Bioresource Technology 101 (2010) S66–S70
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Effect of a buffer mixture system on the activity of lipases during immobilization process Jong Ho Lee a, Sung Bong Kim a, Chulhwan Park b, Seung Wook Kim a,* a b
Department of Chemical and Biological Engineering, Korea University, 1 Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 15 November 2008 Received in revised form 11 February 2009 Accepted 12 March 2009 Available online 9 April 2009 Keywords: Buffer effect Buffer mixture system Ion strength pH
a b s t r a c t In this study, the effects of various buffers and ionic strengths on the immobilization of Candida rugosa and Rhizopus oryzae lipases were investigated to enhance the activities of the immobilized lipases. Among the various buffers, the optimal buffers and ionic strength for the immobilization of C. rugosa and R. oryzae lipases were determined to be a mixture of 0.25 M MOPs and sodium phosphate buffer (pH 6.5). Moreover, the activities of immobilized C. rugosa and R. oryzae lipases under their optimal conditions were 3756.11 and 2845.21 U/g matrix, respectively. Furthermore, the activity of immobilized lipases increased by approximately 4.13 and 3.1 times after 24 h, respectively. Finally, the activities of the immobilized lipases were maintained at levels greater than 90% of their original activities after ten reuses and at levels greater than 60% of their original activities after twenty reuses. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Lipase is the enzyme most often applied in biochemical reactions such as esterification, interesterification and transesterification (Dizge et al., 2009; Lee et al., 2006a,b,c, 2007; Murty et al., 2002; Noor and Omar, 2000; Schoemaker et al., 2003; Yoon et al., 2004). Lipases are enantioselective; therefore, they can be used to synthesize optically active compounds (Shieh et al., 2003). However, lipases are too expensive to be exhausted after usage. Therefore, for industrial applications, they should be immobilized on carriers to allow reuse or use in continuous processes (Dizge et al., 2009). Many studies have reported that the thermal, pH, and mechanical stabilities of lipase can be increased by immobilization (Gao et al., 2009; Lifka and Ondruschka, 2004; Oda et al., 2005). In addition, we previously reported that, the use of covalent bonding for enzyme immobilization could be conducted using glutaraldehyde as a cross-linker and we developed a pretreatment method that prevented the loss of lipase activity during covalent immobilization. Furthermore, immobilized Candida rugosa and Rhizopus oryzae lipases have been used to eliminate the acylmigration step in the production of biodiesel to an increase reaction rate when compared to other production process (Lee et al., 2006a,b,c, 2007; Park et al., 2001, 2002, 2003). It is well known that pH and ionic strength of buffers that are used in the immobilization process are very important factors (Kalisz et al., 1997; Hublik and
* Corresponding author. Tel.: +82 2 3290 3300; fax: +82 2 926 6102. E-mail addresses:
[email protected] (J.H. Lee),
[email protected] (S.B. Kim),
[email protected] (C. Park),
[email protected] (S.W. Kim). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.031
Schinner, 2000; Moly Eldin et al., 2000). However, few studies have been conducted to evaluate the effects of the interaction between pH and the concentration of buffer on the activity of lipases. Therefore, in this study, the effects of various buffers and ionic strengths on the immobilization of C. rugosa and R. oryzae lipases were investigated so the activities and stabilities of the immobilized lipases could be increased. Moreover, synergistic effects between the compounds in several buffer mixture systems were evaluated. 2. Methods 2.1. Materials C. rugosa lipase (700 U/mg) and 3-aminopropyltriethoxysilane was purchased from Sigma Chemical Co. (USA) and R. oryzae lipase (41.6 U/mg) and glutaraldehyde were purchased from Fluka Co. (Switherland). Silica gel was obtained from the Grace Davison Co. (USA). All other chemicals were of reagent grade. 2.2. Preparation of lipase One gram of lipase was suspended in 100 ml of various buffers including sodium phosphate (SP), potassium phosphate (PP), 3(N-morpholino) propane-1-sulfonic acid (MOPs) and 2-amino-2hydroxymethyl-propane-1,3-diol (Tris) buffer. These buffers were chosen because they have various pHs and ionic strengths, which enabled us to test the effect of various buffers on the immobilization of lipases. Following suspension of the lipase in the buffer, the solution was centrifuged at 4000 rpm for 15 min at 4 °C. The
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supernatant was removed and stored at 4 °C for subsequent immobilization studies. 2.3. Pretreatment of lipase Two hundred mg of soybean oil were added to 20 ml of C. rugosa and R. oryzae lipase solution for increase of lipase activity (Lee et al., 2006a,b,c, 2007). The mixtures were then incubated at 40 °C with stirring at 200 rpm for 45 min. Next, 10 ml of the lipase solutions produced were subjected to immobilization. 2.4. Preparation of activated silica gel for lipase immobilization One gram of dry silica gel was mixed with 10% 3-aminopropyltriethoxysilane in 20 ml of acetone and then incubated at 50 °C for 2 h with constant stirring. The silica gel was then washed with water, dried at 60 °C for 2 h, and suspended in 20 ml of 1 mM phosphate buffer solution (pH 7). Two milliliters of 25% (w/v) glutaraldehyde were then added and incubated at 20 °C for 2 h to activate the silica gel. The activated silica gel was then washed with water and dried at 60 °C for 2 h. 2.5. Immobilization of lipase The activated silica gel (500 mg) was mixed with 10 ml of lipase solution and then incubated at 20 °C for 12 h. The immobilized lipase was then recovered by filtration, washed with water and dried overnight at room temperature. The enzymes were then immobilized in a 500 ml immobilization reactor unit. 2.6. Assay of immobilized lipase activity Ten milliliters of isoocatane containing 10% (w/v) soybean oil were added to 10 ml of 50 mM phosphate buffer (pH 7) containing 200 mg of the immobilized lipase. The reaction mixture was then incubated in a shaking water bath at 37 °C and 150 rpm for 30 min. Two milliliters of the upper layer were then transferred to a test tube, after which a cupric acetate-pyridine reagent (0.5 ml) was added. The free fatty acids liberated and dissolved by the isooctane were quantified using a UV spectrophotometer at 715 nm (Lee et al., 2006a,b,c, 2007). One unit of lipase activity was defined as the amount of the enzyme required to liberate 1 lmol of the free fatty acid per min.
Fig. 1 shows the effect of buffers on the activity of the immobilized pretreated C. rugosa lipase. The optimal pH of the buffers was found to be 6.5 and the maximum activity of the immobilized C. rugosa lipase was 760.4 U/g matrix. MOPs buffer was the most effective buffer at pH 6.5. In contrast with the free lipases, the behavior of lipase activity changed gradually at all tested pH range. With the exception of Tris-HCl buffer, buffers with low pHs (6.5) were used. It has been reported that enzyme binding is more efficient at alkaline pHs than acidic pHs when immobilization is conducted using covalent (Lee et al., 2002; Li et al., 2008). Therefore, the decrease in enzyme activity in response to changes in pH is smaller the pH value is low. Fig. 1 shows the effect of various buffers on the activity of immobilized pretreated R. oryzae lipase. The activity of lipase in the MOPs buffers was higher than the activity of the lipase in other buffers at all tested pH ranges. In addition, the optimal pH of the buffers was found to be 7.0. The maximum activity of the immobilized R. oryzae lipase was 763.5 U/g matrix. It has been reported that the activity of enzymes was increased by MOPs and Tris buffer, and MOPs buffer frequently used to induce high protein and enzyme activities. The results of this study suggest that MOPs enhances the activity of enzymes more effectively than other buffers, which is in accordance with the results of previous studies (Fonteh et al., 2005; Mohamed et al., 2007; Ruth et al., 2007; Zhao and Chasteen, 2006). Buffer solutions are generally evaluated in studies designed to compare the effects of buffers with different features on the activity of the enzyme of interest. For example, SP buffer is known to maintain enzymatic activity. Conversely, zwitterionic buffers such as MOPs, Tris, HEPES, TES and PBS buffers contain both positive and negative ionizable groups, which were introduced by Zhao and Chasteen (2006). In zwitterionic buffers, secondary and tertiary amines provide the positive charges, while sulfonic acid and carboxylic acid groups provide the negative charges. In addition, these buffers are highly soluble in water and can maintain a stable pH in a physiological range of 6.5 and 8.0 over a wide range of temperature. However, the stability of zwitterionic buffers is not well defined and aggregation of buffer molecules may occur (Zhao and
900
The immobilization yield was calculated as the ratio of the amount of protein bound to the carrier to the mutual amount of protein and expressed as a percentage. The amount of C. rugosa and R. oryzae lipases was determined using the Folin-Lowry method (Park et al., 2001, 2002, 2003). The amount of C. rugosa and R. oryzae lipases bound to the carriers was determined based on the difference between the initial and residual lipase concentrations.
3. Results and discussion
Activity of immobilized lipase (U/g matrix)
800
2.7. Calculation of immobilization yield
700 600 500 400 300 C. rugosa lipase- MOPs-HCl and NaoH +SP buffer C. rugosa lipase- MOPs-HCl and NaoH +Tris-HCl buffer C. rugosa lipase- Tris-HCl +SP buffer R. oryzae lipase- MOPs-HCl and NaoH +SP buffer R. oryzae lipase- MOPs-HCl and NaoH +Tris-HCl buffer R. oryzae lipase- Tris-HCl +SP buffer
200 100
3.1. Effect of the pH of the various buffers on the activity of the lipases during the immobilization process
0 6.0
6.5
7.0
7.5
8.0
pH
In this study, the effects of the pH of various buffers on the activity of immobilized pretreated lipases were evaluated. Each buffer had an ionic strength of 0.1 M and a pH that ranged from 6.0 to 8.0. C. rugosa and R. oryzae lipases were dissolved in the various buffers and then pretreated with soybean oil. The pretreated lipase solution was immobilized on activated silica gels for 12 h.
Fig. 1. Effect of the pH of various buffers on the activity of immobilized pretreated C. rugosa and R. oryzae lipases. Lipase solutions in various buffers (20 ml) were pretreated with 20 mg of soybean oil at 40 °C with stirring at 200 rpm in 100 ml shake flasks for 45 min. The pretreated lipases were then immobilized on activated silica gels, after which they were used to hydrolyze soybean oil in batch reactions. Soybean oil hydrolysis was performed for 30 min at 37 °C in a shaking water bath.
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Chasteen, 2006). In an attempt to overcome, we evaluated mixtures of zwitterionic buffers to determine if they exerted a synergistic effect on the activity of immobilized lipases. All mixtures evaluated in this study contained a 1:1 ratio of the buffers. Among the various buffer mixture systems, a mixture of SP and MOPs buffers was found to most effectively enhance the activity of the C. rugosa and R. oryzae lipases. In addition, the optimal pH of this buffer system was found to be 6.5. Furthermore, the maximum activity of immobilized C. rugosa and R. oryzae lipase was 850.4 U/g matrix and 832.7 U/g matrix, respectively, when this system was used (Fig. 2). When R. oryzae lipase was evaluated, the optimal pH was transfer from 7.0 to 6.5. Taken together, these findings indicate that mixtures of the buffers resulted in synergistic effect during the immobilization process, which implies that buffer mixture systems have the potential for use in the immobilization of high activity lipases.
1000
Activity of immobilized lipase (U/g matrix)
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900 800 700 600 500 400 300 200 100
C. rugosa - MOPs-HCl and NaOH+PHosphate buffer C. rugosa - MOPs-Tris-HCl and NaOH buffer C. rugosa - Tris-HCl and NaOH+Phosphate buffer R. oryzae lipase- MOPs-HCl and NaoH +SP buffer R. oryzae lipase- MOPs-HCl and NaoH +Tris-HCl buffer R. oryzae lipase- Tris-HCl +SP buffer
0 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Ionic strength(M)
3.2. Effect of the ionic strength of various buffers on the activity of lipases during the immobilization process Many investigators have reported that it is important to adjust the pH of a buffer during immobilization, but that it is more important to determine the proper ionic strength of the buffer (Li et al., 2009; Wang et al., 2008). Therefore, in this study, buffers with ionic strengths ranging from 0.1 M to 1.25 M were evaluated while focusing on the buffer mixture system with the activity that was described in the preceding section. C. rugosa and R. oryzae lipases were dissolved in the various buffers at a pH 6.5. The lipase solutions were then pretreated with soybean oil, after which they were immobilized on activated silica gels for 12 h (Lee et al., 2006a,b,c, 2007). Fig. 3 shows the effect of ionic strength on the activity of immobilized pretreated C. rugosa lipase. Among the various buffers, a mixture of SP and MOPs buffer most effectively increased the activity of the immobilized lipases. The optimal ionic strength for the immobilization of C. rugosa lipase was determined to be 0.25 M. In addition, the optimal buffer and ionic strength for the immobilization of R. oryzae lipase was observed when a mixture of 0.25 M MOPs and 0.25 M SP buffer was used (Fig. 3). Specifically, the activities of C. rugosa and R. oryzae lipases increased the ionic
Fig. 3. The effect of the ionic strength of a mixture of various buffers on the activity of immobilized pretreated C. rugosa and R. oryzae lipases. Lipase solutions of various ionic strengths (20 ml) were pretreated with 20 mg of soybean oil at 40 °C with stirring at 200 rpm in 100 ml shake flasks for 45 min. The pretreated lipases were then immobilized on activated silica gels, after which they were used to hydrolyze soybean oil in batch reactions. Soybean oil hydrolysis was performed for 30 min at 37 °C in a shaking water bath.
strength increased to 0.25 M, but they continuously decreased to 1.25 M. This decrease occurred because the protein became dehydrated at high ionic strengths due to the hydrating effect of the salt molecules (Essa et al., 2007). Despite this effect, other buffer mixture systems were not capable of maintaining a high level of activity (data not shown). Taken together, these results indicate that the activities of lipases in the buffer mixture systems are related to the type of buffers used and the ionic concentrations. Moreover, these finding indicate that a synergistic effect can be obtained through the use of a buffer mixture system. The activities of immobilized C. rugosa and R. oryzae lipases immobilized under their optimal conditions were 910.2 and 880.3 U/g matrix, respectively. 3.3. Effect of immobilization time on the activity of lipases during immobilization process
900
Activity of immobilized lipase (U/g matrix)
800 700 600 500 400 300
C. rugosa lipase- MOPs-HCl and NaoH +SP buffer C. rugosa lipase- MOPs-HCl and NaoH +Tris-HCl buffer C. rugosa lipase- Tris-HCl +SP buffer R. oryzae lipase- MOPs-HCl and NaoH +SP buffer R. oryzae lipase- MOPs-HCl and NaoH +Tris-HCl buffer R. oryzae lipase- Tris-HCl +SP buffer
200 100 0
6.0
6.5
7.0
7.5
8.0
pH Fig. 2. Effect of the pH of a mixture of various buffers on the activity of immobilized pretreated C. rugosa and R. oryzae lipases. Lipase solutions with various pH (20 ml) were pretreated with 20 mg of soybean oil at 40 °C with stirring at 200 rpm in 100 ml shake flasks for 45 min. Pretreated lipases were then immobilized on activated silica gels, after which they were used to hydrolyze soybean oil in batch reactions. Soybean oil hydrolysis was performed for 30 min at 37 °C in a shaking water bath.
In previous studies, a pretreatment system for the immobilization of lipases was established and optimized. The results of previous studies indicated that the immobilized lipases had the highest activities when the immobilization time was 12 h (Lee et al., 2006a,b,c, 2007, 2008). However, in this study, when the optimal immobilization time was applied to the buffer mixture system, the binding yields of C. rugosa and R. oryzae lipases were 67.59 and 69.46%, respectively. These results represent an approximately 20% decrease in binding yield of C. rugosa and R. oryzae lipases (data not shown). Therefore, the effect of the immobilization time was investigated for increase the binding yield of C. rugosa and R. oryzae lipase. As shown in Fig. 4, the activity of C. rugosa and R. oryzae lipases increased in proportion to immobilization time until 24 h and was then maintained until 36 h. The maximum binding yields of the C. rugosa and R. oryzae lipases were 96.83 and 93.58%, respectively, and the maximum activities of the C. rugosa and R. oryzae lipases were 3756.1 and 2721.35 U/g matrix, respectively. As compared with the initial buffer mixture system, the activity of the immobilized C. rugosa and R. oryzae lipases increased to approximately 4.13 and 3.1 times as lipases binding yield was increased. As the distance of the enzymes was approached by increase of lipase binding yield, a shorter diffusional distance was created, which reduced the time required for the substrate to diffuse (Nouaimi-Bachmann et al., 2006; Suman and Pundir, 2003;
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Acknowledgements
100 90
4000
80 70
3000
60 50
2000
40 30
1000
Activity of C. rugosa lipase Activity of R. oryzae lipase Yield of C. rugosa lipase binding Yield of R. oryzae lipase binding
0
Yield of protein binding (%)
Activity of immobilized lipase (U/g matrix)
5000
20 10 0
0
5
10
15
20
25
30
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35
Immobilization time (h) Fig. 4. Effect of immobilization on the activity and yield of protein binding by immobilized C. rugosa and R. oryzae lipases. Lipase solutions (20 ml) were pretreated with 20 mg of soybean oil at 40 °C with stirring at 200 rpm in 100 ml shake flasks for 45 min. Pretreated lipases were then immobilized on activated silica gels for various times, after which they were used to hydrolyze soybean oil in batch reactions. Soybean oil hydrolysis was then performed for 30 min at 37 °C in a shaking water bath.
Torabi et al., 2007). As a result, the activity of the immobilized lipases increased to a greater degree than the yield of enzyme binding. Taken together, these results suggest that immobilization using covalent bonding has the potential for industrial application. 3.4. Reusability of immobilized C. rugosa and R. oryzae lipases Reusability is a crucial feature of immobilized enzyme preparations in most practical applications, and inactivation of immobilized enzyme is the most common problem preventing reusability (Torabi et al., 2007). In this study, reusability of the immobilized C. rugosa and R. oryzae lipases was investigated by a consecutive series of hydrolysis reactions and shows the dependence of relative activities on the number of reuses. The relative activity of the immobilized lipases decreased as the number of reuses increased. Specially, the activities of the immobilized lipases were maintained at levels exceeding 90% and 60% of their original activities after ten reuses and twenty reuses, respectively. After twenty reuses, the activities of C. rugosa and R. oryzae lipases were about 2253.7 and 1632.8 U/g matrix, respectively. Moreover, after fifty reuses, the activities of C. rugosa and R. oryzae lipases were about 751.2 and 544.3 U/g matrix, respectively. When compared with our previous studies, lipases activity and stability were increased markedly (Lee et al., 2006a,b,c, 2007). These results imply that the buffer mixture system has potential for application to industrial immobilized enzymes production and its application. 4. Conclusion In this study, buffer mixture system on the activity of immobilized pretreated C. rugosa and R. oryzae lipases was investigated. The optimal buffer system for the immobilization of C. rugosa and R. oryzae lipases was determined to be a 0.25 M mixture of MOPs and SP (pH 6.5) at 36 h. The maximum activities of the C. rugosa and R. oryzae lipases were 3756.1 and 2721.35 U/g matrix, respectively. Furthermore, the activities of immobilized lipases were maintained at levels exceeding 90% and 60% of their original activities after ten reuses and twenty reuses, respectively.
This study was supported by the Brain Korea 21 program issued by the Ministry of Education, Korea.
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