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Zhu, J., Zhang, J., Lai, Q., Jiang, B., and Gong, R. (2013). "Covalent immobilization of lipase onto citric acid-esterified loofah sponge," BioRes. 8(3), 3289-3298.

Abstract

Loofah sponge was activated by thermochemical esterification between the carboxyl groups of citric acid and hydroxyl groups of cellulose to introduce free carboxyl groups, which were further reacted with lipase amino groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as the condensing reagent. This resulted in loofah sponge–immobilized lipase. Under the optimized immobilization conditions, the highest activity of immobilized lipase per gram of dry carrier was found to be 45.8 U/g. The immobilized lipase exhibited maximum activity at 40 °C, pH 8.0, while the optimal temperature and pH for the free lipase were 37 oC and 7.5, respectively. The immobilized lipase showed better thermal stability, storage stability, and reusability than free lipase.


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Covalent Immobilization of Lipase onto Citric Acid-Esterified Loofah Sponge

Jun Zhu,a,b Jian Zhang,a,b Qiumei Lai,a,b Bo Jiang,c and Renmin Gonga,b,*

Loofah sponge was activated by thermochemical esterification between the carboxyl groups of citric acid and hydroxyl groups of cellulose to introduce free carboxyl groups, which were further reacted with lipase amino groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as the condensing reagent. This resulted in loofah sponge–immobilized lipase. Under the optimized immobilization conditions, the highest activity of immobilized lipase per gram of dry carrier was found to be 45.8 U/g. The immobilized lipase exhibited maximum activity at 40 °C, pH 8.0, while the optimal temperature and pH for the free lipase were 37 oC and 7.5, respectively. The immobilized lipase showed better thermal stability, storage stability, and reusability than free lipase.

Keywords: Lipase; Immobilization; Esterified loofah sponge; Carbodiimide; Amide linkage

Contact information: a: College of Life Science, Anhui Normal University, Wuhu, 241000, P R China; b: Anhui Daily Chemical Engineering Technology Research Center of Traditional Chinese Medicine, Wuhu, 241000, P R China; c: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, P R China; * Corresponding author: rmgong.nju@163.com

Introduction

Enzymes are versatile macromolecular biocatalysts that offer high catalytic efficiency and stereospecificity to biochemical reactions under mild reaction conditions. However, the main problems of using soluble enzymes in industry are the instability of enzymes and the difficulty of their recovery and reutilization (Sheldon 2007). To overcome these problems, enzymes are often immobilized onto insoluble solid supports. Immobilization of enzymes not only can increase the stability of enzymes and facilitate the reuse of enzymes, but also can reduce the cost of biocatalytic production.

There are various methods for enzyme immobilization. They include adsorption onto an insoluble carrier (Zheng et al. 2012; 2013), entrapment or encapsulation in a polymeric matrix (González-Sáiz and Pizarro 2001; Koszelewski et al. 2010), cross-linking with a bifunctional reagent (Šulek et al. 2011), and covalent linking to insoluble solid material (Elnashar et al. 2009). The immobilization of enzyme by covalent binding offers the following potential advantages: because of the strong interaction between the enzyme and carrier, the enzyme does not leak or detach from the carrier in the process of various applications; the stability of the enzyme towards pH, temperature, and organic solvents are increased markedly; at the same time, the immobilized enzyme can easily make contact with substrate because it is localized on the surface of carrier (Varavinit et al. 2001; Kumar et al. 2013).

Cellulose, which is composed of a linear chain of β-1,4-linked D-glucose units, is the most abundant natural polysaccharide in the world. In the search for suitable materials for enzyme immobilization, cellulosic materials have been found to be among the top candidates due to their hydrophility, reproducibility, biocompatibility, biodegradability, and processability (Bryjak et al.2007; Petronijević et al. 2007; Wu and Lia 2008; Villalonga et al. 2008; Huang et al. 2011; Labuset al. 2011). Loofah sponge is an inexpensive, biocompatible, hydrophilic cellulosic material and has been used as a carrier for the immobilization of various biological cells (Ahmadi et al. 2006; Iqbal and Saeed 2007; Saudagar et al. 2008; Meleigy and Khalaf 2009; Pazzetto et al. 2011). But so far there has been no report on the immobilization of enzyme on loofah sponge.

In this paper, loofah sponge was activated by thermochemical esterification between the carboxyl groups of citric acid and hydroxyl groups of cellulose in loofah sponge. Lipase (EC 3.1.1.3), an important biocatalyst in food processing, was covalently immobilized to the activated loofah sponge using carbodiimide as the coupling agent to attach carboxyl groups of the esterified surface of loofah sponge to the amino groups of lipase. The enzymatic characteristics, stabilities, and reusability of immobilized lipase were investigated and compared with free lipase.

Experimental

Materials

Loofah sponge, supplied by a local farm, was cut into discs of about 1 cm height and 7 cm diameter. The loofah sponge discs were soaked in boiling water for 30 min, washed thoroughly with tap water to remove impurities, left in distilled water for 24 h, dried overnight at 50 °C, and then stored in a desiccator. Porcine pancreatic lipase, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma (St. Louis, MO, USA). p-Nitrophenyl palmitate (p-NPP) was obtained from Fluka Chemical Corp. (Ronkonkoma, NY, USA). All other chemicals used were of analytical grade from several trademarks.

Activation of Loofah Sponge

The activation of loofah sponge was carried out according to the following method: Loofah sponge discs were mixed with 0.2 M citric acid (CA) at the ratio of 1:50 (disc:acid, w/v) and stirred for 30 min. The mixture was dried at 50 °C for 24 h, followed by performing the optimal thermochemical esterification between citric acid and cellulose at 100 °C for 60 min (Gong et al.2008). The activated loofah sponge was extensively washed with distilled water to remove unreacted citric acid and recovered as the carrier for lipase immobilization. The esterified loofah sponge with different carboxyl group density on it was prepared by changing the conditions of the thermochemical esterification (temperature and time).

Immobilization of Lipase on Esterified Loofah Sponge

Lipase was immobilized onto esterified loofah sponge by EDC/NHS-mediated amide formation between carboxyl groups of esterified loofah sponge and amino groups of lipase. Briefly, 0.1 g of esterified loofah sponge was immersed in 6 mL of phosphate buffer solution (PBS: 50 mM, pH 5.5) containing 10 mg of EDC and 6 mg of NHS. After stirring gently at room temperature for 1 h, the activated loofah sponge was collected, washed several times with the above PBS, and submerged in 6 mL of enzyme solution (enzyme activity of 5.2 U/mg; 1.0 mg/mL in the above PBS). The immobilization process was carried out at room temperature for 4 h with gentle shaking. Finally, the immobilized lipase was recovered and the unbound lipase was removed by washing with the above PBS and then rinsed with distilled water until no enzymatic activity was detected in the supernatant. The supernatant and washing solutions were pooled for assaying the residual enzyme activity and protein concentration. A reaction scheme for the preparation of the esterified loofah sponge and immobilized lipase is shown in Fig. 1.

Fig. 1. Schematic illustration of the preparation of the esterified loofah sponge and immobilized lipase

Determination of the Immobilized Protein Amount

Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as the standard protein. The amount of bound protein was indirectly calculated from the difference between the amount of protein used for immobilization and the net amount of protein present in the supernatant and washing solutions after immobilization. The immobilization efficiency was defined as the ratio of the total amount of the immobilized protein to the total amount of the used protein.

Measurement of Lipase Activity

The activities of free and immobilized lipases were analyzed spectrophotometric-ally by measuring the increment in the light absorbance at 410 nm caused by the release of p-nitrophenol because of the hydrolysis of p-NPP (Yi et al. 2009). The reaction mixture, which was incubated at 37 °C, consisted of 7.7 mL of PBS (50 mM, pH 7.5) containing 0.4% (w/v) Triton X-100, 0.1% (w/v) gum arabic, and 0.1 mL of free lipase solution (1.0 mg/mL), or 0.1 mL of the above PBS as well as 10 mg of immobilized lipase. The reaction was initiated by adding 0.2 mL of p-NPP substrate solution (40 mM) prepared with 2-propanol followed by incubation for 5 min at 37 °C, and then the reaction was terminated by adding 4 mL of Na2CO(0.1 M). The resultant mixture was centrifuged to obtain supernatant. The amount of released p-nitrophenol in the super-natant was measured by a UV spectrophotometer (Shimadzu UV-1201, Japan) at 410 nm. The lipase activity was calculated from the standard calibration curve of p-nitrophenol. One unit (U) of lipase activity was defined as the amount of enzyme necessary to liberate 1 µM p-nitrophenol per minute under described experimental conditions. The retention activity was defined as the ratio of the activity of the immobilized enzyme to the activity of the same amount of free enzyme.

Effect of pH and Temperature on Lipase Activity

The effect of buffer pH on the free and immobilized lipase activities was estim-ated for reaction mixtures containing 50 mM of the PBS or Gly-NaOH buffer at different pH values (PBS: 6.0 to 8.0; Gly-NaOH: 8.5 to 9.5) at 37 °C. The effect of temperature on both lipase activities was determined at different temperatures in the range of 20 to 50 °C in 50 mM of PBS (pH 7.5) under the same assay conditions.

Stability Detection of Lipases

For determination of thermal stability, both free and immobilized lipases were incubated in substrate-free PBS (50 mM, pH 7.5) at 60 °C for 3 h. The remaining activities of the enzymes were periodically measured according to the above-described method. For the investigation of storage stability, both free and immobilized lipases were stored in a refrigerator at 4 °C for 30 days. The remaining activities of samples were also measured periodically.

Reusability Determination of Immobilized Lipase

The reusability of the immobilized lipase was evaluated in a repeated catalysis process. After each reaction, the immobilized lipase was removed and thoroughly washed with 1% Triton X-100 and 10% 2-propanol in PBS (50 mM, pH 7.5) for the next use. It was then reintroduced into a fresh reaction medium. The residual activity was determined as described above and expressed as a percentage of its residual activity in comparison with the initial activity.

Results and discussion

FTIR Spectra of Loofah Sponge and Esterified Loofah Sponge

The FTIR spectra of loofah sponge and esterified loofah sponge are shown in Fig. 2. Compared with loofah sponge, the characteristic stretching vibration absorption band of carbonyl groups at 1739 cm−1 increased greatly in the FTIR spectrum of esterified loofah sponge. This result demonstrated that the citric acid had been introduced into loofah sponge.

Fig. 2. FTIR spectra of (a) loofah sponge and (b) esterified loofah sponge

Optimal Condition for Lipase Immobilization

To optimize the immobilization conditions of lipase, 0.1 g of esterified loofah sponge with a carboxyl group density of 0.88 mM/g on it was immersed in 6 mL of PBS (50 mM). Then the effect of the lipase amount (6 mL × 0.2 to 2.6 mg/mL), pH value (3.0 to 8.0), EDC amount (2 to 40 mg), and immobilization time (0.5 to 12 h) on the activity of the immobilized lipase was investigated. The optimal conditions for lipase immobilize-ation were established as follows: 6 mg of lipase (6 mL × 1.0 mg/mL), pH 5.5, 10 mg of EDC, and 4 h of immobilization time. Under these conditions, 0.1 g of esterified loofah sponge with different carboxyl group density (0.24 to 1.42 mM/g) on it was used for lipase immobilization. The highest activity of immobilized lipase per gram of dry carrier with carboxyl group density (1.15 mM/g) was found to be 45.8 U/g (Table 1).

Table 1. Influence of Carboxyl Group Density on Lipase Immobilization

Properties of Immobilized Lipase

The influence of pH on the activities of free and immobilized lipases is illustrated in Fig. 3. It was observed that the optimal pH for free lipase was close to 7.5 but was approximately 8.0 for immobilized lipase, shifting 0.5 units towards the alkaline region. This shift in the optimal pH often depends on the immobilization method as well as the structure and charge of the carrier (Costa et al. 2001). Generally, when the enzyme is coupled with a polyanionic support, the optimal pH usually shifts in the alkaline direction (Yi et al. 2009), whereas if the support is polycationic the shift is in the acidic direction. The loofah sponge–immobilized lipase, possibly due to the carboxyl groups introduced by esterification, significantly altered the microenvironment of the enzyme. Furthermore, the immobilized lipase retained a relatively higher activity in the alkaline pH range than the free lipase did, which suggested that the stability of the immobilized lipase was higher than that of the free lipase at the alkaline pH range.

Fig. 3. Influence of pH on the activity of free and immobilized lipases

The effect of temperature on the activities of free and immobilized lipases is shown in Fig. 4. It was found that the optimal temperature of the immobilized lipase was 40 °C, which was 3 °C higher than that of the free lipase. This result was consistent with the literature (Singh et al. 2011); the immobilized lipase often exhibits a higher optimal temperature than its free counterpart. The multipoint covalent attachment of the lipase on the esterified loofah sponge by amide linkage would tend to maintain a more rigid conformation of the enzyme molecule. Thus, the immobilized lipase was much more stable than its free counterpart at higher temperature. In addition, the immobilized lipase retained a relatively higher activity in the high temperature range than the free lipase did.

Fig. 4. Effect of temperature on the activity of free and immobilized lipases

Stabilities of the Free and Immobilized Lipase

The results for the thermal stabilities of the free and immobilized lipases are presented in Fig. 5.

Fig. 5. Thermal stabilities of free and immobilized lipases

The immobilized lipase exhibited smaller thermal inactivation than the free lipase at all time periods. The free lipase lost more than 90% of its initial activity after heat treatment for 3 h at 60 °C, while the immobilized lipase showed significant resistance to thermal inactivation and kept more than 60% of its initial activity under the same conditions. The immobilization of the enzyme to a carrier could prevent the conformation transition of the enzyme at high temperature, thus resulting in increasing the thermal stability of the enzyme towards heat inactivation.

Results for storage stabilities of free and immobilized lipases are shown in Fig. 6. The free lipase lost almost all of its initial activity within 10 days, whereas the immobile-ized lipase retained more than 73% of its activity, and it still preserved more than 50% of its initial activity after the 30 days of the storage period. This result indicated that the immobilized lipase exhibited better storage stability than free lipase. In general, the enzyme is unstable during storage in solution and its activity decreases gradually over time. This decrease in enzyme activity can be explained as a time-dependent natural loss of enzyme activity. It can be prevented to a significant degree by immobilization.

Fig. 6. Storage stabilities of free and immobilized lipases

Fig. 7. Reusability of the immobilized lipase

Reusability of the Immobilized Lipase

The reusability of immobilized enzyme is important for industrial use of enzyme. The main advantage of immobilized enzyme is the easy separation and reusability. The effect of repeated use on activity of immobilized lipase is shown in Fig. 7. The activity of immobilized lipase dropped smoothly during the continuous operation process, and the immobilized lipase retained more than 53% of its initial activity after nine cycles of reuse. The loss of activity for immobilized enzyme with repeated use is a common phenomenon (Arasaratnam et al. 2000), and it can be attributed to the inactivation of the enzyme caused by denaturation.

Conclusions

  1. Cellulose in loofah sponge was thermochemically esterified by citric acid to introduce free carboxyl groups. The esterified loofah sponge then was employed as a carrier for the covalent immobilization of lipase. Loofah sponge–immobilized lipase was successfully obtained by EDC/NHS-mediated amide formation between carboxyl groups of esterified loofah sponge and amino groups of lipase. Loofah sponge–immobilized lipase exhibited excellent thermal stability, storage stability, and reusability.
  2. The lipase immobilized on loofah sponge by amide linkage offers a cheap immobilized biocatalyst suitable for future industrial applications.

Acknowledgements

This work was financially supported by the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University (No. SKLF-KF-201101), the Key Laboratory of Bioresource Protection and Utilization of Anhui Province, the Key Laboratory of Biotic Environment and Ecological Safety of Anhui Province, and the Program for Innovative Research Team at Anhui Normal University.

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Article submitted: March 1, 2013; Peer review completed: April 25, 2013; Revised version received and accepted: May 7, 2013; Published: May 8, 2013.