic activity were also investigated in citrate buffer solution at a pH value of 5.5 on an Ultravioletevisible spectrophotometer and a fluorescence spectrometer, respectively. The results are shown in Figs. 6 and 7. The ultraviolet spectra in Fig. 6 show that the absorbance of the supercritically treated pectinase sample with highest enzymatic activity and stability in citrate buffer solution was notably enhanced in comparison with the control one at a characteristic absorption band from about 250.0 nm to 350.0 nm involving a maximum wavelength at about 280.0 nm, which was assigned to the ultraviolet absorptions of the residual or side groups from some aromatic amino acids such as tryptophan, tyrosine and phenylalanine on the protein chains. However, the absorbance of treated enzyme sample with lowest activity and stability dissolved in a same buffer solution was decreased at the characteristic absorption band compared with that of the control one. It is obvious that the treatment of pectinase sample in supercritical carbon dioxide media presented a notable influence on the conformation of the enzyme structure even after being dissolved in aqueous solution. Moreover, it seems like that the ultraviolet absorption intensity of these aromatic amino acids is sensitive and characteristically relative to enzymatic activity and stability of the treated pectinase
Absorbance (A)
0.9
2
0.8
1
0.7
3
0.6 0.5 0.4 0.3 0.2 0.1
255
270
285
300
315
330
345
Wavelength (nm) Fig. 6. Ultraviolet spectra of pectinase samples for (1) before treatment, and treated at (2) 40.0 C 15.0 MPa 1.0 h, (3) 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide fluid.
Fig. 7. Fluorescence spectra of pectinase samples for (1) before treatment, and treated at (2) 40.0 C 15.0 MPa 1.0 h, (3) 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide fluid.
samples, in which higher absorption intensity indicates a higher enzymatic activity and stability of the pectinase. Theoretically, the primary structure of pectinase is composed with various amino acids linked by amide bonds to form polypeptide chains, including the aromatic amino acids of tryptophan, tyrosine and phenylalanine, etc. An ultraviolet absorption usually occurs in an aqueous solution at 280.0 nm, 275.0 nm and 257.0 nm for the aromatic amino acids of tryptophan, tyrosine and phenylalanine, respectively. Moreover, their absorptions are frequently and notably influenced by medium properties, pH, temperature, rearrangements of the enzyme chains, structure conformations and various interactions among the groups, etc. Therefore, it is evident that the supercritical treatment of pectinase at a favorable condition of 40.0 C 15.0 MPa 1.0 h in the hydrophobic media is helpful to release and expose these residual groups of aromatic acids onto the outer surfaces of enzymatic structure, especially for tryptophan and tyrosine, which is probably relative to the active sites in the enzyme structure and responsible for the enhanced enzymatic activity and stability. However, a worse treatment condition in supercritical carbon dioxide fluid with an overhigh temperature of 90.0 C tends to overshadow these residual groups via chain arrangements and conformational changes, as well as group interactions, etc., which resulted in the reduced enzymatic activity and stability of the pectinase. Fig. 7 is for the recorded fluorescence spectra of pectinase samples in citrate buffer solutions with characteristic fluorescence emission peaks at a range from 280.0 nm to 540.0 nm. It demonstrates that a characteristic emission peak involving a maximum wavelength at 351.0 nm with an evident shift to higher wavelength, namely a red shift, was observed for the treated pectinase sample with highest enzymatic activity and stability, accompanying with a decrease in the peak intensity in comparison with the control one. However, an almost non-shift and a notable increase in the intensity of the characteristic fluorescence emission peak were also obtained for the treated pectinase sample with lowest activity and stability compared with the control one. These clearly indicate that the treatment of pectinase at different conditions in supercritical carbon dioxide media could bring various and significant effects on the tertiary structure or conformation of the polypeptide and side chains, as well as the residual side groups in the enzyme even after being dissolved in aqueous solution, which were probably responsible for the changed enzymatic activity and stability.
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Fig. 8. SEM images of pectinase samples for (a) before treatment, (b) supercritically treated at 40.0 C 15.0 MPa 1.0 h, (c) supercritically treated at 90.0 C 15.0 MPa 1.0 h, with a magnification of 200 and 4000, respectively.
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In principle, the side residual groups from the aromatic amino acids of tryptophan, tyrosine and phenylalanine in the primary structure of pectinase, which involve p-electrons in their structures and are readily subjected to ultraviolet excitation at 280.0 nm, are able to emit intrinsic fluorescence at a longer wavelength of 348.0 nm, 303.0 nm and 282.0 nm, respectively (Yin et al., 2010; Bobone et al., 2014). Usually, the most strong emission intensity of fluorescence is from tryptophan residual group and the lowest is from phenylalanine, and the emission of fluorescence from the tyrosine residual group is frequently quenched readily due to an occurrence of energy transformation to tryptophan residual group in a simultaneous presence of the both in protein. Moreover, the fluorescence emissions from these aromatic side groups are sensitive to their location environment and are usually employed as an indicator to explore the changes of the conformation of proteins. Theoretically, a polar environment tends to reduce the excitation state energy of these aromatic side groups, and to result in a red shift of their fluorescence emission spectra (Yin et al., 2010; Bobone et al., 2014). Therefore, it is obvious that the evident shift of the characteristic emission peak to 351.0 nm, which was from the residual side aromatic group of tryptophan, clearly indicates that an exposure of more side groups of tryptophan into a higher polar environment of aqueous solution was occurred during dissolving the pectinase sample with highest activity and stability which was treated at a condition of 40.0 C 15.0 MPa 1.0 h. Probably, this side group was relative to the active sites of the pectinase, and so it was more readily to combine with the substrate, resulting in the most enhanced enzymatic activity and stability. The decrease of the intensity was also due to an emission quenching from the exposure of the side group to the polar solution. However, as regard to the treated pectinase with lowest activity and stability at a condition of 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide media, the non-shift of the characteristic peak and the increased fluorescence intensity demonstrate that an undesirable and irreversible conformational change, and a deeper burying of the residual side aromatic group of tryptophan into the pectinase structure were occurred during the supercritical treatment by employing the higher system temperature. Moreover, all the investigations from fluorescence spectroscopy analysis are coincident with the analysis from ultraviolet spectroscopy in Fig. 6, as well as with the FT-IR analysis in Fig. 5. These further confirm the role of supercritical treatment for improving the enzymatic activity and stability at an appropriate condition, which is due to the conformational changes in secondary and tertiary structures of the treated pectinase in supercritical carbon dioxide media. Therefore, the supercritical treatment could be employed as an efficient and Ecofriendly method to improve the activity and stability of the pectinase via a conformational change at an appropriate condition. 3.7. Surface morphology analysis of pectinase treated by supercritical carbon dioxide An investigation of the surface morphology of pectinase before and after a supercritical treatment was carried out by employing a scanning electronic microscopy (TM3030, Hitachi, Japan) in order to further explore the influences of the supercritical fluid on the pectinase. The results are depicted in Fig. 8(aec). Fig. 8a shows that many big aggregates of the pectinase before treatment were observed at a low magnification of 200, and a relative smooth and intact surface was also observed for these enzyme aggregates at a high magnification of 5000. Whereas, as shown in Fig. 8b, some broken and more particles in small sizes for the pectinase were obtained at the low magnification of 200 after a treatment in supercritical carbon dioxide media under a condition
339
of 40.0 C 15.0 MPa 1.0 h. Moreover, an evidently fractured surface of the aggregates was observed, and even some fragments and wholes were also visible on the enzyme aggregates with the magnification of 5000 in Fig. 8b. In addition, as depicted in Fig. 8c, more broken and small particles were obtained at the low magnification of 200 for the enzyme sample after a treatment at a higher temperature condition of 90.0 C 15.0 MPa 1.0 h, and also a most seriously fractured surface with many fragments on the aggregates was clearly visible under a higher magnification of 5000. All these above indicate that different etching effects on the surfaces of the pectinase aggregates could be occurred during the treatment in supercritical carbon dioxide media, and a relative low system temperature is more favorable for the enzyme to remain its nature morphology and simultaneously improve its activity and stability. However, an overhigh system temperature could bring a serious deterioration of the enzyme morphology, which is also probably responsible for the reduced enzymatic activity and stability. Therefore, an appropriate treatment condition, especially for the system temperature, is crucial to maintain the natural surface morphology of pectinase and simultaneously improve its enzymatic activity and stability. 4. Conclusions The activity and stability of pectinase were investigated after a treatment in supercritical carbon dioxide. The results reveal that the activity and stability of pectinase could be significantly improved under appropriate conditions. Significant increases in activity and stability of treated pectinase could be available with pressure lower than 15.0 MPa, whereas, temperature tends to reduce enzymatic activity and stability. An excellent stability of pectinase with improved activity was observed with the duration of 4.0 h or longer. Fourier transfer infrared spectra, ultraviolet spectra, fluorescence spectra and scanning electron microscopy analyses demonstrate that some alterations in the secondary and tertiary structures, and surface morphology of treated pectinase were occurred. Therefore, the investigations further indicate that the supercritical treatment could be employed as an efficient and Ecofriendly method to improve the activity and stability of the enzyme, and there is also a feasible to combine the two ecological technologies of the pectinase and supercritical carbon dioxide media for green and environmentally friendly processes, reactions in different industries. Acknowledgment The authors acknowledge gratefully the financial supports from the science and technology support project of Jiangsu province of China (Grant No. BE2013051), and the Priority Academic Program Development of Jiangsu Higher Education Institutions [PAPD] of China. References Andrade, J.M., Oestreicher, E.G., Oliveira, J.V., Oliveira, D., Antunes, O.A.C., Dariva, C., 2008. Effect of treatment with compressed CO2 and propane on d-hydantoinase activity. J. Supercrit. Fluids 46, 342e350. Bobone, S., Van De Weert, M., Stella, L., 2014. A reassessment of synchronous fluorescence in the separation of Trp and Tyr contributions in protein emission and in the determination of conformational changes. J. Mol. Struct. 1077, 68e76. Chand, N., Nateri, A.S., Sajedi, R.H., Mahdavi, A., Rassa, M., 2012. Enzymatic desizing of cotton fabric using a Ca2þ-independent a-amylase with acidic pH profile. J. Mol. Catal. B Enzym. 83, 46e50. Choi, J.-M., Han, S.S., Kim, H.S., 2015. Industrial applications of enzyme biocatalysis: current status and future. Biotechnol. Adv. 33, 1443e1454. Ciftci, D., Saldana, M.D.A., 2012. Enzymatic synthesis of phenolic lipids using flaxseed oil and ferulic acid in supercritical carbon dioxide media. J. Supercrit. Fluids 72, 255e262.
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Dhake, K.P., Deshmukh, K.M., Patil, Y.P., Singhal, R.S., Bhanage, B.M., 2011. Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide. J. Biotechnol. 156, 46e51. Dhake, K.P., Karoyo, A.H., Mohamed, M.H., Wilson, L.D., Bhanage, B.M., 2013. Enzymatic activity studies of Pseudomonas cepacia lipase adsorbed onto copolymer supports containing b-cyclodextrin. J. Mol. Catal. B Enzym. 87, 105e112. Gremos, S., Kekos, D., Kolisis, F., 2012. Supercritical carbon dioxide biocatalysis as a novel and green methodology for the enzymatic acylation of fibrous cellulose in one step. Bioresour. Technol. 115, 96e101. Kalantzi, S., Mamma, D., Christakopoulos, P., Kekos, D., 2008. Effect of pectate lyase bioscouring on physical, chemical and low-stress mechanical properties of cotton fabrics. Bioresour. Technol. 99, 8185e8192. Kashyap, D.R., Vohra, P.K., Chopra, S., Tewari, R., 2001. Applications of pectinases in the commercial sector: a review. Bioresour. Technol. 77, 215e227. Leitgeb, M., Colnik, M., Primozic, M., Zalar, P., Cimerman, N.G., Knez, Z., 2013. Activity of cellulase and a-amylase from Hortaea werneckii after cell treatment with supercritical carbon dioxide. J. Supercrit. Fluids 78, 143e148. Long, J.-J., Ma, Y.-Q., Zhao, J.-P., 2011. Investigations on the level dyeing of fabrics in supercritical carbon dioxide. J. Supercrit. Fluids 57, 80e86. Long, J.-J., Cu, Ch.-L., Zhang, Y.-Q., Yuan, G.-H., 2015. Clean fixation of dye on cotton in supercritical carbon dioxide with a heterogeneous and phase transfer catalytic reaction. Dyes Pigment. 115, 88e95. Manera, A.P., Kuhn, G., Polloni, A., Marangoni, M., Zabot, G., Kalil, S.J., Oliveira, D., Treichel, H., Vladimir, O.J., Mazutti, M.A., Maugeri, F., 2011. Effect of compressed fluids treatment on the activity, stability and enzymatic reaction performance of b-galactosidase. Food Chem. 125, 1235e1240. Melgosa, R., Sanz, M.T., Solaesa, A.G., Bucio, S.L., Beltran, S., 2015. Enzymatic activity and conformational and morphological studies of four commercial lipases treated with supercritical carbon dioxide. J. Supercrit. Fluids 97, 51e62.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426e428. Mukhopadhyay, A., Dutta, N., Chattopadhyay, D., Chakrabarti, K., 2013. Degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented pectate lyase. Bioresour. Technol. 137, 202e208. Natalia, D., Greiner, L., Leitner, W., Ansorge-Schumacher, M.B., 2012. Stability, activity, and selectivity of benzaldehyde lyase in supercritical fluids. J. Supercrit. Fluids 62, 173e177. Oliveira, D., Feihrmann, A.C., Dariva, C., Cunha, A.G., Bevilaqua, J.V., Destain, J., Oliveira, J.V., Freire, D.M.G., 2006. Influence of compressed fluids treatment on the activity of Yarrowia lipolytica lipase. J. Mol. Catal. B Enzym. 39, 117e123. Rezaei, K., Temelli, F., Jenab, E., 2007. Effects of pressure and temperature on enzymatic reactions in supercritical fluids. Biotechnol. Adv. 25, 272e280. Santos, P., Rezende, C.A., Martinez, J., 2016. Activity of immobilized lipase from Candida antarctica (Lipozyme 435) and its performance on the esterification of oleic acid in supercritical carbon dioxide. J. Supercrit. Fluids 107, 170e178. Senyay-Oncel, D., Yesil-Celiktas, O., 2011. Activity and stability enhancement of aamylase treated with sub- and supercritical carbon dioxide. J. Biosci. Bioeng. 112, 435e440. Senyay-Oncel, D., Yesil-Celiktas, O., 2013. Treatment of immobilized a-amylase under supercritical CO2 conditions: can activity be enhanced after consecutive enzymatic reactions? J. Mol. Catal. B Enzym. 91, 72e76. Senyay-Oncel, D., Kazan, A., Yesil-Celiktas, O., 2014. Processing of protease under sub- and supercritical conditions for activity and stability enhancement. Biochem. Eng. J. 92, 83e89. Wohlgemuth, R., 2010. Biocatalysis- key to sustainable industrial chemistry. Curr. Opin. Biotechnol. 21, 713e724. Yin, Y.X., Xiang, B.Q., Tong, L., 2010. The application studying of fluorescence spectroscopy on protein. Exp. Technol. Manag. 27, 33e40.
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Journal of Cleaner Production journal homepage: www.elsevier.com/locate/jclepro
Investigation of enzymatic activity, stability and structure changes of pectinase treated in supercritical carbon dioxide Yi-Kang Peng, Long-Long Sun, Wei Shi, Jia-Jie Long* College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2016 Received in revised form 20 March 2016 Accepted 22 March 2016 Available online 7 April 2016
The activity and stability of enzymes in supercritical carbon dioxide fluid are the crucial points and basis for developing and applying green, environmentally friendly processes and/or reactions in this water free media in different industries, which has attracted increasing interesting recently. The objective of the present work is to investigate the activity and stability of pectinase in supercritical carbon dioxide media, as well as for its structure and conformation changes. The results show that the activity and stability of pectinase were significantly improved under appropriate conditions. Significant increases in activity and stability of treated pectinase could be available with pressure lower than 15.0 MPa, whereas, temperature tends to reduce enzymatic activity and stability. An excellent stability of pectinase with improved activity was observed with duration from 0.5 h to 4.0 h. Fourier transfer infrared spectra, ultraviolet spectra, fluorescence spectra and scanning electron microscopy analyses indicate that alterations in the secondary and tertiary structures, and morphology of treated pectinase, were occurred without conspicuous changes in its primary structure. An exposure of the residual side aromatic groups of tryptophan on polypeptide to outer surface of the enzyme in solution was also detected. Therefore, all the investigations further demonstrate that the supercritical treatment is an efficient method to improve the activity and stability of the enzyme due to conformational changes, and there is also a feasible to perform cleaner and sustainable production processes or reactions with pectinase in supercritical carbon dioxide media. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Supercritical carbon dioxide Pectinase Enzymatic activity Stability Structure Conformation
1. Introduction As a biological catalyst, enzyme has received a growing demand in the last decades as an alternative to most of conventional chemical catalysts in different industries, due to its high catalytic efficiency, specificity and selectivity, minimum side reactions, mild reaction conditions and readily available, as well as industrially economically feasible and more environmentally friendly, etc (Wohlgemuth, 2010; Ciftci and Saldana, 2012; Mukhopadhyay et al., 2013). Up to date, there are many available reports about various enzymes and their applications in various industries, such as aamylase, cellulase, b-galactosidase, protease, lipases, pectinase, Amyloglucosidasee pullanase, glucose oxidase, xylanase, which frequently employed in chemical engineering, organic synthesis, food engineering, textiles, environmental treatments, etc (Choi et al., 2015; Kalantzi et al., 2008; Chand et al., 2012). Among all
* Corresponding author. Tel.: þ86 0512 67164993; fax: þ86 0512 67246786. E-mail address: [email protected] (J.-J. Long). http://dx.doi.org/10.1016/j.jclepro.2016.03.058 0959-6526/© 2016 Elsevier Ltd. All rights reserved.
the employed enzymes, pectinase or pectinolytic enzyme refers to all the species that can catalyze the hydrolysis of pectin substances, and can be divided into protopectinase, pectin esterase and depolymerizing enzymes according to their reaction mechanisms, methods, action patterns, as well as substrates of hydrolysis (Kalantzi et al., 2008; Mukhopadhyay et al., 2013). Pectinase is widely utilized in food industry for extraction and clarification of fruit and vegetable juice, fermentation of red wine and tea, extraction of oil (Kashyap et al., 2001), as well as for scouring of cotton fabric and degumming of ramie fiber in textile industry, etc (Mukhopadhyay et al., 2013). However, all the traditional enzymatic processes for biocatalysis reactions in aqueous bath involve a large amount of water and energy consumptions, and readily cause some concerns of environment pollution by discharging wastewater with high biologic oxygen demand (BOD) and high chemical oxygen demand (COD) values, such as in textile industry. In fact, the conventional enzymatic processes employed for textile treatments usually still require a large bath ratio and long cultivation duration for a
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satisfactory efficiency of enzymatic reactions and commercial production, especially for the biodesizing, biodegumming and bioscouring of natural fiber based substrates by employing various enzymes. Thus, a lot of water resource, energy and effluents with high concentration of pollutants still can't be avoided in the conventional enzymatic processes, although they possess more advantages than traditionally harsh chemical processes. Therefore, the effective, useful and Ecofriendly methodologies or processes with non-aqueous media or in which water is not the predominant content for enzyme applications, are very popular in textile and/or other industries. Fortunately, supercritical carbon dioxide fluid, a green and hydrophobic medium, has received increasing attentions in recent years as an alternative to conventional water and organic solvents in industries, due to its numerous advantages such as nontoxic, environmentally friendly, high diffusion rates, less side reactions, readily downstream treatments, absence of gaseliquid mass transfer limitations (Gremos et al., 2012; Long et al., 2011; Long et al., 2015). Consequently, it is very meaningful and necessary to investigate the activity and stability of enzymes in supercritical carbon dioxide media in order to develop different biocatalytical processes by integrating or combining enzymes with non- or less content aqueous media of supercritical carbon dioxide fluid for cleaner and sustainable production in textile and other industries, with more advantages than conventional enzymatic processes in elimination and/or reduction of the consumption of water resource and the discharge of effluents inherent as well as decreasing total production costs, etc. Theoretically, the activity and stability of enzymes in supercritical carbon dioxide media are the crucial points and basis prior to a development and/or an application of any bio-catalytical methodologies or processes in this water free media. Up to date, part of research for some enzymes is available in literature. Melgosa et al. (2015) investigated the effect of supercritical carbon dioxide treatment on the activity and conformational, morphology of four commercial lipases, and revealed that all the employed enzymes remained activities in the supercritical medium and the activity of some enzymes could be improved, whereas some other enzymes reduced their activity by means of conformational changes and structural alterations according to the treatment conditions. Leitgeb et al. (2013) investigated the activity of cellulase and aamylase from Hortaea werneckii after cell treatment with supercritical carbon dioxide, and found that sufficient residual activity of both the examined enzymes were detected and could be still used as biocatalysts in this medium. Natalia et al. (2012) explored the stability, activity, and selectivity of benzaldehyde lyase in supercritical fluids including carbon dioxide, and revealed that supercritical fluids could be an alternative media to organic solvents. Senyay-Oncel and Yesil-Celiktas (2013) treated immobilized aamylase in supercritical carbon dioxide in order to enhance enzyme stability and activity, and found that the activity of immobilized aamylase after consecutive enzymatic reactions could be enhanced by the retreatment with supercritical carbon dioxide. In addition, an enhancement in activity and stability for free a-amylase was also achieved after being treated in sub- and supercritical carbon dioxide by Senyay-Oncel and Yesil-Celiktas (2011). Moreover, SenyayOncel et al. (2014) investigated the activity and stability of protease in sub- and supercritical carbon dioxide, and demonstrated that enhanced activity and stability were observed with the raise of pressure, and the potential mechanism was also explored. Andrade et al. (2008) assessed the influence of compressed CO2 and propane treatment on the specific activity of partially purified D-hydantoinase from adzuki bean (Vigna angularis), then found that good stability of this enzyme in the two solvents was observed although some activity losses were occurred for resolubilized enzyme extract with compressed CO2. Manera et al. (2011) explored the influences
of pressure, exposure time and depressurization rate on the bgalactosidase activity of permeabilized cells of Kluyveromyces marxianus CCT 7082 submitted to treatment with compressed carbon dioxide, propane and n-butane, and showed that the activities of this biocatalyst were always higher than those of the nontreated ones. Furthermore, Rezaei et al. (2007) reviewed effects of pressure and temperature on enzymatic reactions in supercritical fluids including carbon dioxide, and demonstrated that most of the enzymes were active in supercritical fluids, and their activity could be improved with temperature within limits. However, some negative effects from treatments with supercritical carbon dioxide on the activity and stability of some enzymes were also reported. Santos et al. (2016) investigated the activity of immobilized lipase from Candida antarctica (Lipozyme 435) and its performance on the esterification of oleic acid in supercritical carbon dioxide, and showed that the activity of Lipozyme 435 decreased with the increase of pressure, temperature, exposure time and the number of pressurization/depressurization cycles. Oliveira et al. (2006) also investigated the influence of temperature, pressure, exposure time and depressurization rate on the activity of an immobilized lipase from Yarrowia lipolytica in compressed carbon dioxide, propane and n-butane, and showed that significant activity losses were obtained in carbon dioxide in comparison with other organic solvents. Moreover, the effects of treatment parameters on enzyme activity depended on its nature and source, as well as its forms. However, Dhake et al. (2011) investigated the activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide, and revealed that the immobilization method by using the blended polymer of hydroxylpropyl methyl cellulose and polyvinyl alcohol could greatly overcome the drawback by enhancing the catalytic activity of R. oryzae lipase for synthesis in supercritical carbon dioxide. It is obvious that most of the reported research works about the activity and stability of enzymes in supercritical carbon dioxide are mainly concentrated on a-amylase, cellulase, b-galactosidase, protease, free and/or immobilized lipases, etc. Very few works have been reported about the activity and stability of pectinase in supercritical carbon dioxide; especially, it is very lack of investigations about the alterations of pectinase in primary, secondary and tertiary structures, as well as its conformational and morphology changes induced by the supercritical media, although it is very important as a crucial basis for developing or performing green and environmentally friendly processes, reactions by combination of the two ecological technologies in different industries. The objective of the present work is to investigate the activity and stability of pectinase in supercritical carbon dioxide media, and also to explore some information about its structural, conformational and morphology alterations during treatment. The effects of system pressure, temperature and treatment duration on the activity and stability of pectinase were evaluated. Moreover, Fourier transfer infrared spectra (FT-IR), ultraviolet (UV) spectra, fluorescence spectra and scanning electron microscopy (SEM) analyses were performed for investigating the structural, conformational and morphology information of treated pectinase. 2. Material and methods 2.1. Materials A pectinase (from aspergillus Niger) in an analytical pure grade was purchased from Beijing Solarbio Technology Co., Ltd. (Beijing of China), and was used without further purification. A pectin (Galacturonic acid > 74.0%, dried basis) from citrus peel was obtained from SigmaeAldrich Co., Ltd. (Shanghai of China), and used as a
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1.2
(a) 0.25 /g L 0.30 0.35 0.40 0.60 0.80 1.20 1.60 2.00
1.0
Absorbance (A)
substrate for enzymatic hydrolysis reaction. Glucose anhydrouse was purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai of China). Dinitrosalicylic acid (DNS; 3, 5-dinitro-2-hydroxybenzoic acid) in a chemical pure grade was supplied by Chinasun specialty products Co., Ltd. (Changsu of China), and employed as a reagent for measuring the content of reducing sugar. Other chemicals utilized in enzymatic activity determination, such as sodium hydroxide, citric acid monohydrate, potassium sodium tartrate tetrahydrate, etc., were commercially available products in an analytical pure grade. A pure carbon dioxide gas (99.6 vol.%) was employed for supercritical carbon dioxide treatment of pectinase samples.
333
0.8 0.6
-1
0.4 0.2
2.2. Treatment of pectinase in supercritical carbon dioxide system
2.3. Evaluation of pectinase activity and stability The evaluation of pectinase activity was carried out on the basis of the determination of reducing sugars produced from pectin hydrolyzation. An aliquot of 1.0 mL pectinase sample solution at a concentration of 0.5 g L1 was added into substrate solution involving 10.0 g L1 pectin and 0.05 mol L1 citrate buffers at a pH value of 5.5. Then the enzymatic reaction was incubated at 50 C for 0.5 h, and terminated by boiling the mixed solution for 6.0 min. The produced reducing sugars were measured with the dinitrosalicylic acid (DNS) method declared by Miller (1959) employing galacturonic acid as a reference. Moreover, a construction of a calibration curve for determination of reducing sugars was carried out by employing standard addition method, in which glucose was used as a reference of reducing sugar with concentration ranging from 0.0 to 2.0 g L1 in a citrate buffer solution at a pH value of 5.5. After a complete reducing reaction between the glucose and the DNS indicator at boiling temperature for 5.0 min, the mixture solution was dilute with citrate buffer and its absorbance was monitored on an ultravioletevisible spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd, Beijing of China) at wavelength from 450.0 nm to 700.0 nm. Simultaneously, a control experiment was also carried out. Then, the calibration curve was constructed and regressed in a linear tendency with characteristic absorbance at maximum wavenumber against reducing sugar concentrations of glucose, as shown in Fig. 1(a, b) and Eq. (1):
C ¼ k DA þ b
(1)
where C refers to the concentration of reducing sugar of glucose; k is for the slope value of the calibration curve for activity determination; DA represents the difference of the absorbance at maximum wavenumber between the sample solution and the control; b is for the constant value from curve regression.
0.0 450
500
550
600
650
700
Wavelength (nm) 2.1
Concentration of glucose (g L-1)
The pectinase sample with a weight about 0.5 g were treated in the same batch system and the reactor as described elsewhere (Long et al., 2011; Long et al., 2015) at system temperature ranging from 30.0 C to 90.0 C, system pressure from 6.0 MPa to 23.0 MPa and treatment duration from 0.5 h to 4.0 h in supercritical carbon dioxide fluid, respectively. The solid sample of pectinase was packed with a metal mesh, and set at the up section of the reactor as described in detail elsewhere (Long et al., 2015). Then pure CO2 gas was pumped into the system after being preheated. As the system pressure and temperature were attained a request condition, the treatment process was begun by circulating the supercritical fluid with a syringe pump. After request treatment duration, the enzyme was took out and stored in refrigerator for further analysis.
(b)
1.8 1.5 1.2 0.9 0.6 0.3 0.0
0.2
0.4
0.6
0.8
1.0
1.2
Absorbance (A) Fig. 1. The visible spectra (a) and calibration curve (b) for pectinase determination with DNS method from the standard solution with different concentration of glucose.
Accordingly, the activity of pectinase was described by Unit (U) against per gram of pectinase, and one unit of its activity was defined as the amount of pectinase that catalyze the degradation of pectin to release 1.0 mg galacturonic acid within the duration of per hour at the incubation conditions above. Consequently, the activity of pectinase (AP) could be evaluated according to the following Eq. (2):
AP ¼
ðk DA þ bÞ V ð194=180Þ m t
(2)
where AP refers to the activity of pectinase with a unit of U g1; k is for the slope value of the calibration curve for activity determination; DA represents the difference of the absorbance between the sample solution and the control; V is for the volume of the enzymatic reaction solution after a centrifugation, 10.0 mL was employed in this work; 194/180 refer to the equivalent conversion of the reducing sugar of glucose to galacturonic acid in terms of their individually relative molecular mass; m is for the diluted times of the reaction solution; t is for the enzymatic reaction time with a unit of hour in this work; b is for the constant value from curve regression. Furthermore, the stability of pectinase subjected to various conditions in supercritical carbon dioxide media, was also characterized by the residual activity of pectinase (APRe.) in comparison
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with its enzymatic activity before and after the treatment, and assessed according to Eq. (3):
APRe: ¼
AP1 100% AP0
(3)
where APRe. refers to the residual activity of pectinase; AP0, AP1 are for the activity of pectinase before and after a treatment in supercritical carbon dioxide media, respectively. 2.4. FT-IR analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions, were also analyzed on an FT-IR instrument (Nicolet 5700, Thermo electron Co., Ltd., US) with traditional KBr pellet sampling method under an identical measurement condition with a same quantity at 0.58 mg of the samples. The transmittance of the infrared in individual powder sample of pectinase was recorded from 400.0 cm1 to 3800.0 cm1 at a resolution of 2.0 cm1 for infrared spectra. 2.5. Ultraviolet spectroscopy analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were dissolved, respectively, in citrate buffer solution at a pH value of 5.5 with a concentration of 2.50 g L1. Then the prepared solutions were measured by employing an ultravioletevisible spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co., Ltd, Beijing of China) at wavelength ranging from 250.0 nm to 350.0 nm at room temperature for scanning three times, in order to explore some information about the tertiary structure variations of pectinase samples after being subjected to supercritical carbon dioxide media. 2.6. Fluorescence spectroscopy analysis of pectinase samples The pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were dissolved, respectively, in citrate buffer solution at a pH value of 5.5 with a concentration of 2.50 g L1. Then the prepared solutions were measured on a spectrofluorometer (FM4P-TCSPC, Horiba Jobin Yvon Co., Ltd, USA) with an excitation wavelength at 280.0 nm and an emission wavelength ranging from 300.0 nm to 540.0 nm at room temperature for scanning three times, in order to further explore some information about the tertiary structure variations of pectinase samples after being subjected to supercritical carbon dioxide media. 2.7. Investigation of surface morphology for pectinase samples The surface morphological investigations of pectinase samples before and after a supercritical treatment were carried out by employing a scanning electronic microscopy (SEM) (TM3030, Hitachi, Japan) with an acceleration voltage of 15.0 kV at 200 and 5000 magnifications, respectively. Before investigation, samples were sputtered with gold on an ion sputter (E-1045 Hitachi, Japan) at 10.0 mA for 60.0 s. 3. Results and discussion 3.1. Calibration curve for determination of the activity of pectinase samples By employing the standard addition method and utilizing DNS as an indicator, a calibration curve for determination of reducing
sugars was constructed by employing glucose as a reference in a citrate buffer solution at a pH value of 5.5. The obtained results are depicted in Fig. 1(a, b). Fig. 1(a) shows that good absorption curves with a main and single characteristic peak at a maximum wavenumber (lmax) of 490.0 nm for the reduced colorful product of DNS were obtained in a citrate buffer solution with a pH value of 5.5, by accompanying with a proportional increase of the peak height as glucose concentration ranging from 0.25 g L1 to 2.00 g L1. Furthermore, Fig. 1(b) indicates that a good linear relationship (r2 ¼ 0.9995) was obtained between the glucose concentration (C) and the peak absorbance (DA), as expressed in Eq. (1). The linear dynamic range for reducing sugar determination in the predetermined conditions was 0.25e2.00 g L1 with a satisfactory sensitivity (the slop of k is 1.679 g L1. DA1 and the intercept b is 0.142 g L1). The relative standard deviation was 2.65% (n ¼ 2) at 0.25 g L1 of glucose in the employed determination conditions. These notably indicate that the proposed standard addition method and the obtained calibration curve with the DNS indicator are feasible and adequate to quantitatively determine reducing sugars in the employed system and conditions, as well as proved by our previous experiments. In principle, the reducing sugars which are added or produced from the hydrolyzation of pectins by pectinase, can react with the indicator of DNS in an appropriate condition to reduce the DNS to 3amino-5-nitrosalicylic acid, which is a stable brownish red color and proportionally increased with the concentration of reducing sugars in a linear dynamic range in a favorable buffer solution. Consequently, according to this calibration curve and a further equivalent conversion of the reducing sugars of glucoses to galacturonic acids, the activities of all the pectinase samples before or after being subjected to supercritical carbon dioxide media at different conditions were evaluated, as well as for the enzymatic stability in the following experiments. 3.2. Effect of system pressure on the supercritical treatment of pectinase An effect of system pressure on the activity and stability of pectinase in supercritical carbon dioxide media was investigated at a system temperature of 40.0 C for treatment duration of 1.0 h as system pressure ranging from 6.0 MPa to 23.0 MPa. The obtained results are shown in Fig. 2. Fig. 2 shows that a notable enhancement in the enzymatic activity and residual activity of pectinase was obtained with system pressure increased from 6.0 MPa to 15.0 MPa after a treatment in supercritical carbon dioxide media, and then a sharply decrease was also observed as system pressure further increased from 15.0 MPa to 23.0 MPa. Moreover, Fig. 2 further declare that the residual activities higher than 100% were achieved with the system pressure from 6.0 MPa to 20.0 MPa with a maximum value of 135% at 15.0 MPa in comparison with the one before treatment in supercritical carbon dioxide. These clearly indicate that the pectinase showed highly catalytical activity and stability in supercritical carbon dioxide media, and the supercritical treatment is benefit to improve its enzymatic activity during a favorable pressure range over 6.0 MPae20.0 MPa. Theoretically, supercritical carbon dioxide is a hydrophobic fluid medium and its density increases with system pressure at a fixed system temperature, which leads to enhanced interactions, or influences on the solutes. Therefore, an appropriate higher pressure probably shows more benefits to modify and/or rearrange the conformations of the protein structures such as the secondary and tertiary structures, via the interactions among the polypeptides, side chains and groups, and the solvent molecules, as well as by extracting some bound water and impurities, etc. It is important
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Fig. 3. Effect of system temperature on enzymatic activity (B) and residual activity (D) of pectinase treated at a system pressure of 15.0 MPa for 1.0 h in supercritical carbon dioxide fluid by employing DNS determination method.
that these interactions present a positive effect on the enzymatic activity and stability. However, the decrease tendencies of the enzymatic activity and stability as the system pressure higher than 15.0 MPa are probably due to some irreversible and/or destructive changes in the secondary and tertiary structures at an overhigh pressure of supercritical fluid. Accordingly, by consideration of the effects of system pressures on the activity and stability of pectinase, a system pressure of 15.0 MPa was recommended in further experiments for the supercritical treatment of pectinase. Moreover, an overhigh system pressure such as higher than 23.0 MPa should be avoided during a design and control of the enzymatic processes and/or reactions in supercritical carbon dioxide media, in order to maintain an adequate activity and enough lifetime of the pectinase.
and stability of pectinase. Consequently, a relative lower treatment temperature is favorable for pectinase applications in supercritical carbon dioxide media, and a temperature higher than 80.0 C should be avoided.
3.3. Effect of temperature on the supercritical treatment of pectinase An effect of system temperature on the enzymatic activity and residual activity of pectinase in supercritical carbon dioxide was explored at a system pressure of 15.0 MPa for treatment duration of 1.0 h with temperature ranging from 30.0 C to 90.0 C. The results are depicted in Fig. 3. Fig. 3 shows that evident enhancements in the enzymatic activity and residual activity of pectinase were achieved after being treated in supercritical carbon dioxide media as system temperature over 30.0 Ce80.0 C in comparison with the activity of untreated free pectinase at 1.88 104 U g1, although a gradual decrease tendency was also observed by accompanying with the whole tested temperatures. Moreover, serious reduces were also obtained in enzymatic activity and residual activity with lower values than the untreated samples as the system temperature higher than 80.0 C. These results demonstrate that there is feasible for pectinase to remain its enhanced catalytic activity and stability at a system temperature lower than 80.0 C for substrates in comparison with its untreated one. However, it is also obvious that the system temperature imposed a negative effect on the enzymatic activity and stability, especially for the system temperature higher than 80.0 C. Theoretically, system temperature usually presents notable affects on the polypeptide chain arrangements and irreversible variations in conformation of protein in secondary and tertiary structures, and readily makes it denaturation, restriction and even deactivation in catalytic activities. Therefore, the increase of the system temperature in supercritical carbon dioxide media reasonably resulted in the decrease of the enzymatic activity
3.4. Effect of treatment duration on the supercritical treatment of pectinase An effect of treatment duration on the enzymatic activity and residual activity of pectinase in supercritical carbon dioxide was investigated at a system pressure of 15.0 MPa and a temperature of 40.0 C with treatment duration from 0.5 h to 4.0 h. The results are depicted in Fig. 4. Fig. 4 shows that an evident enhancement in the enzymatic activity and residual activity was achieved, respectively, as the pectinase sample treated in supercritical carbon dioxide media only for 0.5 h, and then a platform was observed in the both curves of enzymatic activity and residual activity with prolonged treatment duration up to 4.0 h. In addition, for all the supercritically treated samples, the enzymatic activities were notably improved in comparison with the untreated one, and the residual activities were all higher than 100% for all the treatment durations. These clearly disclose a fact that the treatment of pectinase in supercritical carbon dioxide media is helpful and highly efficient to improve its enzymatic activity and stability by employing an appropriate
Fig. 4. Effect of treatment duration on enzymatic activity (B) and residual activity (D) of pectinase treated at a system pressure of 15.0 MPa and a temperature of 40 C in supercritical carbon dioxide fluid by employing DNS determination method.
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system temperature and pressure. Probably, the treatment of pectinase in the hydrophobic media at a favorable condition is benefit to present positive modifications in the secondary and tertiary structures of the protein via the rearrangements, interactions of its polypeptides, side chains and the solvent molecules. Therefore, it makes the pectinase become more suitable and efficient to combine with the substrate at its active centers, as well as to stimulate a higher catalytic performance of the enzyme than the untreated one. Consequently, treatment duration over 0.5 he4.0 h, and even longer durations are applicable for catalytic reactions and/ or processes with pectinase in supercritical carbon dioxide media by employing appropriate system pressures and temperatures. 3.5. FT-IR analysis of pectinase treated by supercritical carbon dioxide According to the above investigations, the treatment of pectinase with supercritical carbon dioxide media brought a significant influence on the enzymatic activity and stability. In order to further explore the influences of the supercritical treatment on the primary structure and conformation of the enzyme, the treated pectinase samples with highest and lowest enzymatic activity in supercritical carbon dioxide media were investigated by employing an FT-IR instrument. The results are shown in Fig. 5. Fig. 5 indicates that some significant changes in the infrared spectra of treated pectinase in supercritical carbon dioxide media were observed in comparison with the control sample, especially for the absorption intensities of the main and characteristic peaks. The infrared spectrum of pectinase sample with highest enzymatic activity and stability after a treatment in supercritical carbon dioxide media at conditions of 40.0 C 15.0 MPa 1.0 h shows the highest absorption intensities for all the characteristic peaks among all the samples. These indicate a structural change occurred during the treatment, which was responsible for the enhanced enzymatic activity and stability. Moreover, the characteristically strong peak at 3394.7 cm1 was attributed to the combination of the stretching vibrations of NeH and OeH (nNeH, nOeH), which was shifted to the lower location from 3410.7 cm1 for the control sample, accompanying with more evident peak splits at 3368.4 cm1 and
3293.1 cm1. These obviously demonstrate that more and stronger hydrogen bonds were formed between eOH, eNHe and eNH2 groups among inter- and/or intra-macrochains of the enzyme after the treatment in supercritical carbon dioxide fluid under the favorable conditions. The absorption bands at 2930.6 cm1 was assigned to the symmetrical stretching vibration of CeH in eCH3 and/or eCH2e groups, which was a slight shift from 2928.4 cm1 for the control sample, indicating a change occurred in the chemical environment of the aliphatic groups or chains. Furthermore, the characteristically strong and increased peak at 1647.1 cm1 was due to the stretching vibration of C]O from amide І (nC]O), which was closely relative and more sensitive than other amide bands to the change of the secondary structure of the main chains, such as the ahelix and b-sheet conformations in the treated pectinase (SenyayOncel and Yesil-Celiktas, 2013; Senyay-Oncel et al., 2014). It is obvious that the increased absorption intensity of the amide І indicates a significant change and/or a conformation transformation occurred in the secondary structure of the main chains, which is probably responsible for improving the enzymatic activity and stability. The medium intensity peaks at 1542.4 cm1 and 1451.9 cm1 were assigned to the bending vibration of NeH (dNeH) from amide ІІ and the stretching vibration of CeN (nCeN) from amide ІІІ, respectively (Dhake et al., 2013). In addition, the absorption band at 1405.7 cm1 was assigned to the bending vibration within plane of NeH (dNeH), and CeC (nCeC), CeN (nCeN) stretching vibrations (Dhake et al., 2013). Moreover, the absorption bands at 1154.9 cm1, 1079.9 cm1 and 1021.8 cm1 were due to the stretching vibrations of CeO (nCeO) and NeO (nNeO), etc. The weak absorption bands at 576.5 cm1 is attributed to CeS stretching vibrations of sulfides and disulfides (Senyay-Oncel and Yesil-Celiktas, 2013). However, the infrared spectrum of treated pectinase sample with lowest activity and stability at higher temperature condition of 90.0 C 15.0 MPa 1.0 h, exhibits notably reduced absorption intensities for all the corresponding peaks in comparison with the sample possessing highest enzymatic activity and stability, although their peak intensity are higher than those of the control sample. Moreover, the predominant peak for the combination of the stretching vibrations of NeH and OeH (nNeH, nOeH) was also
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8.0 Fluorescence intensity (cps.E6)
shifted to a higher wavenumber at 3410.1 cm1 compared with the highest activity sample. These indicate that a change and/or a weakening of the hydrogen bonds or interactions were occurred among inter- and/or intra-macrochains of the enzyme. Moreover, the symmetrical stretching vibration of CeH in eCH3 and/or eCH2e groups was slightly shifted to a lower wavenumber at 2929.5 cm1, as well as for the amide ІІ band. Probably, it is due to irreversible variations occurred in secondary structure of the main chains of pectinase, such as unfolding actions, rearrangements of the chains, which was induced by the increased and overhigh system temperature for the enzyme. In addition, there were no new characteristic peaks observed for all the treated enzyme samples in the FT-IR spectra, which indicates that no conspicuous change in its primary structure was occurred during the supercritical treatment.
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3.6. Ultraviolet and fluorescence spectroscopy analysis of pectinase treated by supercritical carbon dioxide In order to further explore the effect of supercritical treatment on the tertiary structure of pectinase samples, the treated pectinase samples with highest and lowest enzymatic activity were also investigated in citrate buffer solution at a pH value of 5.5 on an Ultravioletevisible spectrophotometer and a fluorescence spectrometer, respectively. The results are shown in Figs. 6 and 7. The ultraviolet spectra in Fig. 6 show that the absorbance of the supercritically treated pectinase sample with highest enzymatic activity and stability in citrate buffer solution was notably enhanced in comparison with the control one at a characteristic absorption band from about 250.0 nm to 350.0 nm involving a maximum wavelength at about 280.0 nm, which was assigned to the ultraviolet absorptions of the residual or side groups from some aromatic amino acids such as tryptophan, tyrosine and phenylalanine on the protein chains. However, the absorbance of treated enzyme sample with lowest activity and stability dissolved in a same buffer solution was decreased at the characteristic absorption band compared with that of the control one. It is obvious that the treatment of pectinase sample in supercritical carbon dioxide media presented a notable influence on the conformation of the enzyme structure even after being dissolved in aqueous solution. Moreover, it seems like that the ultraviolet absorption intensity of these aromatic amino acids is sensitive and characteristically relative to enzymatic activity and stability of the treated pectinase
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Wavelength (nm) Fig. 6. Ultraviolet spectra of pectinase samples for (1) before treatment, and treated at (2) 40.0 C 15.0 MPa 1.0 h, (3) 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide fluid.
Fig. 7. Fluorescence spectra of pectinase samples for (1) before treatment, and treated at (2) 40.0 C 15.0 MPa 1.0 h, (3) 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide fluid.
samples, in which higher absorption intensity indicates a higher enzymatic activity and stability of the pectinase. Theoretically, the primary structure of pectinase is composed with various amino acids linked by amide bonds to form polypeptide chains, including the aromatic amino acids of tryptophan, tyrosine and phenylalanine, etc. An ultraviolet absorption usually occurs in an aqueous solution at 280.0 nm, 275.0 nm and 257.0 nm for the aromatic amino acids of tryptophan, tyrosine and phenylalanine, respectively. Moreover, their absorptions are frequently and notably influenced by medium properties, pH, temperature, rearrangements of the enzyme chains, structure conformations and various interactions among the groups, etc. Therefore, it is evident that the supercritical treatment of pectinase at a favorable condition of 40.0 C 15.0 MPa 1.0 h in the hydrophobic media is helpful to release and expose these residual groups of aromatic acids onto the outer surfaces of enzymatic structure, especially for tryptophan and tyrosine, which is probably relative to the active sites in the enzyme structure and responsible for the enhanced enzymatic activity and stability. However, a worse treatment condition in supercritical carbon dioxide fluid with an overhigh temperature of 90.0 C tends to overshadow these residual groups via chain arrangements and conformational changes, as well as group interactions, etc., which resulted in the reduced enzymatic activity and stability of the pectinase. Fig. 7 is for the recorded fluorescence spectra of pectinase samples in citrate buffer solutions with characteristic fluorescence emission peaks at a range from 280.0 nm to 540.0 nm. It demonstrates that a characteristic emission peak involving a maximum wavelength at 351.0 nm with an evident shift to higher wavelength, namely a red shift, was observed for the treated pectinase sample with highest enzymatic activity and stability, accompanying with a decrease in the peak intensity in comparison with the control one. However, an almost non-shift and a notable increase in the intensity of the characteristic fluorescence emission peak were also obtained for the treated pectinase sample with lowest activity and stability compared with the control one. These clearly indicate that the treatment of pectinase at different conditions in supercritical carbon dioxide media could bring various and significant effects on the tertiary structure or conformation of the polypeptide and side chains, as well as the residual side groups in the enzyme even after being dissolved in aqueous solution, which were probably responsible for the changed enzymatic activity and stability.
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Fig. 8. SEM images of pectinase samples for (a) before treatment, (b) supercritically treated at 40.0 C 15.0 MPa 1.0 h, (c) supercritically treated at 90.0 C 15.0 MPa 1.0 h, with a magnification of 200 and 4000, respectively.
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In principle, the side residual groups from the aromatic amino acids of tryptophan, tyrosine and phenylalanine in the primary structure of pectinase, which involve p-electrons in their structures and are readily subjected to ultraviolet excitation at 280.0 nm, are able to emit intrinsic fluorescence at a longer wavelength of 348.0 nm, 303.0 nm and 282.0 nm, respectively (Yin et al., 2010; Bobone et al., 2014). Usually, the most strong emission intensity of fluorescence is from tryptophan residual group and the lowest is from phenylalanine, and the emission of fluorescence from the tyrosine residual group is frequently quenched readily due to an occurrence of energy transformation to tryptophan residual group in a simultaneous presence of the both in protein. Moreover, the fluorescence emissions from these aromatic side groups are sensitive to their location environment and are usually employed as an indicator to explore the changes of the conformation of proteins. Theoretically, a polar environment tends to reduce the excitation state energy of these aromatic side groups, and to result in a red shift of their fluorescence emission spectra (Yin et al., 2010; Bobone et al., 2014). Therefore, it is obvious that the evident shift of the characteristic emission peak to 351.0 nm, which was from the residual side aromatic group of tryptophan, clearly indicates that an exposure of more side groups of tryptophan into a higher polar environment of aqueous solution was occurred during dissolving the pectinase sample with highest activity and stability which was treated at a condition of 40.0 C 15.0 MPa 1.0 h. Probably, this side group was relative to the active sites of the pectinase, and so it was more readily to combine with the substrate, resulting in the most enhanced enzymatic activity and stability. The decrease of the intensity was also due to an emission quenching from the exposure of the side group to the polar solution. However, as regard to the treated pectinase with lowest activity and stability at a condition of 90.0 C 15.0 MPa 1.0 h in supercritical carbon dioxide media, the non-shift of the characteristic peak and the increased fluorescence intensity demonstrate that an undesirable and irreversible conformational change, and a deeper burying of the residual side aromatic group of tryptophan into the pectinase structure were occurred during the supercritical treatment by employing the higher system temperature. Moreover, all the investigations from fluorescence spectroscopy analysis are coincident with the analysis from ultraviolet spectroscopy in Fig. 6, as well as with the FT-IR analysis in Fig. 5. These further confirm the role of supercritical treatment for improving the enzymatic activity and stability at an appropriate condition, which is due to the conformational changes in secondary and tertiary structures of the treated pectinase in supercritical carbon dioxide media. Therefore, the supercritical treatment could be employed as an efficient and Ecofriendly method to improve the activity and stability of the pectinase via a conformational change at an appropriate condition. 3.7. Surface morphology analysis of pectinase treated by supercritical carbon dioxide An investigation of the surface morphology of pectinase before and after a supercritical treatment was carried out by employing a scanning electronic microscopy (TM3030, Hitachi, Japan) in order to further explore the influences of the supercritical fluid on the pectinase. The results are depicted in Fig. 8(aec). Fig. 8a shows that many big aggregates of the pectinase before treatment were observed at a low magnification of 200, and a relative smooth and intact surface was also observed for these enzyme aggregates at a high magnification of 5000. Whereas, as shown in Fig. 8b, some broken and more particles in small sizes for the pectinase were obtained at the low magnification of 200 after a treatment in supercritical carbon dioxide media under a condition
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of 40.0 C 15.0 MPa 1.0 h. Moreover, an evidently fractured surface of the aggregates was observed, and even some fragments and wholes were also visible on the enzyme aggregates with the magnification of 5000 in Fig. 8b. In addition, as depicted in Fig. 8c, more broken and small particles were obtained at the low magnification of 200 for the enzyme sample after a treatment at a higher temperature condition of 90.0 C 15.0 MPa 1.0 h, and also a most seriously fractured surface with many fragments on the aggregates was clearly visible under a higher magnification of 5000. All these above indicate that different etching effects on the surfaces of the pectinase aggregates could be occurred during the treatment in supercritical carbon dioxide media, and a relative low system temperature is more favorable for the enzyme to remain its nature morphology and simultaneously improve its activity and stability. However, an overhigh system temperature could bring a serious deterioration of the enzyme morphology, which is also probably responsible for the reduced enzymatic activity and stability. Therefore, an appropriate treatment condition, especially for the system temperature, is crucial to maintain the natural surface morphology of pectinase and simultaneously improve its enzymatic activity and stability. 4. Conclusions The activity and stability of pectinase were investigated after a treatment in supercritical carbon dioxide. The results reveal that the activity and stability of pectinase could be significantly improved under appropriate conditions. Significant increases in activity and stability of treated pectinase could be available with pressure lower than 15.0 MPa, whereas, temperature tends to reduce enzymatic activity and stability. An excellent stability of pectinase with improved activity was observed with the duration of 4.0 h or longer. Fourier transfer infrared spectra, ultraviolet spectra, fluorescence spectra and scanning electron microscopy analyses demonstrate that some alterations in the secondary and tertiary structures, and surface morphology of treated pectinase were occurred. Therefore, the investigations further indicate that the supercritical treatment could be employed as an efficient and Ecofriendly method to improve the activity and stability of the enzyme, and there is also a feasible to combine the two ecological technologies of the pectinase and supercritical carbon dioxide media for green and environmentally friendly processes, reactions in different industries. Acknowledgment The authors acknowledge gratefully the financial supports from the science and technology support project of Jiangsu province of China (Grant No. BE2013051), and the Priority Academic Program Development of Jiangsu Higher Education Institutions [PAPD] of China. References Andrade, J.M., Oestreicher, E.G., Oliveira, J.V., Oliveira, D., Antunes, O.A.C., Dariva, C., 2008. Effect of treatment with compressed CO2 and propane on d-hydantoinase activity. J. Supercrit. Fluids 46, 342e350. Bobone, S., Van De Weert, M., Stella, L., 2014. A reassessment of synchronous fluorescence in the separation of Trp and Tyr contributions in protein emission and in the determination of conformational changes. J. Mol. Struct. 1077, 68e76. Chand, N., Nateri, A.S., Sajedi, R.H., Mahdavi, A., Rassa, M., 2012. Enzymatic desizing of cotton fabric using a Ca2þ-independent a-amylase with acidic pH profile. J. Mol. Catal. B Enzym. 83, 46e50. Choi, J.-M., Han, S.S., Kim, H.S., 2015. Industrial applications of enzyme biocatalysis: current status and future. Biotechnol. Adv. 33, 1443e1454. Ciftci, D., Saldana, M.D.A., 2012. Enzymatic synthesis of phenolic lipids using flaxseed oil and ferulic acid in supercritical carbon dioxide media. J. Supercrit. Fluids 72, 255e262.
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Dhake, K.P., Deshmukh, K.M., Patil, Y.P., Singhal, R.S., Bhanage, B.M., 2011. Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide. J. Biotechnol. 156, 46e51. Dhake, K.P., Karoyo, A.H., Mohamed, M.H., Wilson, L.D., Bhanage, B.M., 2013. Enzymatic activity studies of Pseudomonas cepacia lipase adsorbed onto copolymer supports containing b-cyclodextrin. J. Mol. Catal. B Enzym. 87, 105e112. Gremos, S., Kekos, D., Kolisis, F., 2012. Supercritical carbon dioxide biocatalysis as a novel and green methodology for the enzymatic acylation of fibrous cellulose in one step. Bioresour. Technol. 115, 96e101. Kalantzi, S., Mamma, D., Christakopoulos, P., Kekos, D., 2008. Effect of pectate lyase bioscouring on physical, chemical and low-stress mechanical properties of cotton fabrics. Bioresour. Technol. 99, 8185e8192. Kashyap, D.R., Vohra, P.K., Chopra, S., Tewari, R., 2001. Applications of pectinases in the commercial sector: a review. Bioresour. Technol. 77, 215e227. Leitgeb, M., Colnik, M., Primozic, M., Zalar, P., Cimerman, N.G., Knez, Z., 2013. Activity of cellulase and a-amylase from Hortaea werneckii after cell treatment with supercritical carbon dioxide. J. Supercrit. Fluids 78, 143e148. Long, J.-J., Ma, Y.-Q., Zhao, J.-P., 2011. Investigations on the level dyeing of fabrics in supercritical carbon dioxide. J. Supercrit. Fluids 57, 80e86. Long, J.-J., Cu, Ch.-L., Zhang, Y.-Q., Yuan, G.-H., 2015. Clean fixation of dye on cotton in supercritical carbon dioxide with a heterogeneous and phase transfer catalytic reaction. Dyes Pigment. 115, 88e95. Manera, A.P., Kuhn, G., Polloni, A., Marangoni, M., Zabot, G., Kalil, S.J., Oliveira, D., Treichel, H., Vladimir, O.J., Mazutti, M.A., Maugeri, F., 2011. Effect of compressed fluids treatment on the activity, stability and enzymatic reaction performance of b-galactosidase. Food Chem. 125, 1235e1240. Melgosa, R., Sanz, M.T., Solaesa, A.G., Bucio, S.L., Beltran, S., 2015. Enzymatic activity and conformational and morphological studies of four commercial lipases treated with supercritical carbon dioxide. J. Supercrit. Fluids 97, 51e62.
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