Functionalized Magnetic Bacterial Cellulose Beads as Carrier for Lecitase® Ultra Immobilization
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Bacterial cellulose spheres subjected to amination and inlaid modification with superparamagnetic molecules were analyzed with regard to possibility of their application as an immobilization carrier of Lecitase® Ultra (LU) enzyme. The starting point to obtain the carrier was synthesis of bacterial cellulose spheres performed in shaking cultures of Komagataeibacter xylinus. These spheres were subsequently subjected to a multi-stage modification to increase the efficiency of the immobilization process and to separate product from the reaction medium. Maximal yield of Lecitase® Ultra immobilization equaled 70%. It was also found that immobilization process did not affect the pH and LU temperature optimum. Moreover, immobilized enzyme exhibited similar temperature stability profile as its native form. The immobilization process did not significantly affect the enzyme KM value. The immobilized enzyme retained over 70% of its initial activity after 8 cycles of use. The immobilized enzyme displayed good storage stability and retained 80% of its initial activity after 4 weeks at 4 °C. The potential application of such modified cellulose-based carrier may be correlated with lower costs of process thanks to higher enzyme’s reusability in comparison to unbound enzyme. Moreover, data presented in the current study may serve as proof of a concept of cellulose-based carrier utilization for immobilization of enzymes other than LU and of high industrial importance.
KeywordsBacterial cellulose Modification Immobilization Lecitase® Ultra
Immobilization allows to use enzymes in many industrial branches thanks to the possibility of their repeated use (it lowers costs of new enzymes’ production) and increased stability related with severe for biological macromolecules conditions of reactions, especially in industrial settings . The process of enzymes’ immobilization is referred by some authors as “an art,” and it requires, more than anything else, using a suitable carrier that meets all pre-designed requirements [2, 3]. In the era of searching for environment-friendly technologies, natural biopolymers are gaining more and more recognition . One of such biopolymers, with such unique properties, as high homogeneity or high Young’s modulus, is bacterial cellulose (BC) . Depending on the bacterial culturing conditions, BC takes form of flat membranes (in static cultures), or spheres (when shaking is performed). The BC size, mechanical properties, degree of crystallinity, or polymerization also depends on the culture conditions [6, 7]. Despite many advantages, purified BC lacks specific functional groups that allow to permanently bind enzymes to its fibrils. Interactions between enzyme and BC are result of hydrophobic or hydrogen bond interactions which are susceptible to temperature changes, pH, or ionic strength. The BC matrix for enzyme immobilization may be modified in situ by supplementation of the culture medium with, e.g., carboxymethylcellulose, chitosan, alginate, or lignin derivatives [8, 9, 10, 11]. Another way to modify the physicochemical properties of BC is to modify the conditions of drying of BC membranes, which affect its porosity and the ability to adsorb the enzyme . BC can also be modified by introduction of epoxide groups using epichlorohydrine or 1,4-butanediol diglycidyl ether and further amination or oxidization using 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) to introduce carboxyl groups on its surface [13, 14, 15, 16]. These treatments considerably increase the possibilities of using BC as enzyme or other active substances’ carrier.
Lipolytic enzymes, despite many years of research on their properties and applicability, still attract great attention thanks to their high biotechnological potential. For example, in the food industry, lipolytic enzymes are increasingly used as replacements or additives to traditional emulsifiers [17, 18]. Lipases have also become one of the most important groups of enzymes used in organic syntheses. The ability to catalyze ester synthesis and transesterification reactions allows the use of this enzyme as biocatalysts in the production of biodegradable polyesters [19, 20]. Thus, methods increasing lipases’ efficiency and operational stability, including enzymes’ immobilization, are constantly developed .
The aim of the present study was to analyze the properties of BC spheres obtained from a Komagataeibacter xylinus shaking culture, modified with polyethyleneimine and ferromagnetic particles, for use as a carrier for the immobilization of enzymes of lipolytic activity, namely, Lecitase® Ultra.
Material and Methods
An enzymatic preparation displaying activity of phospholipase A and lipase (trade name Lecitase® Ultra; E.C.184.108.40.206, LU, Sigma-Aldrich) was used. K. xylinus ATCC 53582 strain was used for the production of BC. All reagents used were of analytical quality and were purchased from Sigma-Aldrich (Poland) or Chempur (Poland).
Bacterial Cellulose Beads Preparation
The culture of K. xylinus was carried out in a 25-ml Erlenmeyer flask with Herstin-Schramm (HS) medium containing glucose 20 g/l, yeast extract 2.0 g/l, peptone 2 g/l, citric acid 1.15 g/l, Na2HPO4 2.7 g/l, and MgSO4 ∙7H2O 0.06 g/l with 1% ethanol. Prepared medium was inoculated with 1 ml of 2-week-old starter culture. The cultivation was carried out at 28 °C, on a laboratory shaker at 180 rpm for 24 h. After this time, the formed “spheres,” hereinafter referred to as “beads” of BC (BCB), were picked up using a laboratory strainer and rinsed in deionized H2O (dH2O) to remove culture medium. The BCB was then digested with 0.1 M NaOH at 80 °C for 30 min (3×) to remove bacterial cells and residual nutrient components. Finally, the cellulose was rinsed again with dH2O until the pH stabilization at 7.0. Cellulose beads prepared in this way were stored at 4 °C until use.
Modification of Bacterial Cellulose Beads and Lecitase® Ultra Immobilization
Initially, the BCB-PEI-Fe were activated with 1% glutaraldehyde in 100 mM phosphate buffer at pH 7.0 by adding 2 volumes of solution in relation to the BC volume and mixed using a roller shaker at room temperature for 1 h. The activated BCB-PEI-Fe was rinsed three times in 100 mM phosphate buffer of pH 7.0 with 100 mM NaCl to remove unbound glutaraldehyde. Immobilization of the enzyme was performed by adding 2 volumes of the enzyme solution to 1 volume of BCB-PEI-Fe activated with glutaraldehyde and incubation at 4 °C for 24 h. After the incubation, the supernatant was removed, and the obtained matrix was washed twice with 100 mM phosphate buffer pH 7.0. Then, 4 volumes of sodium borohydride (1 mg/ml in 100 mM phosphate buffer of pH 7.0) solution were added to the immobilized enzyme and incubated at 4 °C for 1 h. In the next stage, BCB-PEI-Fe was rinsed once in 100 mM phosphate buffer of pH 7.0 containing 100 mM NaCl and 0.25% Triton-X100, and then twice in 100 mM phosphate buffer of pH 7.0.
Determination of the Activity of the Native and Immobilized Enzyme
As substrate for the determination of LU activity, 4-nitrophenol palmitate (pNPP) was used at a concentration of 0.5 mM in Tris-Cl 50 mM pH 8.5 in the presence of 0.25% Triton X-100. The stock solution of pNPP (3.3 mg per ml) was prepared in 2-propanol, mixed with 10 volumes of assay buffer, and heated to 60 °C for 15 min to obtain transparent solution. Activity was determined by measuring the absorbance changes for 5 min at 30 °C at wavelength λ = 348 nm (ε = 5.400 mM−1 cm−1) using a microplate reader. One Lecitase® Ultra unit releases 1 μmol pNPP per minute. The specific activity of the enzyme activities was expressed as units per mg protein, immobilized enzyme as units per g of wet weight of the carrier.
Protein Concentration Determination
Protein concentrations were assayed by Bradford method with bovine serum albumin as a standard .
Efficiency of Binding of the Enzyme to the Carrier
In order to test the ability of binding the enzyme with the carrier, Lecitase® Ultra formulation was diluted in phosphate buffer of pH 7.0 in the range of activity of 500 to 1200 mU/ml and protein concentration range of 0.45 to 1.8 mg/ml. The prepared dilutions were then mixed with the activated carrier. Next, prepared samples were incubated overnight at 4 °C. After this time, activity of LU was measured in each of the trials according to the methodology given above.
Optimum pH of Free and Immobilized Enzymes
In order to check the pH optimum for free and immobilized enzymes, the enzyme activity was measured at pH 6.0, 6.5, 7.0, 7.5 (50 mM phosphate buffer), and at pH 8.0, 8.5, and 9.0 (50 mM Tris-Cl buffer).
Temperature Optimum of Free and Immobilized Enzymes
The temperature optimum of the free enzyme was measured at 25, 30, 35, 40, 45, 50, 55, and 60 °C. Before adding the enzyme, substrate solution was equilibrated and next 10 ml of the enzyme solution in 50 mM phosphate buffer of pH 7.5 to 300 μl of substrate and incubated for 5 min at suitable temperatures. Determination of the optimum temperature of the immobilized enzyme was carried out by transferring 300 μl of the heated substrate to the tube with buffer containing ~25 mg of the immobilized enzyme. The mixture was then incubated for 5 min at appropriate temperatures. The activity was expressed in relative terms taking the highest activity at a given temperature for 100%.
Thermal Stability of Free and Immobilized Enzymes
The thermal stability of the free and immobilized enzymes was determined at a selected temperature 40, 50, and 60 °C by incubation in 1 ml of 50 mM phosphate buffer pH 7.5 for 10–60 min. The residual activity of free and immobilized enzyme was expressed in relative terms taking initial activity as 100%.
Determination of Kinetic Parameters Free and Immobilized Lecitase® Ultra
Kinetic parameters KM and Vmax of immobilized and free LU were determined by measuring the rate of hydrolysis of pNPP. Initial velocities were determined for substrate concentrations in the range from 0.05 to 1.5 mM in 50 mM Tris-Cl of pH 8.5 in the presence of 0.25% Triton X-100. The kinetic constants were determined according to the Michaelis-Menten kinetics model using a non-linear regression model using the Origin8pro program.
Effect of Reusability on Immobilized Enzyme Stability
The reusability of immobilized enzyme was determined by using the immobilized beads for 10 times. After each cycle of reaction, the BCB-PEI-Fe beads were removed and washed with phosphate buffer 100 mM (pH 7.5) to clean it from residual substrate and products of reaction from immobilized beads. Next, the immobilized beads were transferred into fresh reaction medium to start reaction. The initial activity was considered as 100%.
Determination of Storage Stability Immobilized Lecitase® Ultra
The immobilized enzyme was stored as suspension in 50 mM phosphate buffer of pH 7.0, and activity was determined several times during 28 days of storage at 4 °C. At this time, an equal amount of carrier with immobilized enzyme was collected every 2–3 days and its activity was measured. The initial activity was considered as 100%.
Carrier Property Determination
Scanning Electron Microscopy
Scanning electron microscopy (SEM) was performed using a high-resolution field emission gun scanning electron microscope (ZEISS EVO MA 25, Oberkochen, Germany). The samples of modified BCB were firstly fixed by 3% glutaraldehyde solution in phosphate buffer of pH 7.0 by 30 min. Next, samples were flushed by deionized water to remove extended amount of glutaraldehyde. Then, fixed BCB were dehydrated in graded series of ethanol dilution 10–100% (5 min each) and finally dried with the hexamethyldisilazane (HMDS) chemical drying series, ethanol:HMDS 1:1, 2:1, and 100% HMDS two times 20 min and allow to air dry overnight under a fume hood. Afterwards, prepared beads were used to characterize the morphology of the functionalized BCB. Prior to the SEM, all the samples were fixed onto SEM by the sputtering with Au/Pd (60:40) using Q150R ES device (Quorum Technologies, Laughton, UK).
Attenuated Total Reflectance Fourier Transform Infrared Spectral Studies of Modified Cellulose Beads
Samples before attenuated total reflectance Fourier transform infrared (ATR-FTIR) analysis were dried at room temperature for 24 h. The analysis was carried out using a Bruker spectrophotometer with an ATR-FTIR adapter. The spectra were collected in the range of 4000–400 cm−1 with a resolution of 8 cm−1 (64 scans). The obtained ATR-FTIR spectra were analyzed using the Omnics and Origin8pro software.
Results and Discussion
SEM Morphology of Modified BCB
Effectiveness of Immobilization Process on Modified BCB
Optimum pH for Immobilized Lecitase® Ultra
Influence of Temperature on Activity and Stability of Immobilized Lecitase® Ultra
Effect of Immobilization on Catalytic Constants of Lecitase® Ultra
Influence of immobilization process on KM and Vmax and catalytic efficiency Kcat/KM of Lecitase® Ultra
Vmax (μmol l−1 min−1)
Kcat × 103 (s−1)
Kcat/KM (M−1 s−1)
8.7 ± 0.7
0.40 ± 0.07
1.9 ± 0.15
4.8 ± 0.8
49.6 ± 4.7
0.45 ± 0.05
9.3 ± 0.9
20.9 ± 2.3
Reusability of Immobilized Lecitase® Ultra
Storage Stability of Immobilized Lecitase® Ultra
Effective immobilization of Lecitase® Ultra on functionalized spheres of bacterial cellulose extends the possibility of its use in various types of bioprocesses. Moreover, the immobilized LU on the carrier sensitive to the external magnetic field has the main advantage of being easily separable from the reaction medium. As a result, utilizing these obvious benefits, the enzyme immobilized on this type of carrier can be used in processes carried out using bioreactors supported with various types of magnetic field . In this type of reactors, the use of carriers’ sensitivity to magnetic field gives the opportunity of mixing without use of traditional mechanical stirrers, thus increasing the stability of the carrier due to minimizing the mechanical shear, while mixing and also improving the mass transfer . One of the frequent uses of lipases and also LU is their ability to synthesize various types of esters that can be used as biofuels or precursors of substances desired in the pharmaceutical or cosmetic industries. The utilization of magnetic field in processes catalyzed by immobilized enzymes can potentially affect the reaction mode and in this way giving the possibility of obtaining new compounds or increasing the process effectiveness .
Bacterial cellulose beads modified by polyethyleneimine and ferromagnetic material were used as a new support material for the immobilization of Lecitase® Ultra. The important advantage of using a natural biopolymer as BC is also significant reduction in costs associated with its purification and preparation of the carrier for further modification. Properties of this new carrier allow for efficient immobilization of analyzed enzyme and also simplification during its manipulation by easy separation form reaction medium with use a regular magnet only. Immobilization process did not significantly influence on main catalytic parameters of LU (pH, temperature optima). However, immobilized Lecitase® Ultra showed fourfold lower catalytic efficiency, which on the other hand was compensated by high resistance to reuse.
The resulting carrier is characterized by unique properties allowing its further modification to be used for the immobilization of many other enzymes that can be applied in biotechnological process, in which magnetic field is applied as a force for a biocatalyst separation and modification of its properties.
The research was supported partially by the National Centre for Research and Development in Poland (Grant No. LIDER/011/221/L-5/13/NCBR/2014).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflicts of interest.
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