The study on effective immobilization of lipase on functionalized bentonites and their properties

https://doi.org/10.1016/j.molcatb.2013.05.018Get rights and content

Highlights

  • The immobilized lipase on composite bentonite shows the highest activity.

  • Hydrophobicity and enlarged catalytic interface of the supports activate lipase.

  • The immobilized lipase shows excellent storage and operational stabilities.

  • The lower Km of immobilized lipase confirmed the improved affinity for olive oil.

Abstract

Three different functionalized bentonites including acid activated bentonite (Ba), organically modified bentonite with cetyltrimethyl ammonium bromide (BCTMAB) and the composite by acid activation and organo-modification (Ba-CTMAB) were prepared, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. The amount of lipase adsorbed on the functionalized bentonites was in the following sequence: Ba > BCTMAB > Ba-CTMAB, showing the strongest affinity of Ba for lipase among the three supports. However, the immobilized lipase on Ba-CTMAB showed the highest activity in the hydrolysis of olive oil by 1.67 times of activity of free lipase due to the hydrophobically interfacial activation and enlarged catalytic interface. While, the activity of immobilized lipase on Ba was lower than 20% of free lipase’s activity due to the absence of hydrophobic activation and negative impact of excessive hydrogen ions on the surface. The Km values for the immobilized lipase on Ba-CTMAB (0.054 g/mL) and BCTMAB (0.074 g/mL) were both lower than that of free lipase (0.115 g/mL), and the Vmax values were higher for the immobilized lipases, exhibiting a higher affinity of the immobilized lipase toward olive oil than free lipase. In comparison to free lipase, the better resistance to heating inactivation, storage stability and reusability of the immobilized lipases on Ba-CTMAB and BCTMAB were also obtained. The results show that the efficient and stable biocatalysts for industrial application can be prepared by using the low-cost bentonite mineral as the supports.

Introduction

Enzyme-mediated reactions always have been gained an enormous attention depending on their higher catalytic efficiency and specificity under milder conditions in comparison with chemical catalysts [1]. Lipase is one class of the important enzymes with practical significance, and widely applied in a variety of industries, such as food, fine chemistry and pharmaceutical industries [2], [3]. However, their widespread application is often hampered by low catalytic and operational stabilities. Among the methods for stabilization of enzymatic activity [4], [5], [6], [7], the immobilization of lipase has been extensively studied because it also can provide the following crucial advantages for the continuous fabrication on a large-scale: reutilization of lipase, facilitating the separation and preparation of aim products, enhancing the lipase’s stability under changeable and extreme conditions [7], [8], [9]. Of the main immobilization methods including adsorption, entrapment, covalent bonding and cross-linking [10], [11], [12], physical adsorption is universally acknowledged as a simpler one without introducing redundant and toxic reagents [13], [14]. More importantly, physical adsorption shows less negative affect on the conformation of lipase, and a high catalytic activity is always obtained [13], [15], [16]. Numerous supports for the immobilization of lipases from different sources by adsorption have been investigated [15], [16], [17]. Comparative studies have indicated that the marked differences in the activities of immobilized lipases on different supports are observed [15], [16], [17], [18]. The catalytic performance of immobilized enzyme can be affected by the support through the following ways: diffusion of substrate and product, the conformation of enzyme and the interaction between the support and the reaction medium [19], [20].

Nowadays, relying on the advantages of mechanical stability, high adsorption and availability of reactive functional groups, various inorganic support materials are more and more popular in the application of enzyme immobilization [3], [16], [18], [19], [20], [21]. In the past decade, functionalizations of inorganic materials for lipase immobilization have been frequently performed by different reactive compounds with unique properties [13], [15], [16], [20]. Some studies have showed that preparation of composite supports with hydrophilicity–hydrophobicity balanced surfaces could adjust the structures of supports to be appropriate for certain biocatalytic applications [14], [18], [22], and promote the “interfacial activation” by shifting the lipase’s active center from closed form (inactive form) toward open form (active form) [15], [16], [23]. Furthermore, a hydrophilicity/hydrophobicity balanced support with high specific surface can enlarge the effectively catalytic area of immobilized lipase, improve the affinity between hydrophobic substrate and lipase’s active center, and decrease the diffusion resistance of substrate [16]. Therefore, preparation of suitable supports for enzyme immobilization has become an important subject.

Bentonite, a layered material constituting primarily of montmorillonite, has the potentially interesting properties including high specific surface, thermal stability, natural abundance, chemical and bacterial resistance, and electrostatic interactions [24], [25]. The original bentonites are easily obtainable and cheaper than other inorganic materials such as carbon nanotubes and nano silica. Moreover, the presence of silanol groups provides the active sites for structural and functional adjustment to tailor-make suitable supports for enzyme immobilization [13], [14], [26]. The original bentonites have been applied to support the enzymes to improve the reusability and thermal stability of lipase and α-amylase [24], [27]. However, their activities were observed to decrease markedly mainly resulting from the absence of hydrophobic interfacial activation for lipase, the block of the active sites, and low affinity to hydrophobic substrates [16], [28]. Ghiaci et al. prepared the organo-bentonites for enzyme immobilization, showing better biocompatibility with enzymes than original bentonites [29]. However, the catalytic activities of immobilized lipases were still low, possibly due to the absence of effectively adjusting the balance of hydrophilicity/hydrophobicity, surface property and structure of bentonite for the catalytic performance of guest lipase. To the best of our knowledge, there has been no report concerning the application of functionalized bentonites by acid activation, organo-modification and combination of both methods in the immobilization of lipase by adsorption, for the purpose of its catalytic performance enhancement.

In this study, the different functionalized bentonites were prepared including organo-bentonite, acid activated bentonite and the composite bentonite by acid activation and organo-modification, and used for immobilization of lipase from bovine pancreatic lipase by adsorption. Olive oil was used as a model substrate to investigate the catalytic activities of the free and immobilized lipases. The adsorptions of lipases on these prepared supports were investigated to evaluate and compare the affinity between lipase and the selected supports. The catalytic efficiencies of the immobilized lipases were compared to get the truth how the organobentonite and composite affected the activity of lipase. The catalytic kinetics of the free and immobilized lipases were investigated, and their reusability and storage stability were also studied.

Section snippets

Materials

The bovine pancreatic lipase (15–35 U mg−1), olive oil and polyvinyl alcohol were purchased from Aladdin Chemicals, Shanghai, China. Bovine serum albumin and cetyltrimethyl ammonium bromide (CTMAB) were obtained from Aldrich. All other chemicals were of analytical grade. The original bentonite (denoted Na-bentonite) consisting primarily of montmorillonite was purchased from Inner Mongolia, China. The cationic exchange capacity (CEC) of dried Na-bentonite (NB) was determined to be 1.15 mmol g−1 by

Characterization of modified bentonites

The structures of original bentonite, Ba, BCTMAB and Ba-CTMAB were characterized by XRD and FTIR. XRD is a powerful tool to understand the changes in the interior of the support. As shown in Fig. 1a, the d0 0 1-spacing of original bentonite was calculated to be 1.23 nm, and the d0 0 1 values of Ba, BCTMAB and Ba-CTMAB increased to be 1.51, 1.62 and 1.78 nm, respectively. This result showed that acid treatment and the intercalated surfactant ions enlarged the interlayer space of bentonite, and more

Conclusion

Three different functionalized bentonites were prepared and used to support the lipase. The immobilized lipases including Ba-CTMAB-lipase and BCTMAB-lipase showed the improved activity, thermal and storage stabilities than free lipase, exhibiting an improved biocompatibility of hydrophobically functionalized bentonite with lipase due to the interfacial activation. The lower Km and higher Vmax for the immobilized lipases than that of free lipase also confirmed the improved catalytic efficiency.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (20977063), the Natural Science Foundation of Zhejiang Province, China (LY12B06003), and the Science and Technology Program of Shaoxing (2011A21056).

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