Ya Li,Jicheng Hu ,Pingfang Han ,*

1 College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 210009,China

2 Department of Environment and Resource,Nantong Science and Technology College,Nantong 226007,China

Keywords:Magnetically modified palygorskite Immobilized lipase Adsorption Stability Reusability

A B S T R A C T Magnetically modified palygorskite composites were synthesized with γ-Fe2O3 dispersing on the external surface of clay mineral.The magnetic clay was characterized with Fourier transform infrared,X-ray diffraction,transmission electron microscopy,and vibrating sample magnetometer.Candida sp.99–125 lipase was immobilized on magnetic palygorskite composites by physical adsorption with enzyme loading of 41.5 mg·g?1 support and enzyme activity of 2631.6 U·(g support)?1.The immobilized lipase exhibit better thermal and broader pH stability and excellent reusability compared with free lipase.

1.Introduction

In the last decade,immobilized enzymes were widely used in the production of food,pharmaceuticals,and other biologically important fine products[1–5].Immobilized enzymes can offer many advantages over their free forms,making this a topic of active research in the area of biotechnology.In order to enhance their reusability,operational stability and recovery,enzymes are immobilized on different carriers[6–14].An important area of interest is the immobilization of lipase on magnetic materials.The use of magnetic supports can reduce the capital and operation costs[15,16].They can be more easily separated from a reaction system and stabilized in a fluidized bed reactor by applying an external magnetic field[17].Magnetic support based separation has several advantages in comparison with conventional separation methods[18].Moreover,the capability and efficiency of magnetic separation are especially useful for large-scale operations[19,20].Several magnetic carriers,such as microspheres of various biomaterials encapsulating magnetic particles and copolymers with magnetic particles,have been used with good results[21].How ever,due to hydrophilic constraints,these micro-spheres could not contact with enzymes preferably in a solution,reducing enzyme loading on the carrier and decreasing the activity yield[22].Therefore,it is preferable and significant to develop high hydrophilic magnetic materials with active groups,which can be employed as immobilization carriers with better performance.

Palygorskite[PA,formerly called attapulgite,with an ideal formula of(Mg,Al)5Si8O20(OH)2(H2O)4·4H2O],is a form of crystalline hydrated magnesium aluminum silicate mineral with unique three-dimensional structure,presenting a fibrous morphology with exchangeable cations and reactive–OH groups on its surface.Palygorskite is widely used as lipase carriers because of its unique structure,special sorptivity and excellent mechanical resistance.Its natural origin and the low cost make it more attractive.In this study,magnetically modified palygorskite composite is prepared and characterized.Then palygorskite modified with magnetic iron oxide phases is utilized as carrier for the immobilization of Candida sp.99–125 lipase via physical adsorption.The stability and reusability of immobilized and free lipases are investigated.

2.Materials and Methods

2.1.Materials

Candida sp.99–125 lipase was purchased from Beijing CTA New Century Biotechnology Co.Ltd.Bovine serum albumin was purchased from Sigma.The palygorskite was supplied by Jiangsu Xuyi Anhalt Nonmetallic Mining Ltd.with an average diameter of 0.075 mm(200 mesh).For the iron-containing palygorskite product,the salt FeCl3·6H2O,AR grade,was used without any pretreatment.All other reagents were of analytical grade.Distilled water was used in all experiments.

2.2.Synthesis of magnetically modified palygorskite composite

The magnetically modified palygorskite composite was obtained with the method by Bourlinos et al.[23].First,100 ml of 0.02 mol·L?1FeCl3·6H2O solution was added drop wise to the palygorskite suspension(20 g·L?1)under stirring.The resulting mixture was centrifuged and w ashed with water.The solid was collected and redispersed into 20 ml of water and dried under vacuum for 24 h(P–Fe).After being crashed into pow der,the dried sample was exposed in glacial acetic acid vapor at 80°C for 2 h,and then dried at the same temperature for a few minutes in order to remove the surface-adsorbed acetic acid(P-FeAc).The powder was placed in a quartz tube and calcinated at 400°C for 2 h in nitrogen atmosphere,and then it became magnetic.The pale reddish powder is the magnetically modified palygorskite composite,denoted as MPC.

2.3.Immobilization of lipase onto MPC

For the immobilization of lipase,200 mg of MPC was added to 40 ml phosphate buffer(0.1 mol·L?1)containing 20 mg Candida sp.99–125 lipase.The adsorption process was performed at 25°C with shaking at 110 r·min?1.Then the suspension was separated by magnet and w ashed with phosphate buffer(pH 7.0,0.01 mol·L?1)three times.The amount of Candida sp.99–125 immobilized on the MPC was determined by measuring the concentration of protein in the supernatant using the Bradford protein assay method[24].

w here Pais the amount of immobilized lipase on carriers(mg·g?1,mg enzyme per gram MPC),Ciand Cfare the initial and final concentrations of enzyme in the reaction medium(mg·ml?1),respectively,V is the volume of reaction medium(ml),and W is the mass of the carrier(g).

2.4.Assay of enzyme activity

The activity of free and immobilized Candida sp.99–125 lipases was determined by using olive oil as a substrate according to reported assay method[25].Lipase was added in the 20%olive oil emulsification solution,which was a pH stabilized mixture of pure olive oil,deionized water and pH 7.0 phosphate buffer solutions.With 15 min of incubation at 40°C,the reaction was stopped by adding 15.0 ml of 95%alcohol solution.The fatty acid liberated from the hydrolysis of olive oil was titrated with 0.02 mol·L?1of NaOH.Oneunit of lipase activity is defined as the amount of enzyme that liberates 1 μmol of fatty acid per min under the assay condition.

2.5.Characterization

The samples were characterized by a Nicolet corporation VATAR-360 Fourier transform infrared(FT-IR)spectrophotometer(USA).X-ray diffraction(XRD)analysis was conducted using an ARL corporation ARL/X'TRA with Cu Kαradiation,operated at 45 k V and 40 m A over 5°＜ 2θ ＜ 70°.The transmission electron microscopy(TEM)images were taken on a FEI Tecnai20 electron microscope operating at 120 kV accelerating voltage.Magnetic measurements were obtained at room temperature with a Lake Shore 7410 vibrating sample magnetometer(VSM).

2.6.Stability of lipase

To investigate thermal and pH stabilities,free or immobilized lipase was incubated in buffers with different pH values or different temperatures for 1 h.The stability of lipases was obtained by measuring residual activity.The relative activity of the lipase is calculated by

where R is the relative activity(%),and A and A0are the residual and initial activity of lipase(U),respectively.

2.7.Reusability assay

Immobilized lipase was recovered by magnet assistance,washing and drying after each hydrolysis reaction.There usability was characterized by residual enzyme activity relative to initial activity.

3.Results and Discussion

3.1.FT-IR analysis

Fig.1 shows the FT-IR spectra of samples P–Fe(a)and P-FeAc(b).As shown in spectrum(a),the bands at 3612 cm?1,3547 cm?1,3415 cm?1and 1030 cm?1are associated with hydroxyl bending of structural OH2,and the band at 472 cm?1is assigned to the bending vibrations of Si–O–Si.The spectrum of P-FeAc exhibits two strong absorptions at 1590 cm?1and 1448 cm?1,due to the symmetric and asymmetric stretching vibrations of the–COO–bonds of acetate anion in a bridging mode of coordination[26].Obviously,P–Fe is transformed to P-FeAC due to the interaction of iron ion in the interlamellar space of palygorskite in acetic acid vapor[27].Moreover,the peak at about 1653 cm?1in spectrum(a)disappears in spectrum(b),which corresponds to hydroxyl bending of zeolitic H2O,which is removed after calcination[28].

Fig.1.FT-IR spectra of samples P-Fe(a)and P-FeAc(b).

3.2.XRD analysis

Fig.2 shows the crystal structures of the bare and magnetically modified palygorskite from XRD.The characteristic diffraction peaks of palygorskite in curves a and b can be assigned to the(110),(200),(130)and(040)planes according to literature[29].Moreover,curve b,apart from the palygorskite reflections,presents some additional peaks originated from iron oxides.Comparison of the observed peak positions with those in the database of Joint Committee on Powder Diffraction Standards reveals the presence of γ-Fe2O3with reflections at about 2θ=30.24°,35.63°,43.28°and 53.73°(card 39–1346).Besides,the reflections at about 2θ =33.15°(card 33–0664)indicate the existence of α-Fe2O3in the MPC.

Fig.2.XRD patterns of palygorskite(a)and MPC(b).

3.3.TEM analysis

Fig.3 show s the morphology of magnetic clay composite from TEM.γ-Fe2O3particles(black spots)disperse quite uniformly over the layered support.The surface coverage of magnetic nanoparticles on palygorskite is high.The nanoparticles exhibit a broad distribution with sizes ranging from 4 nm to 15 nm and an average size of about 9 nm.

3.4.VSM measurement

To investigate the magnetic properties,the magnetization of magnetic clay composite MPC versus the applied field was recorded at ambient temperature.From Fig.4,the saturation magnetization of the composite is found to be 13.4 emu·g?1,suggesting that the magnetic modification is successful.In addition,the coercivity and remanence magnetization are very low and could be ignored.The typical superparamagnetic behavior can intensify the separation and recovery of magnetically modified clay mineral composites[30].

Fig.4.Magnetization curve for the MPC sample at room temperature.

3.5.Optimum pH for lipase immobilization

Table 1 show s the effect of pH of solution on the amount of lipase immobilized and activity of immobilized lipase.The adsorbed amounts of lipase do not present significant difference at pH values of 5.0–9.0.How ever,the activity of lipase bound to the MPC changes with the increase of pH value and attains a maximum value of 963.2 U·g?1at pH 7.0 with activity recovery of 35.37%.It is reported that the optimal pH for the immobilization of lipase is pH 7.0[25].

Table 1Effect of pH on the amount adsorbed and activity of immobilized lipase

Fig.3.TEM images for the MPC.

3.6.Optimum immobilizing time for lipase immobilization

The effect of immobilizing time on lipase immobilization is shown in Table 2 in terms of the amount adsorbed and enzyme activity.The amount of lipase immobilized increases with immobilizing time,reaching 46.3 mg·g?1support with 6 h of immobilizing time.The enzyme activity and activity recovery of immobilized lipase first increase and then decrease with immobilizing time,reaching the maximum values of 2631.6 U·g?1and 96.64%,respectively.It can be explained that with the immobilizing time more than 4 h,Candida sp.99–125 lipase forms a multilayer adsorption and adsorbed amount increases accordingly,while some of enzyme activities cannot be expressed[31].Thus the optimum immobilizing time for the immobilization process is 4 h.

Table 2Effect of time on the amount adsorbed and activity of immobilized lipase

3.7.pH stability of free and immobilized lipases

Fig.5 show s the effect of medium pH on the enzyme activity of free and immobilized lipases after incubating in the pH range of 5.0–9.0 at 40°C for 1 h.The immobilized lipase is stable in the pH range from 5 to 8 while free lipase is stable in the pH range from 7 to 8,indicating that the immobilization appreciably improves the stability of lipase in the acidic region.A similar observation was reported[32].It may be a result of conformation change(exposure of catalytic site)of lipase molecules after immobilization,making the catalytic site more easily accessible to H+or OH?ions[15].

Fig.5.pH stability of free and immobilized Candida sp.99–125 lipases.

3.8.Thermal stability of free and immobilized lipases

It is well know n that enzymes in solution are not stable and their activities decrease gradually in the usage[33].To investigate the thermal stability of immobilized lipase,the residual activities of immobilized and free lipases were determined by measuring the hydrolysis of olive oil after the lipase exposed to temperatures ranging from 20 to 60°C in phosphate buffer(0.1 mol·L?1,pH 7.5)for 1 h.Fig.6 show s that the immobilized lipase is stable and maintains 81.2%of enzyme activity at 40°C,while the activity of free lipase decreases drastically and only 32.7%of the activity is left.The immobilized lipase presents more than 70.4%activity at 50°C,while the free lipase has only 20.9%activity.The thermal stability of immobilized sample is better than that of free sample.The interlayer space of supports can keep lipase from injury from direct exposure to environmental changes.

Fig.6.Thermal stability of free and immobilized Candida sp.99–125 lipases.

3.9.Reuse of immobilized lipase

In general,it is difficult to recover and reuse enzymes.The recovery and reusability of immobilized lipase are important aspects that deserve further investigation.In this study,the immobilized lipase was repeatedly separated and recovered with a magnet in the reaction system,and reused as the biocatalyst for the hydrolysis reaction.The reusability of immobilized lipase is shown in Fig.7.After 8 consecutive operations,the immobilized lipase still retained 75.6%of their original activities.The result show s that the immobilized lipase presents good reusability.Fig.8 indicates that the lipase immobilized on MPC could be separated and recovered easily with a magnet outside the aqueous solution without being centrifuged.

Fig.7.The reusability of immobilized lipase.

Fig.8.The magnetic separation of immobilized lipase by external magnet.

4.Conclusions

In this study,magnetic clay composites with γ-Fe2O3nanoparticles dispersing in clay matrix were prepared,based on the reaction of acetic acid vapor with the iron sites in an iron-exchanged palygorskite to form iron acetate precursor species in the interlayer space of the mineral and further produce magnetic particles with pyrolysis.The magnetically modified palygorskite composite,with saturation magnetization of 13.4 emu·g?1,was used as the support for Candida sp.99–125 lipase immobilization by physical adsorption.It is shown that pH and immobilizing time have significant effects on the adsorption.The immobilized lipase can be separated from the reaction medium by a magnetic field and then reused.The ability of immobilized lipase against temperature and pH is much better than that of free lipase.The immobilized lipase maintains 75.6%of its activity even after 8 cycles of use.