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Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity
Colloids and Surfaces B: Biointerfaces 133 (2015) 51–57
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity Caixia Cui, Yifeng Tao, Chunling Ge, Yueju Zhen, Biqiang Chen ∗ , Tianwei Tan ∗ National Energy R&D Center for Biorefinery, Beijing Key Laboratory of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 15 May 2015 Accepted 26 May 2015 Available online 4 June 2015 Keywords: Lipase Immobilization Biocompatibility Hydrophobic Ionic Balance
a b s t r a c t We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA ) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420 U/g support) and the biocompatible gelatin modified support (600 U/g support) when compared to the support without modification (240 U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180 U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lipases are a group of enzymes that have great applications in food, pharmaceutical, cosmetic, and chemical industries [1–4]. However, lipase-based catalysts are usually expensive, unstable, and are difficult to be efficiently separated from reaction systems used in industrial production [5]. To address this, various immobilization techniques have been developed to improve their stabilization, reutilization [6,7] and other properties [8–10]. For example, the enzyme aggregation reduced in the immobilization processes compared to free enzyme and generated favorable environment [11,12]. Multipoint covalent attachment of enzyme on highly activated pre-existing supports via short spacer arms involving many residues placed on the enzyme surface promotes rigidification of structure of the immobilized enzyme [13]. For some enzymes with several subunits, the immobilization process may protect the stabilization and prevent subunit dissociation [14]. However, a simple protocol for effective immobilization system is not easily accomplished because small changes in variables such
∗ Corresponding author. Tel.: +86 10 64416691; fax: +86 10 64416691. E-mail addresses: [email protected] (B. Chen), [email protected] (T. Tan). http://dx.doi.org/10.1016/j.colsurfb.2015.05.045 0927-7765/© 2015 Elsevier B.V. All rights reserved.
as nature and characteristics of the immobilization support as well as age in vs coupling chemistry can have a significant impact on biocatalyst activity and stability [15,16]. Therefore, it is very important to select a suitable support and design proper modification protocols, especially to create the appropriate micro-environment for enzymes in the supports, and it is still challenging to develop optimized immobilization techniques. Currently, there are four main immobilization techniques: covalent binding, adsorption, cross-linking, and entrapment [5]. Importantly, immobilized lipases can become more active than their native form. This is due to the fact that the lipase can exist in two forms: an open, active form in which the lid displaces and allows substrate access to the catalytic site and a closed, inactive form in which the lid shuts, thereby hiding the catalytic site [17–19]. In aqueous media, the lid exists in a closed conformation. However, in the presence of an insoluble substrate, the lipase becomes active by interfacial activation, shifting lid conformation from a closed to an open state [17]. Lipase becomes more active if the interactions between them and their support stabilize their open form which generates a higher activity [20]. Immobilization techniques utilizing hydrophobic matrices have attracted much attention, since the interaction between the lipase and its hydrophobic support is similar to the phenomenon observed with insoluble substrates [21]. Moreover, the accessibility of
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the substrate to the active site of the lipase lid was promoted by employing hydrophobic support [22]. Lipase immobilized on hydrophobic supports involves its open form of the lipase, and the hydrophobic support makes lipase more stable [23]. Thus far, there are two strategies to make hydrophobic supports. One strategy is that the support itself may be reagent (e.g. acccurel, divinylbencene, etc.) [24,25]. The other is to decorate the support with a hydrophobic reagent [26]. For instance, Jin et al. [27] modified magnetic siliceous mesocellular foam (MCF) with organosilane to improve the support surface’s hydrophobicity. They found that lipase derived from Pseudomonas cepacia (PCL) that was immobilized on a support of hydrophobic MCF-Ph obtained the highest activity, which was eight times that of native MCF. Taken together, results obtained from time-resolved fluorescence catalytic activity experiments showed that the hydrophobic activation drove the improvement in catalytic activity [16]. However, the excessive support resulting from this hydrophobic interaction can destroy the structure of the enzyme, leading to rapid denaturation and a sharp decrease in the activity of surface-bond enzymes [28]. Ultimately, this excess would decrease the enzyme properties [25]. When compared with hydrophobic supports, lipase immobilized on hydrophilic supports can acquire a higher efficiency of immobilization. When a lipase which was immobilized on a hydrophilic support was used in organic solution, water was able to concentrate around the lipase, thus promoting the catalysis of the reaction [29,30]. In addition, the process of tethering enzymes to carriers could cause inevitable conformational changes of the enzymes, resulting in decreased bioactivities of enzymes [31]. To overcome this limitation, researchers have adopted many methods, one of which is to modify the support surface with biofriendly molecules to obtain the biocompatible surface to reduce the enzyme denaturation during the immobilization [32–34]. There are many methods to form such biocompatible (“bio-friendly”) support surfaces, including coating, adsorption, and self-assembly [35,36]. Among these methods, modifications to support structures, such as natural, molecular-like gelatin, collagen, alginate, or biotin on the support have often been used in tissue engineering due to their non-toxic nature [37]. In this work, the support PGMA-TAIC-EGDMA with epoxy group was used as the immobilized support while the epoxy group could be modified by thiols, amino, hydroxyl and carboxyl groups easily, and the final bonds are very stable. However, the epoxy group exhibits a relative low reactivity [13]. Thus, many support modifications have been researched [38–40]. Although there are many hydrophobic and biocompatible modifications for supports, most have only used hydrophobic or bio-friendly reagents to regulate the support environment. In our study, different types of amines and proteins were introduced to investigate their respective activities in our first work. Lipase could be immobilized on support by different modifications such as ionic exchange, hydrophobic adsorption, covalent binding, and biocompatible adsorption. Here we present work in which we prepared an amine and protein immobilized matrix in order to regulate the properties of the support surface. The ratios of gelatin to BA, the concentrations of BA/gelatin and the sequences of BA and gelatin modified support were studied, and the synergistic effects of the hydrophobic, ionic and biocompatible modifications on the activity of the immobilized lipase were analyzed.
Fig. 1. SDS-PAGE of Yarrowia lipolytica lipase lip2.
albumin, BA, methylamine, and n-octylamine were purchased from Beijing Chemical Factory (Beijing, China). All of the experimental reagents and solvents used were of analytical grade. 2.2. The preparation of lipase solution The fermentation of YLIP2 lipase in our laboratory was carried out in a 5 L reactor at 26 ◦ C with stirring at 500 rpm and with an air input of 1 VVM (air volume/culture volume/min). The culture medium was composed of 4% (wt.) soybean powder, 4% (wt.) soybean oil, 0.1% (wt.) KH2 PO4 and 0.1% (wt.) (NH4 )2 SO4 . The lipase produced reached 8000 U/ml after 96 h fermentation. The cells were removed by centrifugation, and the lipase in the supernatant was precipitated by addition of three volumes of acetone. The precipitate was washed with acetone and dried at room temperature. The activity of the lipase powder is 67000 U/g. The lipase powder was dissolved into distilled water getting the lipase solution (10 mg/ml). The purity of YLIP2 lipase is higher than 90%, which was confirmed by SDS-PAGE via Quantity-One (Fig. 1).
2. Materials and methods 2.3. Electrophoresis 2.1. Materials Both the Yarrowia lipolytica lipase YLIP2 lipase and the epoxy support PGMA-TAIC-EGDMA were previously produced in our laboratory [41,42]. Gelatin, fibrion, casein, ovalbumin, bovine serum
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on protein using a Mini Protein Electrophoresis Cell (Bio-Rad Laboratories, California, USA) and a 12% acrylamide separating and a 5% acrylamide stacking. Protein
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solutions (1 mg/ml) were dissolved 1:3 with sample buffer solution (4% SDS, 20% glycerol, 10% -mercaptoethanol, 0.125 M Tris, pH 6.8). After heating in boiling water for 3 min, a 10 L of protein per lane were loaded onto the gel and electrophoresed in a Mini-PROTEAN 3 apparatus (Bio-Rad Laboratories, Hercules, CA). A mixture of molecular weight standards (10–170 kDa) was also run along with the samples. After electrophoretic separation, the gels were stained with a solution of Coomassie Brilliant Blue containing trichloroacetic acid for fixation and destained with acetic acid. 2.4. Procedure of lipase immobilization
and 200 rpm for 6 h. The modified support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on different BA/gelatin supports through the same procedures described in Section 2.4.1. The immobilization yield was defined as YI% = Uact /Uads × 100, where Uads are the absorbed units evaluated as the difference between initial activity and the remaining activity in the supernatant at the end of the adsorption procedure; Uact is the activity present on the support [43]. The expressed activity was the same as the activity of the immobilized lipase.
2.4.1. Hydrophobic adsorption followed by covalent attachment on epoxy-support The native epoxy-support (1 g) was immersed in 5 ml of lipase solution and agitated at 15 ◦ C and 200 rpm for 2 h. The immobilized lipase was washed several times with distilled water to remove any unbound lipase and then recovered by filtration. The immobilized lipase was stored at 4 ◦ C until later use.
2.5. Protein determination
2.4.2. Lipase immobilized on protein or/and amine modified support 2.4.2.1. Adsorption on protein modified support. The protein modifications were conducted as follows: the native epoxy-support (1 g) was immersed in 5 ml of 1% (w/v) different kinds of protein solution: gelatin, fibrion, casein, ovalbumin or bovine serum albumin solution and agitated at 60 ◦ C and 200 rpm for 6 h, respectively. After modification, the support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on protein-support through the same procedures described in Section 2.4.1.
2.6. BA determination
2.4.2.2. Ionic exchange or/and hydrophobic adsorption on amine modified support. The amine modifications were conducted as follows: the native epoxy-support (1 g) was immersed in 5 ml of 1% (w/v) different kinds of amine solution: BA, methylamine, or noctylamine solution and agitated at 60 ◦ C and 200 rpm for 6 h, respectively. After modification, the support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on amine-support through the same procedures described in Section 2.4.1. 2.4.2.3. Synergistic effect of BA and gelatin modifications. The gelatin and BA modifications were conducted as follows: The ratios of BA to gelatin (w/w) used in modification were as follows: the native epoxy-support (1 g) was modified by 5 ml 1% (w/v) BA and gelatin solution with one of three different ratios of gelatin:BA (2:1, 1:1 and 1:2) before being agitated at 60 ◦ C and 200 rpm for 6 h. The concentrations of BA and gelatin (BA/gelatin) used in modification were as follows: the native epoxy-support (1 g) was immersed in 5 ml of BA and gelatin solution at one of the following concentrations: 0%, 0.5%, 1%, 2%, 5%, 10% or 20% (w/v) and agitated at 60 ◦ C and 200 rpm for 6 h. The sequences of BA and gelatin used in modifications were as follows: (i) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) gelatin solution and agitated at 60 ◦ C and 200 rpm for 6 h, and the support was washed three times with distilled water to remove the residual gelatin solution, and then incubated in 5 ml 2% (w/v) BA solution; (ii) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) BA solution and agitated at 60 ◦ C and 200 rpm for 6 h, and then washed three times with distilled water to remove the residual BA solution before being incubated in 5 ml 2% (w/v) gelatin solution; (iii) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) BA/gelatin (1:1) solution and agitated at 60 ◦ C
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The amount of gelatin immobilized on epoxy-support and lipase immobilized on the support structure was determined via Bradford protein assay by measuring the initial and final concentrations of protein in the modified solution and immobilization lipase medium [44].
The amount of BA linked on the epoxy-support was quantified via gas chromatography (GC) by measuring the initial and final concentrations of BA in the modified solution. GC analysis was performed using GC-2010 (Shimadzu Japan) equipped with a DB-FFAP capillary column (30.0 m × 0.32 mm × 0.5 m; J&W Scientific, USA) and a flame ionizing detector. The injector temperature was 200 ◦ C. The column oven temperature was 50 ◦ C, and then increased to 200 ◦ C at a heating rate of 10 ◦ C/min and maintained for 10 min. The detector temperature was 220 ◦ C. 2.7. Activity of free and immobilized lipases The activity of free and immobilized lipases was measured via olive oil hydrolysis according to a previously published report [45]. Briefly, an olive oil emulsion (200 ml) was prepared by mixing olive oil (50 ml) and 2% PVA (150 ml) under vigorous stirring for 6 min. 5 ml of this emulsion and 4 ml 0.1 M phosphate buffer (pH 8.0) were then warmed for 5 min at 35 ◦ C. Either the free or immobilized lipase was added to the mixture, incubated at 35 ◦ C and agitated at 100 rpm for 5 min in a water bath shaker. The reaction was then terminated by addition of 15 ml ethanol. We then determined the amount of released fatty acids by titration with 50 mM NaOH solution. Following convention, one unit of lipase hydrolytic activity was defined as the release of 1 m fatty acids per min at 35 ◦ C. All the results shown were the averages of experiments conducted in triplicate. 3. Results and discussion 3.1. Effect of amine modifications of support structure on the activity of immobilized lipase A critical issue in lipase immobilization is the support structure. In particular, the hydrophobic character of the support surface is imperative, as it can have a large effect on enzyme immobilization [46–48]. The PGMA-TAIC-EGDMA support itself is hydrophobic in some extent due to the usage of hydrophobic reagents in the synthesized processes. Lipase was immobilized on the native epoxy-support under neutral pH values and low ionic strength, via interfacial activation on the support [49]. In this work, in order to adjust the property of the support more reasonable, different chain lengths of amine were used to modify the support. As shown in Fig. 2, amine modification could significantly affect the activity of immobilized YLIP2. The activity of immobilized lipase on methylamine-modified
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Fig. 2. Lipase immobilized on support with different hydrophobic modifications.
support (340 U/g support), BA-modified support (450 U/g support) and n-octylamine-modified support (280 U/g support) was 1.4-, 1.8- and 1.2-fold greater than that on the native support (240 U/g support), respectively, which suggesting that the amines could contribution to improving the lipase immobilization by regulating the support surface property. However, the main cause of immobilization by amines might be different. When using methylamine, they have a secondary amino group below the short alkane chain, the main immobilization cause was anionic exchange (secondary amino). When the carbon chain of amine increased to eight (octylamine), they have steric hindrances for the enzyme ion exchange, and a hydrophobic layer that can produce the enzyme immobilization via interfacial activation. When using butyl-amine, perhaps they have something in between, after interfacial activation the enzyme may become ionically exchanged. Thus, the length of the amines has a great influence on immobilization. Such results reinforce past work that has shown that the length of the carbon chain can significantly affect the activity of the immobilized lipase [50]. To this end, Miletic´ et al. [50] reported that the lipase activity dependeded on support modifications, including supports with different lengths of hydrophobic carbon chains (e.g. 1,2-diaminoethane, 1,4diaminobutane, 1,6-diaminohexane, and 1,8-diaminooctane). They showed that lipase immobilized on resin 3 obtained the highest activity with a 1,4-diaminobutane modification, while lipase immobilized on resin 6 obtained the highest activity with a 1,2diaminoethane modification. BA modification could regulate the support surface to be more appropriate via the combination of hydrophobic adsorption and ionic exchange for YLIP2 immobilization. They also demonstrated that the proper modification for lipase immobilization was different for different support structures; an excess of hydrophobicity within the immobilized matrix would destroy the enzymatic structure and partially reduce its overall surface biocompatibility [51] and the balance of ionic and hydrophobic could improve the property in some extent. 3.2. Effect of biocompatible modifications of support structures on the activity of immobilized lipase A hydrophobic interface does not always produce higher activity, as excessive hydrophobicity will destroy enzymatic structure [28]. A protein that has good biocompatibility is usually used in the immobilization process via adsorption to regulate the properties of the support surface [27]. As such, the protein used during the immobilization processes has attracted much interest [52]. To this end, Fig. 3 presents our data relevant to YLIP2 immobilization onto
Fig. 3. Lipase immobilized on support with different protein modifications.
support structures with or without protein modification. We found that the activity of lipase immobilized on support with ovalbumin, casin, BSA, gelatin, and fibroin modifications was 2.1-, 1.1-, 1.6-, 2.5, and 2-fold greater, respectively, when compared to that on native support. Our results demonstrated that the activity of lipase immobilized on different protein-modified supports was improved by different degrees. When the epoxy support immobilized lipase by covalent binding, the exited protein layer could protect the lipase against inactivation by the covalent reaction. For instance, lipase immobilized on a gelatin-modified support obtained the highest activity (600 U/g support), which was 2.5-fold greater when compared to that on an unmodified support structure. At present, we can only speculate on the mechanism behind such results. For one, it is possible that the biocompatible gelatin layer on the support surface created a good micro-environment for the lipase, which has been shown to result in a retention of the high activity of the immobilized enzyme [53]. Secondly, gelatin derived from collagen is composed of polypeptides of various sizes, which possess a molecular weight distribution in the range of 15,000–250,000 Da [54]. It also contains free groups (e.g. amino acid) that can immobilize the lipase, while the non-polar amino acid chain of gelatin can further immobilize the lipase via an adsorption interaction [54]. Finally, it is possible that as a protein, gelatin could protect lipase against inactivation, since additional protein has been shown to improve enzymatic activity and stability [55]. 3.3. Effect of BA and gelatin modifications of support structures on the activity of immobilized lipase All of the techniques used in this study were advantageous to improving the immobilized lipase properties with either BA or gelatin modification. Taking into account these two categories, BA and gelatin were used to decorate the support structure in order to obtain a support with simultaneous hydrophobic, ionic and biocompatible characteristics (Scheme 1). As shown in Fig. 4, it is evident that the activity of the immobilized lipase on BA/gelatinsupport (850 U/g support) was higher than that either on the BA-support (450 U/g support) or on the gelatin-support (600 U/g support) alone. Furthermore, this effect was far greater than that on an unmodified support (240 U/g support). Since the protein attachment of these immobilized lipases was almost identical (10.4 mg protein/g support) (Table 1), these results suggest that protein attachment is not a critical factor influencing increases in lipase activity. The specific activity of lipase immobilized on BA/gelatin-support (81.7 U/mg protein), which is higher than
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Scheme 1. Lipase immobilized on support with different modifications.
Table 1 Free lipase and lipase immobilization on support modified with BA, gelatin and BA/gelatin: protein attachment, hydrolytic activity, specific activity and immobilization yield. Different lipases
Activity (U/g support or lipase)
Protein loading (mg/g support)
Specific activity (U/mg protein)
Immobilization yield (%)
Free lipase Lipase immobilized on support directly Lipase immobilized on BA-support Lipase immobilized on gelatin-support Lipase immobilized on BA/gelatin (1:1)-support
67,000 240 450 600 850
– 8.2 8.9 9.4 10.4
67 29.3 50.6 63.8 81.7
– 48.6 70.6 90.3 125.4
that of the free lipase (67 U/mg protein), is higher than that on gelatin-support (63.8 U/mg protein) and BA-support (50.6 U/mg protein), and is much higher than that on native epoxy-support (29.3 U/mg protein). Considering the hydrophobic nature of the native epoxy-support, the interfacial activation of lipase on the native support could be achieved and thus the immobilized preparations get hyperactivated [56]. However, the specific activity of lipase immobilized on native epoxy-support was lower than that of the free lipase. It indicated that lipase was inactivated in the processes of immobilizing on native support in some extent. The improved specific activity of lipase immobilized on BA,
Fig. 4. Effect of BA and gelatin modification on activity.
gelatin and BA/gelatin modified supports proved that the modifications could improve the micro-environment of the support surface, and the BA/gelatin-support provided an optimal microenvironment. This could also explain the immobilization yield of native support (48.6%), BA-support (70.6%), gelatin-support (90.3%), and BA/gelatin-support (125.4%), respectively. There are several aspects which could explain this phenomenon. Firstly, BA-support provided ionic exchange with the secondary amino group and hydrophobic adsorption of the hydrophobic support and alkane chain for immobilizing lipase. Secondly, gelatin could provide a biocompatible support surface for immobilization, which could protect the enzyme conformation. Zheng et al. [57] reported the immobilization of hemoglobin on gelatin/carbon nanotubes to make electrodes, and the secondary structure of hemoglobin immobilized on gelatin/carbon nanotubes was almost the same as that of the native hemoglobin. Thus, the increase in enzymatic activity suggests that immobilization might establish a complex attachment between lipase and the supports [7]. 3.3.1. Effect of different ratios of gelatin to BA modifications on lipase activity In order to investigate the immobilization behavior of lipase on BA/gelatin-support, the ratio of gelatin to BA was altered to conduct a BA/gelatin equilibrium experiment. Table 2 shows the effect of different gelatin-to-BA ratios on activity of the immobilized lipase, gelatin loading amount, BA loading amount and BA:gelatin on the support. The results indicated that a 1:1 ratio of gelatin to BA was more suitable for immobilizing the lipase, seen in its high activity (3.5-fold greater when compared to the lipase directly immobilized on the support). As the ratios of gelatin to BA were altered, with increases in concentrations of either BA or gelatin, the lipase activity was shown to decrease. Similar results from other groups have
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Table 2 The effects of BA/gelatin ratios used for immobilization activity, gelatin loading amount, butylamine loading amount and the ratio of BA and gelatin loading amount. Ratios of gelatin to BA (%)
Activity (U/g support)
Gelatin loading amount (mg/g support)
Butylamine loading amount (mg/g support)
BA:gelatin (w/w)
1:2 1:1 2:1
630 850 560
7.6 12.3 16.0
15.9 13.15 7.3
1:2.1 1:0.94 2.2:1
Table 3 The effects of BA/gelatin concentration used for immobilization on activity, epoxy group content, gelatin loading amount, butylamine loading amount and the ratio of BA and gelatin loading amount. Concentration (%)
Activity (U/g support)
Epoxy group content (%)
Gelatin loading amount (mg/g support)
Butylamine loading amount (mg/g support)
0 0.5 1 2 5 10 20
240 360 850 1180 1150 1200 1180
100 61 38 20 15 17 20
– 5.65 12.3 17.1 17.4 16.8 17
– 5.85 13.15 18.63 19.55 18.05 18.24
shown that support with proper property might provide feasible contact between the substrates and the immobilized lipase, thereby improving lipase activity [58]. In addition, the loading ratio of BA to gelatin on the support was almost the same with the addition of BA and gelatin. We propose three possible hypotheses to explain our results. First, when the lipase is immobilized onto the hydrophobic support, the hydrophobic environment could induce the opening of the lid structure, thus activating the lipase by interaction activation. Second, when the lipase is immobilized on BA modified support, the secondary amino group could immobilize lipase by ionic exchange. Third, when the lipase is immobilized onto the biocompatible support, the lipase conformation is almost entirely maintained, which accelerates its catalytic efficiency. The addition of BA and gelatin can balance the hydrophobicity/ionicity/biocompatibility characteristics of the support structure, simultaneously protecting against lipase inactivity. Taking these factors together, our results provide support for a synergistic efficacy for lipase immobilization on the BA/gelatinsupport structure, leading to stable lipase activity. In short, the BA/gelatin-support structure was suitable for immobilizing YLIP2. 3.3.2. Effect of BA/gelatin concentrations on the activity of immobilized lipase The effect of BA/gelatin concentrations on the activity of immobilized enzyme was evaluated from a range of 0–20%. Not surprisingly, we observed an increase in activity with an increase in the modified BA/gelatin concentration. As shown in Table 3, the activity of the immobilized lipase reached approximate 1180 U/g support at 2% BA/gelatin concentration, which was close to its equilibrium activity (1200 U/g support) and 4.9-fold greater when compared to lipase directly immobilized onto the support structure. When the concentration of BA/gelatin increased from 0% to 2%, the content of epoxy group on the support decreased from 100% to 20%, the gelatin loading amount increased to 17.1 mg/g support, and the BA loading amount increased to 18.63 mg/g support. However, all the parameters reached a plateau when the modifier concentration was at 2% or greater (Table 3). It proved that the support surface modification reached saturation when the modifier concentration was at 2%. And further increases to the concentration of BA/gelatin did not lead to higher surface modifications. It is worth mentioning that the ratio of BA to gelatin was stable maintained at 1:0.93, which was similar with the addition of BA and gelatin. Collectively, these data indicate that the main contribution to lipase activity is the concentration of BA/gelatin on the support surface.
BA:gelatin (w/w)
1:0.97 1:0.94 1:0.92 1:0.89 1:0.93 1:0.93
3.3.3. Effect of modified sequences of BA and gelatin on the activity of immobilized lipase We used three additional methods to further validate the synergistic effect of BA and gelatin on immobilized lipase performance: (i) modification of the support surface with 2% BA followed by 2% gelatin; (ii) gelatin (2%) was first used to modify the support and then the gelatin-support was modified with 2% BA; (iii) gelatin (2%) and BA (2%) were concurrently used to modify the support structure. As shown in Fig. 5, the last method of modification with gelatin and BA had a significant influence on immobilized lipase activity. Overall, immobilized lipase activity was 705, 485, and 1180 U/g support for the first, second, and third modified method, respectively. Lipase immobilized on the support with the first method (705 U/g support) has a similar effect on lipase activity as that immobilized on a gelatin (1%) modified support (600 U/g support). Likewise, the activity of lipase immobilized on the support with the second method (485 U/g support) was similar to that on BA (1%) modified support (400 U/g support). These results show that the increase in lipase activity is most likely due to an enrichment of the modifier. Moreover, when gelatin and BA are used to modify the support in sequence, the latter one works. Only when these two modifiers were used simultaneously was the activity the highest. These results provide further evidence for the synergism of hydrophobicity, ionicity and biocompatibility and how using the three can improve the properties of the immobilized lipase.
Fig. 5. Effect of BA and gelatin modified sequences on activity.
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4. Conclusion In the present study, a series of modifications with different proteins and amines were used in the lipase immobilizing processes. When compared to immobilizing the lipase directly onto the support, lipase immobilized on the support with gelatin or BA modifications resulted in an elevation of its activity. Furthermore, when the lipase was immobilized onto a BA/gelatin support, the activity also greatly increased. These results showed that the synergistic usage of hydrophobic, ionic and biocompatible modifications could significantly improve lipase activity. Ultimately, this study demonstrated that a moderate micro-environment of support surface was important for increasing immobilized lipase activity. Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (2013CB733600, 2012CB725200), the National Nature Science Foundation of China (21436002, 21106005), and the National High-Tech R&D Program of China (863 Program) (2014AA022101, 2012AA022205). References [1] A.J. Straathof, S. Panke, A. Schmid, Curr. Opin. Biotechnol. 13 (2002) 548. [2] R. Aravindan, P. Anbumathi, T. Viruthagiri, Indian J. Biotechnol. 6 (2007) 141. [3] W. Alloue-Boraud, E.A. Mireille, K. Amighi, F.K. N’Guessan, R. Koffi-Nevry, J. Destain, Afr. J. Pharm. Pharmacol. 8 (2014) 206. [4] N.F.A. Rahman, M. Basri, M.B.A. Rahman, R.N.Z.R.A. Rahman, A.B. Salleh, Bioresour. Technol. 102 (2011) 2168. [5] R.A. Sheldon, S. van Pelt, Chem. Soc. Rev. 42 (2013) 6223. [6] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biotechnol. Adv. 28 (2010) 628. [7] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R. FernandezLafuente, Enzyme Microb. Technol. 40 (2007) 1451. [8] P. Tielmann, H. Kierkels, A. Zonta, A. Ilie, M.T. Reetz, Nanoscale 6 (2014) 6220. [9] Y. Ren, J.G. Rivera, L. He, H. Kulkarni, D.-K. Lee, P.B. Messersmith, BMC Biotechnol. 11 (2011) 63. [10] T. Dong, L. Zhao, Y. Huang, X. Tan, Bioresour. Technol. 101 (2010) 6569. [11] X.C. Liu, W.H. Scouten, J. Mol. Recognit. 9 (1996) 462. [12] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chem. Soc. Rev. 42 (2013) 6290–6317. [13] O. Barbosa, R. Torres, C. Ortiz, A.n. Berenguer-Murcia, R.C. Rodrigues, R. Fernandez-Lafuente, Biomacromolecules 14 (2013) 2433. [14] S. Liu, B. Lin, X. Yang, Q. Zhang, J. Phys. Chem. B 111 (2007) 1182. [15] C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues, Adv. Synth. Catal. 353 (2011) 2885. [16] R.C. Rodrigues, Á. Berenguer-Murcia, R. Fernandez-Lafuente, Adv. Synth. Catal. 353 (2011) 2216. [17] A. Brzozowski, U. Derewenda, Z. Derewenda, G. Dodson, D. Lawson, J. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. Patkar, L. Thim, Nature 351 (1991) 491. [18] R. Verger, Trends Biotechnol. 15 (1997) 32. [19] R.D. Schmid, R. Verger, Angew. Chem. Int. Ed. 37 (1998) 1608.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity Caixia Cui, Yifeng Tao, Chunling Ge, Yueju Zhen, Biqiang Chen ∗ , Tianwei Tan ∗ National Energy R&D Center for Biorefinery, Beijing Key Laboratory of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 15 May 2015 Accepted 26 May 2015 Available online 4 June 2015 Keywords: Lipase Immobilization Biocompatibility Hydrophobic Ionic Balance
a b s t r a c t We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA ) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420 U/g support) and the biocompatible gelatin modified support (600 U/g support) when compared to the support without modification (240 U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180 U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lipases are a group of enzymes that have great applications in food, pharmaceutical, cosmetic, and chemical industries [1–4]. However, lipase-based catalysts are usually expensive, unstable, and are difficult to be efficiently separated from reaction systems used in industrial production [5]. To address this, various immobilization techniques have been developed to improve their stabilization, reutilization [6,7] and other properties [8–10]. For example, the enzyme aggregation reduced in the immobilization processes compared to free enzyme and generated favorable environment [11,12]. Multipoint covalent attachment of enzyme on highly activated pre-existing supports via short spacer arms involving many residues placed on the enzyme surface promotes rigidification of structure of the immobilized enzyme [13]. For some enzymes with several subunits, the immobilization process may protect the stabilization and prevent subunit dissociation [14]. However, a simple protocol for effective immobilization system is not easily accomplished because small changes in variables such
∗ Corresponding author. Tel.: +86 10 64416691; fax: +86 10 64416691. E-mail addresses: [email protected] (B. Chen), [email protected] (T. Tan). http://dx.doi.org/10.1016/j.colsurfb.2015.05.045 0927-7765/© 2015 Elsevier B.V. All rights reserved.
as nature and characteristics of the immobilization support as well as age in vs coupling chemistry can have a significant impact on biocatalyst activity and stability [15,16]. Therefore, it is very important to select a suitable support and design proper modification protocols, especially to create the appropriate micro-environment for enzymes in the supports, and it is still challenging to develop optimized immobilization techniques. Currently, there are four main immobilization techniques: covalent binding, adsorption, cross-linking, and entrapment [5]. Importantly, immobilized lipases can become more active than their native form. This is due to the fact that the lipase can exist in two forms: an open, active form in which the lid displaces and allows substrate access to the catalytic site and a closed, inactive form in which the lid shuts, thereby hiding the catalytic site [17–19]. In aqueous media, the lid exists in a closed conformation. However, in the presence of an insoluble substrate, the lipase becomes active by interfacial activation, shifting lid conformation from a closed to an open state [17]. Lipase becomes more active if the interactions between them and their support stabilize their open form which generates a higher activity [20]. Immobilization techniques utilizing hydrophobic matrices have attracted much attention, since the interaction between the lipase and its hydrophobic support is similar to the phenomenon observed with insoluble substrates [21]. Moreover, the accessibility of
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the substrate to the active site of the lipase lid was promoted by employing hydrophobic support [22]. Lipase immobilized on hydrophobic supports involves its open form of the lipase, and the hydrophobic support makes lipase more stable [23]. Thus far, there are two strategies to make hydrophobic supports. One strategy is that the support itself may be reagent (e.g. acccurel, divinylbencene, etc.) [24,25]. The other is to decorate the support with a hydrophobic reagent [26]. For instance, Jin et al. [27] modified magnetic siliceous mesocellular foam (MCF) with organosilane to improve the support surface’s hydrophobicity. They found that lipase derived from Pseudomonas cepacia (PCL) that was immobilized on a support of hydrophobic MCF-Ph obtained the highest activity, which was eight times that of native MCF. Taken together, results obtained from time-resolved fluorescence catalytic activity experiments showed that the hydrophobic activation drove the improvement in catalytic activity [16]. However, the excessive support resulting from this hydrophobic interaction can destroy the structure of the enzyme, leading to rapid denaturation and a sharp decrease in the activity of surface-bond enzymes [28]. Ultimately, this excess would decrease the enzyme properties [25]. When compared with hydrophobic supports, lipase immobilized on hydrophilic supports can acquire a higher efficiency of immobilization. When a lipase which was immobilized on a hydrophilic support was used in organic solution, water was able to concentrate around the lipase, thus promoting the catalysis of the reaction [29,30]. In addition, the process of tethering enzymes to carriers could cause inevitable conformational changes of the enzymes, resulting in decreased bioactivities of enzymes [31]. To overcome this limitation, researchers have adopted many methods, one of which is to modify the support surface with biofriendly molecules to obtain the biocompatible surface to reduce the enzyme denaturation during the immobilization [32–34]. There are many methods to form such biocompatible (“bio-friendly”) support surfaces, including coating, adsorption, and self-assembly [35,36]. Among these methods, modifications to support structures, such as natural, molecular-like gelatin, collagen, alginate, or biotin on the support have often been used in tissue engineering due to their non-toxic nature [37]. In this work, the support PGMA-TAIC-EGDMA with epoxy group was used as the immobilized support while the epoxy group could be modified by thiols, amino, hydroxyl and carboxyl groups easily, and the final bonds are very stable. However, the epoxy group exhibits a relative low reactivity [13]. Thus, many support modifications have been researched [38–40]. Although there are many hydrophobic and biocompatible modifications for supports, most have only used hydrophobic or bio-friendly reagents to regulate the support environment. In our study, different types of amines and proteins were introduced to investigate their respective activities in our first work. Lipase could be immobilized on support by different modifications such as ionic exchange, hydrophobic adsorption, covalent binding, and biocompatible adsorption. Here we present work in which we prepared an amine and protein immobilized matrix in order to regulate the properties of the support surface. The ratios of gelatin to BA, the concentrations of BA/gelatin and the sequences of BA and gelatin modified support were studied, and the synergistic effects of the hydrophobic, ionic and biocompatible modifications on the activity of the immobilized lipase were analyzed.
Fig. 1. SDS-PAGE of Yarrowia lipolytica lipase lip2.
albumin, BA, methylamine, and n-octylamine were purchased from Beijing Chemical Factory (Beijing, China). All of the experimental reagents and solvents used were of analytical grade. 2.2. The preparation of lipase solution The fermentation of YLIP2 lipase in our laboratory was carried out in a 5 L reactor at 26 ◦ C with stirring at 500 rpm and with an air input of 1 VVM (air volume/culture volume/min). The culture medium was composed of 4% (wt.) soybean powder, 4% (wt.) soybean oil, 0.1% (wt.) KH2 PO4 and 0.1% (wt.) (NH4 )2 SO4 . The lipase produced reached 8000 U/ml after 96 h fermentation. The cells were removed by centrifugation, and the lipase in the supernatant was precipitated by addition of three volumes of acetone. The precipitate was washed with acetone and dried at room temperature. The activity of the lipase powder is 67000 U/g. The lipase powder was dissolved into distilled water getting the lipase solution (10 mg/ml). The purity of YLIP2 lipase is higher than 90%, which was confirmed by SDS-PAGE via Quantity-One (Fig. 1).
2. Materials and methods 2.3. Electrophoresis 2.1. Materials Both the Yarrowia lipolytica lipase YLIP2 lipase and the epoxy support PGMA-TAIC-EGDMA were previously produced in our laboratory [41,42]. Gelatin, fibrion, casein, ovalbumin, bovine serum
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on protein using a Mini Protein Electrophoresis Cell (Bio-Rad Laboratories, California, USA) and a 12% acrylamide separating and a 5% acrylamide stacking. Protein
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solutions (1 mg/ml) were dissolved 1:3 with sample buffer solution (4% SDS, 20% glycerol, 10% -mercaptoethanol, 0.125 M Tris, pH 6.8). After heating in boiling water for 3 min, a 10 L of protein per lane were loaded onto the gel and electrophoresed in a Mini-PROTEAN 3 apparatus (Bio-Rad Laboratories, Hercules, CA). A mixture of molecular weight standards (10–170 kDa) was also run along with the samples. After electrophoretic separation, the gels were stained with a solution of Coomassie Brilliant Blue containing trichloroacetic acid for fixation and destained with acetic acid. 2.4. Procedure of lipase immobilization
and 200 rpm for 6 h. The modified support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on different BA/gelatin supports through the same procedures described in Section 2.4.1. The immobilization yield was defined as YI% = Uact /Uads × 100, where Uads are the absorbed units evaluated as the difference between initial activity and the remaining activity in the supernatant at the end of the adsorption procedure; Uact is the activity present on the support [43]. The expressed activity was the same as the activity of the immobilized lipase.
2.4.1. Hydrophobic adsorption followed by covalent attachment on epoxy-support The native epoxy-support (1 g) was immersed in 5 ml of lipase solution and agitated at 15 ◦ C and 200 rpm for 2 h. The immobilized lipase was washed several times with distilled water to remove any unbound lipase and then recovered by filtration. The immobilized lipase was stored at 4 ◦ C until later use.
2.5. Protein determination
2.4.2. Lipase immobilized on protein or/and amine modified support 2.4.2.1. Adsorption on protein modified support. The protein modifications were conducted as follows: the native epoxy-support (1 g) was immersed in 5 ml of 1% (w/v) different kinds of protein solution: gelatin, fibrion, casein, ovalbumin or bovine serum albumin solution and agitated at 60 ◦ C and 200 rpm for 6 h, respectively. After modification, the support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on protein-support through the same procedures described in Section 2.4.1.
2.6. BA determination
2.4.2.2. Ionic exchange or/and hydrophobic adsorption on amine modified support. The amine modifications were conducted as follows: the native epoxy-support (1 g) was immersed in 5 ml of 1% (w/v) different kinds of amine solution: BA, methylamine, or noctylamine solution and agitated at 60 ◦ C and 200 rpm for 6 h, respectively. After modification, the support was washed with distilled water to remove any residual modifying solution before being stored at 4 ◦ C for later immobilization. Lipase immobilized on amine-support through the same procedures described in Section 2.4.1. 2.4.2.3. Synergistic effect of BA and gelatin modifications. The gelatin and BA modifications were conducted as follows: The ratios of BA to gelatin (w/w) used in modification were as follows: the native epoxy-support (1 g) was modified by 5 ml 1% (w/v) BA and gelatin solution with one of three different ratios of gelatin:BA (2:1, 1:1 and 1:2) before being agitated at 60 ◦ C and 200 rpm for 6 h. The concentrations of BA and gelatin (BA/gelatin) used in modification were as follows: the native epoxy-support (1 g) was immersed in 5 ml of BA and gelatin solution at one of the following concentrations: 0%, 0.5%, 1%, 2%, 5%, 10% or 20% (w/v) and agitated at 60 ◦ C and 200 rpm for 6 h. The sequences of BA and gelatin used in modifications were as follows: (i) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) gelatin solution and agitated at 60 ◦ C and 200 rpm for 6 h, and the support was washed three times with distilled water to remove the residual gelatin solution, and then incubated in 5 ml 2% (w/v) BA solution; (ii) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) BA solution and agitated at 60 ◦ C and 200 rpm for 6 h, and then washed three times with distilled water to remove the residual BA solution before being incubated in 5 ml 2% (w/v) gelatin solution; (iii) the native epoxy-support (1 g) was immersed in 5 ml 2% (w/v) BA/gelatin (1:1) solution and agitated at 60 ◦ C
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The amount of gelatin immobilized on epoxy-support and lipase immobilized on the support structure was determined via Bradford protein assay by measuring the initial and final concentrations of protein in the modified solution and immobilization lipase medium [44].
The amount of BA linked on the epoxy-support was quantified via gas chromatography (GC) by measuring the initial and final concentrations of BA in the modified solution. GC analysis was performed using GC-2010 (Shimadzu Japan) equipped with a DB-FFAP capillary column (30.0 m × 0.32 mm × 0.5 m; J&W Scientific, USA) and a flame ionizing detector. The injector temperature was 200 ◦ C. The column oven temperature was 50 ◦ C, and then increased to 200 ◦ C at a heating rate of 10 ◦ C/min and maintained for 10 min. The detector temperature was 220 ◦ C. 2.7. Activity of free and immobilized lipases The activity of free and immobilized lipases was measured via olive oil hydrolysis according to a previously published report [45]. Briefly, an olive oil emulsion (200 ml) was prepared by mixing olive oil (50 ml) and 2% PVA (150 ml) under vigorous stirring for 6 min. 5 ml of this emulsion and 4 ml 0.1 M phosphate buffer (pH 8.0) were then warmed for 5 min at 35 ◦ C. Either the free or immobilized lipase was added to the mixture, incubated at 35 ◦ C and agitated at 100 rpm for 5 min in a water bath shaker. The reaction was then terminated by addition of 15 ml ethanol. We then determined the amount of released fatty acids by titration with 50 mM NaOH solution. Following convention, one unit of lipase hydrolytic activity was defined as the release of 1 m fatty acids per min at 35 ◦ C. All the results shown were the averages of experiments conducted in triplicate. 3. Results and discussion 3.1. Effect of amine modifications of support structure on the activity of immobilized lipase A critical issue in lipase immobilization is the support structure. In particular, the hydrophobic character of the support surface is imperative, as it can have a large effect on enzyme immobilization [46–48]. The PGMA-TAIC-EGDMA support itself is hydrophobic in some extent due to the usage of hydrophobic reagents in the synthesized processes. Lipase was immobilized on the native epoxy-support under neutral pH values and low ionic strength, via interfacial activation on the support [49]. In this work, in order to adjust the property of the support more reasonable, different chain lengths of amine were used to modify the support. As shown in Fig. 2, amine modification could significantly affect the activity of immobilized YLIP2. The activity of immobilized lipase on methylamine-modified
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Fig. 2. Lipase immobilized on support with different hydrophobic modifications.
support (340 U/g support), BA-modified support (450 U/g support) and n-octylamine-modified support (280 U/g support) was 1.4-, 1.8- and 1.2-fold greater than that on the native support (240 U/g support), respectively, which suggesting that the amines could contribution to improving the lipase immobilization by regulating the support surface property. However, the main cause of immobilization by amines might be different. When using methylamine, they have a secondary amino group below the short alkane chain, the main immobilization cause was anionic exchange (secondary amino). When the carbon chain of amine increased to eight (octylamine), they have steric hindrances for the enzyme ion exchange, and a hydrophobic layer that can produce the enzyme immobilization via interfacial activation. When using butyl-amine, perhaps they have something in between, after interfacial activation the enzyme may become ionically exchanged. Thus, the length of the amines has a great influence on immobilization. Such results reinforce past work that has shown that the length of the carbon chain can significantly affect the activity of the immobilized lipase [50]. To this end, Miletic´ et al. [50] reported that the lipase activity dependeded on support modifications, including supports with different lengths of hydrophobic carbon chains (e.g. 1,2-diaminoethane, 1,4diaminobutane, 1,6-diaminohexane, and 1,8-diaminooctane). They showed that lipase immobilized on resin 3 obtained the highest activity with a 1,4-diaminobutane modification, while lipase immobilized on resin 6 obtained the highest activity with a 1,2diaminoethane modification. BA modification could regulate the support surface to be more appropriate via the combination of hydrophobic adsorption and ionic exchange for YLIP2 immobilization. They also demonstrated that the proper modification for lipase immobilization was different for different support structures; an excess of hydrophobicity within the immobilized matrix would destroy the enzymatic structure and partially reduce its overall surface biocompatibility [51] and the balance of ionic and hydrophobic could improve the property in some extent. 3.2. Effect of biocompatible modifications of support structures on the activity of immobilized lipase A hydrophobic interface does not always produce higher activity, as excessive hydrophobicity will destroy enzymatic structure [28]. A protein that has good biocompatibility is usually used in the immobilization process via adsorption to regulate the properties of the support surface [27]. As such, the protein used during the immobilization processes has attracted much interest [52]. To this end, Fig. 3 presents our data relevant to YLIP2 immobilization onto
Fig. 3. Lipase immobilized on support with different protein modifications.
support structures with or without protein modification. We found that the activity of lipase immobilized on support with ovalbumin, casin, BSA, gelatin, and fibroin modifications was 2.1-, 1.1-, 1.6-, 2.5, and 2-fold greater, respectively, when compared to that on native support. Our results demonstrated that the activity of lipase immobilized on different protein-modified supports was improved by different degrees. When the epoxy support immobilized lipase by covalent binding, the exited protein layer could protect the lipase against inactivation by the covalent reaction. For instance, lipase immobilized on a gelatin-modified support obtained the highest activity (600 U/g support), which was 2.5-fold greater when compared to that on an unmodified support structure. At present, we can only speculate on the mechanism behind such results. For one, it is possible that the biocompatible gelatin layer on the support surface created a good micro-environment for the lipase, which has been shown to result in a retention of the high activity of the immobilized enzyme [53]. Secondly, gelatin derived from collagen is composed of polypeptides of various sizes, which possess a molecular weight distribution in the range of 15,000–250,000 Da [54]. It also contains free groups (e.g. amino acid) that can immobilize the lipase, while the non-polar amino acid chain of gelatin can further immobilize the lipase via an adsorption interaction [54]. Finally, it is possible that as a protein, gelatin could protect lipase against inactivation, since additional protein has been shown to improve enzymatic activity and stability [55]. 3.3. Effect of BA and gelatin modifications of support structures on the activity of immobilized lipase All of the techniques used in this study were advantageous to improving the immobilized lipase properties with either BA or gelatin modification. Taking into account these two categories, BA and gelatin were used to decorate the support structure in order to obtain a support with simultaneous hydrophobic, ionic and biocompatible characteristics (Scheme 1). As shown in Fig. 4, it is evident that the activity of the immobilized lipase on BA/gelatinsupport (850 U/g support) was higher than that either on the BA-support (450 U/g support) or on the gelatin-support (600 U/g support) alone. Furthermore, this effect was far greater than that on an unmodified support (240 U/g support). Since the protein attachment of these immobilized lipases was almost identical (10.4 mg protein/g support) (Table 1), these results suggest that protein attachment is not a critical factor influencing increases in lipase activity. The specific activity of lipase immobilized on BA/gelatin-support (81.7 U/mg protein), which is higher than
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Scheme 1. Lipase immobilized on support with different modifications.
Table 1 Free lipase and lipase immobilization on support modified with BA, gelatin and BA/gelatin: protein attachment, hydrolytic activity, specific activity and immobilization yield. Different lipases
Activity (U/g support or lipase)
Protein loading (mg/g support)
Specific activity (U/mg protein)
Immobilization yield (%)
Free lipase Lipase immobilized on support directly Lipase immobilized on BA-support Lipase immobilized on gelatin-support Lipase immobilized on BA/gelatin (1:1)-support
67,000 240 450 600 850
– 8.2 8.9 9.4 10.4
67 29.3 50.6 63.8 81.7
– 48.6 70.6 90.3 125.4
that of the free lipase (67 U/mg protein), is higher than that on gelatin-support (63.8 U/mg protein) and BA-support (50.6 U/mg protein), and is much higher than that on native epoxy-support (29.3 U/mg protein). Considering the hydrophobic nature of the native epoxy-support, the interfacial activation of lipase on the native support could be achieved and thus the immobilized preparations get hyperactivated [56]. However, the specific activity of lipase immobilized on native epoxy-support was lower than that of the free lipase. It indicated that lipase was inactivated in the processes of immobilizing on native support in some extent. The improved specific activity of lipase immobilized on BA,
Fig. 4. Effect of BA and gelatin modification on activity.
gelatin and BA/gelatin modified supports proved that the modifications could improve the micro-environment of the support surface, and the BA/gelatin-support provided an optimal microenvironment. This could also explain the immobilization yield of native support (48.6%), BA-support (70.6%), gelatin-support (90.3%), and BA/gelatin-support (125.4%), respectively. There are several aspects which could explain this phenomenon. Firstly, BA-support provided ionic exchange with the secondary amino group and hydrophobic adsorption of the hydrophobic support and alkane chain for immobilizing lipase. Secondly, gelatin could provide a biocompatible support surface for immobilization, which could protect the enzyme conformation. Zheng et al. [57] reported the immobilization of hemoglobin on gelatin/carbon nanotubes to make electrodes, and the secondary structure of hemoglobin immobilized on gelatin/carbon nanotubes was almost the same as that of the native hemoglobin. Thus, the increase in enzymatic activity suggests that immobilization might establish a complex attachment between lipase and the supports [7]. 3.3.1. Effect of different ratios of gelatin to BA modifications on lipase activity In order to investigate the immobilization behavior of lipase on BA/gelatin-support, the ratio of gelatin to BA was altered to conduct a BA/gelatin equilibrium experiment. Table 2 shows the effect of different gelatin-to-BA ratios on activity of the immobilized lipase, gelatin loading amount, BA loading amount and BA:gelatin on the support. The results indicated that a 1:1 ratio of gelatin to BA was more suitable for immobilizing the lipase, seen in its high activity (3.5-fold greater when compared to the lipase directly immobilized on the support). As the ratios of gelatin to BA were altered, with increases in concentrations of either BA or gelatin, the lipase activity was shown to decrease. Similar results from other groups have
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Table 2 The effects of BA/gelatin ratios used for immobilization activity, gelatin loading amount, butylamine loading amount and the ratio of BA and gelatin loading amount. Ratios of gelatin to BA (%)
Activity (U/g support)
Gelatin loading amount (mg/g support)
Butylamine loading amount (mg/g support)
BA:gelatin (w/w)
1:2 1:1 2:1
630 850 560
7.6 12.3 16.0
15.9 13.15 7.3
1:2.1 1:0.94 2.2:1
Table 3 The effects of BA/gelatin concentration used for immobilization on activity, epoxy group content, gelatin loading amount, butylamine loading amount and the ratio of BA and gelatin loading amount. Concentration (%)
Activity (U/g support)
Epoxy group content (%)
Gelatin loading amount (mg/g support)
Butylamine loading amount (mg/g support)
0 0.5 1 2 5 10 20
240 360 850 1180 1150 1200 1180
100 61 38 20 15 17 20
– 5.65 12.3 17.1 17.4 16.8 17
– 5.85 13.15 18.63 19.55 18.05 18.24
shown that support with proper property might provide feasible contact between the substrates and the immobilized lipase, thereby improving lipase activity [58]. In addition, the loading ratio of BA to gelatin on the support was almost the same with the addition of BA and gelatin. We propose three possible hypotheses to explain our results. First, when the lipase is immobilized onto the hydrophobic support, the hydrophobic environment could induce the opening of the lid structure, thus activating the lipase by interaction activation. Second, when the lipase is immobilized on BA modified support, the secondary amino group could immobilize lipase by ionic exchange. Third, when the lipase is immobilized onto the biocompatible support, the lipase conformation is almost entirely maintained, which accelerates its catalytic efficiency. The addition of BA and gelatin can balance the hydrophobicity/ionicity/biocompatibility characteristics of the support structure, simultaneously protecting against lipase inactivity. Taking these factors together, our results provide support for a synergistic efficacy for lipase immobilization on the BA/gelatinsupport structure, leading to stable lipase activity. In short, the BA/gelatin-support structure was suitable for immobilizing YLIP2. 3.3.2. Effect of BA/gelatin concentrations on the activity of immobilized lipase The effect of BA/gelatin concentrations on the activity of immobilized enzyme was evaluated from a range of 0–20%. Not surprisingly, we observed an increase in activity with an increase in the modified BA/gelatin concentration. As shown in Table 3, the activity of the immobilized lipase reached approximate 1180 U/g support at 2% BA/gelatin concentration, which was close to its equilibrium activity (1200 U/g support) and 4.9-fold greater when compared to lipase directly immobilized onto the support structure. When the concentration of BA/gelatin increased from 0% to 2%, the content of epoxy group on the support decreased from 100% to 20%, the gelatin loading amount increased to 17.1 mg/g support, and the BA loading amount increased to 18.63 mg/g support. However, all the parameters reached a plateau when the modifier concentration was at 2% or greater (Table 3). It proved that the support surface modification reached saturation when the modifier concentration was at 2%. And further increases to the concentration of BA/gelatin did not lead to higher surface modifications. It is worth mentioning that the ratio of BA to gelatin was stable maintained at 1:0.93, which was similar with the addition of BA and gelatin. Collectively, these data indicate that the main contribution to lipase activity is the concentration of BA/gelatin on the support surface.
BA:gelatin (w/w)
1:0.97 1:0.94 1:0.92 1:0.89 1:0.93 1:0.93
3.3.3. Effect of modified sequences of BA and gelatin on the activity of immobilized lipase We used three additional methods to further validate the synergistic effect of BA and gelatin on immobilized lipase performance: (i) modification of the support surface with 2% BA followed by 2% gelatin; (ii) gelatin (2%) was first used to modify the support and then the gelatin-support was modified with 2% BA; (iii) gelatin (2%) and BA (2%) were concurrently used to modify the support structure. As shown in Fig. 5, the last method of modification with gelatin and BA had a significant influence on immobilized lipase activity. Overall, immobilized lipase activity was 705, 485, and 1180 U/g support for the first, second, and third modified method, respectively. Lipase immobilized on the support with the first method (705 U/g support) has a similar effect on lipase activity as that immobilized on a gelatin (1%) modified support (600 U/g support). Likewise, the activity of lipase immobilized on the support with the second method (485 U/g support) was similar to that on BA (1%) modified support (400 U/g support). These results show that the increase in lipase activity is most likely due to an enrichment of the modifier. Moreover, when gelatin and BA are used to modify the support in sequence, the latter one works. Only when these two modifiers were used simultaneously was the activity the highest. These results provide further evidence for the synergism of hydrophobicity, ionicity and biocompatibility and how using the three can improve the properties of the immobilized lipase.
Fig. 5. Effect of BA and gelatin modified sequences on activity.
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