Immobilization of bile salt hydrolase enzyme on mesoporous SBA-15 for co-precipitation of cholesterol

International Journal of Biological Macromolecules 63 (2014) 218–224

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Immobilization of bile salt hydrolase enzyme on mesoporous SBA-15 for co-precipitation of cholesterol Pallavi Bhange a,∗,1 , N. Sridevi b,1 , Deu S. Bhange c , Asmita Prabhune b , Veda Ramaswamy a a

Catalysis Division, National Chemical Laboratory, Pune 411008, India Biochemical Sciences Division, National Chemical Laboratory, Pune 411008, India c Department of Chemistry, Shivaji University, Kolhapur 416004, Maharashtra, India b

a r t i c l e

i n f o

Article history: Received 16 July 2013 Received in revised form 15 November 2013 Accepted 18 November 2013 Available online 25 November 2013 Keywords: SBA-15 Bile salt hydrolase Co-precipitation of cholesterol

a b s t r a c t We describe herein a simple and effective strategy for immobilization of bile salt hydrolase enzyme by grafting glutaraldehyde groups inside channels of APTES functionalized SBA-15. The increase in glutaraldehyde concentration prevents leakage of enzyme but showed a steep decrease in enzyme activity in the immobilized matrix. So the degree of cross-linking should be the minimum possible to ensure sufficient stability without loss of activity. Cross-linking carried out with 0.1% glutaraldehyde concentration showed the highest activity, so this was used in all further experiments. Physico-chemical characterizations of the immobilized enzyme were carried out by XRD, N2 adsorption, TEM, FTIR and 29 Si CP-MAS NMR techniques. Immobilized BSH exhibits enhanced stability over a wide pH (3–11) and temperature range (40–80 ◦ C) and retains an activity even after recycling experiments and six months of storage. From our in vivo research experiment toward co-precipitation of cholesterol, we have shown that immobilized BSH enzyme may be the promising catalyst for the reduction of serum cholesterol levels in our preliminary investigation. Enhancement in pH stability at the extreme side of pH may favor the use of immobilized BSH enzyme for drug delivery purpose to with stand extreme pH conditions in the gastrointestinal conditions. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Bile salt hydrolase (BSH) (cholylglycine hydrolase; EC (3.5.1.24), which belong to Ntn hydrolase super family is considered as an important enzyme as it reduces serum cholesterol levels with its bile salt deconjugation property (Structure 1). Hypercholesterolemia (elevated blood cholesterol levels) is considered a major risk factor for the development of coronary heart disease.

Pharmacologic agents such as fibrates, nicotinic acid, bile acid sequestrants, and statins were generally used for the treatment of high cholesterol. Although these drugs effectively reduce cholesterol levels but they are expensive and are known to have severe side effects [1,2]. In recent years, interest has risen in the possibility of using bile salt deconjugation property to lower serum cholesterol level in hypercholesterolemic patients and prevent hypercholesterolemia in normal people.

∗ Corresponding author. Present address: Department of Chemistry, Shivaji University, Kolhapur 416004, Maharashtra, India. Tel.: +91 231 2609164. E-mail addresses: [email protected], [email protected] (P. Bhange). 1 These authors contributed equally to this work. 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.11.008

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Bile salt hydrolase (BSH) enzyme is well known for its activity in this aspect, as it catalyzes the hydrolysis of glycine- and taurineconjugated bile salts into amino acid residues and bile acids. Bile salts are the water-soluble end products of cholesterol synthesized in the liver. Bile salts play an important role in lipid digestion and absorption in the small intestine [3]. After absorption, the mixture of bile salts is partly returned to the liver by the hepatic portal circulation in the process known as enterohepatic circulation. Deconjugation of bile salts by the bacterial BSH enzyme results in the production of free bile acids, which are less efficiently reabsorbed than their conjugated counterpart results in the excretion of larger amounts of free bile acids in feces. Therefore, deconjugation of bile salts could lead to a reduction in serum cholesterol by increasing the demand for cholesterol for de novo synthesis of bile acids to replace those lost in feces [3,4]. In recent years, silica based mesoporous materials with unique structural properties have inspired the usage of this type of materials for controlled delivery systems of biologically active molecules [5]. The silica matrices offer a number of advantages over conventional organic polymers for immobilization owing to their high surface area and pore volume to host the required amount of biomolecules/drugs, higher mechanical strength, enhanced stability, and openness to wide variety of chemical modification. Besides this they are biocompatible, non-toxic and highly resistant against microbial attacks and organic solvents. The regular repeating mesoporous structures of these silica-based solid supports has motivated the adsorption in their pores and subsequent release of a large variety of biologically active species such as proteins, polypeptides or amino acids [6–8]. Several proteins have been demonstrated to retain their functionality without being denatured inside the frameworks of silica-based mesoporous materials [9–19]. These findings open up many expectations for the research in the adsorption of proteins and other biologically active agents into mesoporous silicas. Valler-Regi et al. reported in 2001 for the first time the possibility of using MCM-41 matrices as a delivery system of Ibuprofen [5]. The structure of MCM-41 with cylindrical mesopores and free silanol groups facilitated the controlled adsorption and liberation of variety of pharmaceutical compounds [20–22]. Apart from this, other mesoporous silica materials with different pore structure and surface properties were also being used for drug delivery purpose include SBA-15, SBA-16, SBA-1, SBA-6 [23–25]. During the last several years, SBA-15, which is a polymertemplated silica with hexagonally ordered mesopores, has been extensively studied and is being evaluated for numerous applications in the field of biocatalysis [26,27]. Among all the inorganic supports, the mesoporous silicas SBA-15 with high surface area, tunable pore diameter, high degree of structural ordering, ease of synthesis, thicker pore walls and high hydrothermal stability compared to MCM-41 render the opportunity of loading and releasing large quantities of biomedical agents [28,29]. It is well known that the two factors are important for enzyme immobilization inside the porous matrices, one is the design of the support materials and the other is choice of immobilization method. Surface functionalization of the silica surface by grafting organic groups selective to the nature of drug molecules to be adsorbed (to allow better control over drug loading and release) has been the key factor in the designing of advanced drug delivery systems as it creates a beneficial microenvironment for optimum catalytic activity of the enzyme [30–33]. In this work, we present for the first time the immobilization of bile salt hydrolase enzyme by grafting glutaraldehyde groups inside the channels of amino-functionalized SBA-15 materials and the effect of silica as host matrix on the enzyme kinetics. We report in detail our studies on the effects of glutaraldehyde concentration on enzyme loading, biocatalytic activity, reusability and storage stability of the immobilized BSH were tested.

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2. Experimental 2.1. Synthesis of the support material SBA-15 mesoporous molecular sieve and amino functionalized SBA-15 (Am-SBA-15) were prepared and characterized as described by us earlier (Supplementary information, S1.1 and 1.2) [34]. 2.2. Immobilization of bile salt hydrolase Bile salt hydrolase enzyme has been linked onto amino functionalized SBA-15 through a molecular coupling agent, glutaraldehyde (50% stock solution). Cross-linking of bile salt hydrolase enzyme with Am-SBA-15 was carried out by varying the glutaraldehyde concentrations from 0.025 to 1%. Glutaraldehyde was added to 0.5 g of functionalized SBA-15 silica powder containing bile salt hydrolase enzyme (17.89 U) in 0.1 M potassium phosphate buffer pH 6.5. The reaction mixture was stirred for 24 h at 5–10 ◦ C followed by centrifugation at 10,000 rpm for 2 min. These samples were centrifuged to separately analyze the supernatant for any enzyme activity (to check the amount of the enzyme released from the material) and to determine the actual enzyme-loaded materials on the functionalized SBA15 silica support. The immobilized matrix was washed with 0.1 M potassium phosphate buffer pH 6.5. The amount of enzyme immobilized on SBA-15 was measured by determining the difference in the protein concentration of the enzyme before and after the immobilization reaction by Micro Lowry method [36]. The BSH immobilized SBA-15 sample is designated as BSH-SBA15. 2.3. Characterization of immobilized BSH enzyme The powder X-ray diffraction (XRD) patterns of SBA-15, Am-SBA-15 and BSH-Am-SBA-15 samples were collected on X’Pert Pro (M/s Panalytical) diffractometer using Cu Ka radiation and proportional counter as detector. The BET surface area of the samples was determined by N2 adsorption at 77 K by using Autosorb-1 instrument. The specific surface area, SBET , was determined from the linear part of the BET equation (p/p0 = 0.05–0.31). The pore size distributions were obtained from the Barrett–Joyner–Halenda (BJH) method applied to the desorption part of the isotherm. Scanning electron micrographs of the samples were recorded on a JEOL-JSM-5200 microscope to observe the morphology of the particles. TEM of the samples were recorded on a JEOL Model 1200EX microscope operating at 100 kV. Immobilized BSH enzyme sample was dispersed in isopropyl alcohol and deposited and dried on a Cu grid on 400-mesh size. 29 Si CP-MAS experiment were performed on a Bruker MSL-300 NMR spectrometer at the Larmor frequencies of 59.595 MHz for 29 Si. The chemical shift values (in ppm) were calculated with TMS as reference for 29 Si measurements. The FTIR spectra were recorded in the 400–4000 cm−1 region in diffuse reflectance mode (spectral resolution = 4 cm−1 ; number of scans = 100) using FT-IR spectroscopy (Shimadzu 8201 PC spectrophotometer). 2.4. Biocatalytic activity measurement/evaluation of the enzymatic activity BSH assay was carried out by determining the amounts of the amino acids liberated from conjugated bile salts. To the portions (20 mg) of BSH-SBA-15 immobilized enzyme and (10 ␮l) of free enzyme 20–30 ␮l of reaction buffer (0.1 M sodium phosphate, pH 6.5), 10 ␮l of 1 mM glycodeoxycholic acid (GDCA) substrate and 10 ␮l DTT (100 mM) was added. The reaction mixture was

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incubated at 40 ◦ C for 10 min. Samples (50 ␮l) were removed after 10 min of incubation and mixed immediately with 50 ␮l of 15% trichloroacetic acid (TCA). Subsequently, the samples were centrifuged at 10,000 rpm for 2 min to remove the precipitate. An aliquot of 50 ␮l was removed from the reaction mixture and to this 50 ␮l of 2% Ninhydrin reagent was added, and the total reaction mixture was boiled for 14 min. Then the tube was cooled and the reaction mixture was diluted to 1 ml with distilled water and the absorbance was measured at 570 nm. A blank experiment was also carried out by the above assay procedure without adding soluble free enzyme or immobilized enzyme to the mixture. One unit of BSH activity was defined as the amount of enzyme that liberated 1 ␮mol of amino acid from the substrate per minute. The specific activity was defined as the number of activity units per mg of protein. The protein concentration was determined by the Folin Lowry method. A UV-1601 PC spectrophotometer (Shimadzu) was used to determine protein concentration in the immobilization matrix at a wavelength of 750 nm. The difference in the protein concentration of the free enzyme before addition of the SBA-15 support and after adsorption of the enzyme on the support yielded the enzyme loading on the support (mg of immobilized enzyme per gram of SBA-15). The activity as well as protein concentration was also determined in the supernatants liquid. The activity of the BSH immobilized SBA-15 was compared with the free enzyme as a function of the degree of cross-linking. 2.5. Effect of temperature and pH and determination of kinetic parameters Conditions such as temperature and pH are tested for the immobilized BSH enzyme and compared to the enzyme in solution. pH optima was done by assaying the enzyme activity in buffer between pH 3.0 and pH 11.0 under standard assay conditions. The temperature optimum was determined in 0.1 M sodium phosphate buffer at pH 6.5 by using the standard reaction mixture. In addition, thermostability and pH stability of the immobilized enzyme was also compared to that of the soluble form of the enzyme. The pH dependent variation in the biocatalytic activity of BSH in free as well as encapsulated form was evaluated in the pH range of 3.0–11.0. Samples were incubated at the different pH for 1 h at RT and then the residual activity was determined at pH 6.5 by the standard assay. All the reactions were carried out after pre-incubating the free and immobilized enzyme for 1 h at different pH and measuring the biocatalytic activity at pH 6.5 and 40 ◦ C as described earlier. The temperature stability of the immobilized BSH enzyme was checked by pre-incubating it for 1 h at different temperatures in the range 40–80 ◦ C and was compared with an identical amount of free enzyme under similar conditions. All the reactions were carried out after pre-incubation for 1 h at different temperatures and measuring the biocatalytic activity at pH 6.5 and 40 ◦ C as described earlier. The stability of immobilized enzymes was tested by storing the BSH immobilized enzyme sample for 6 month at 4 ◦ C. 2.6. Co-precipitation of cholesterol with immobilized BSH enzyme The cholesterol (70–100 ␮g/ml) was added to 100 mg of BSHSBA-15 in buffer at pH 6.5 and incubated at 37 ◦ C and the samples were collected after 30 min and 1 h respectively [37]. After the incubation period, the mixture was centrifuged and the remaining cholesterol concentration in the supernatant was determined using a modified colorimetric method as described by Rudel and Morris (Supplementary information, S1.3) [38]. Blank and standard experiments were also carried out at the same time along with immobilized enzyme for cholesterol precipitation.

Activity of the supernant liquid, units/ml/min

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Glutaraldehyde Conc (%) Fig. 1. The degree of cross-linking carried out using various glutaraldehyde concentrations from 0.025, 0.05, 0.1, 0.25, 0.5 and 1% on Am-SBA-15 sample.

3. Results and discussion 3.1. Enzyme adsorption and effect of cross-linking on immobilized BSH enzyme After 24 h in an aqueous BSH solution during immobilization, the glutaraldehyde modified materials showed an enzyme loading almost 3–4 times higher than that of the unmodified and aminofunctionalized (Am-SBA-15) materials. Cross-linking of bile salt hydrolase with Am-SBA-15 was carried out with different glutaraldehyde concentrations. During cross-linking, glutaraldehyde reacted with the amino groups of functionalized SBA-15. Now the other free aldehyde group of the functional supports ensures strong binding and negligible leaching into the surrounding solution (in the supernatant) because an imine binding may form easily between the free aldehyde groups of the support and the amino groups on the protein molecules. The amount of enzyme immobilized onto the functionalized silica, Am-SBA-15 was monitored by UV–vis spectrophotometry, that is, by measuring the enzyme absorbance before and after immobilization (after separating the supernatant via centrifugation). The band used to monitor the immobilization of BSH was 570 nm. The activity in the supernatant was used to quantify the amount of enzyme bound to the functionalized silica support for specific activity determination. With increase in glutaraldehyde concentration, we observed almost complete binding of the enzyme to the functionalized silica support which was determined by monitoring the decrease in the absorbance at 570 nm in the supernatant. This electrostatic attracting interaction seems to be stronger and higher enzyme retention can be expected with increase in the glutaraldehyde concentration. As a consequence, the loading increases and the release slow down, with the subsequent control in the release kinetics. The effect of degree of cross-linking indicates that increase in glutaraldehyde concentration (from 0.025 to 1%) prevent leakage of enzyme (in the supernatant) but showed a steep decrease in enzyme activity in the immobilized matrix after 0.1% glutaraldehyde concentration (Fig. 1). So the degree of cross-linking should be the minimum possible to ensure sufficient stability without loss of activity. Cross-linking carried out with 0.1% glutaraldehyde concentration showed the highest activity (Fig. 2), so this was used in all further experiments. The immobilization and the activity measurement were performed at pH 6.5, which indicate that 0.1% glutaraldehyde provided

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10 Acivity of the matrix, units/mg/min

9 8 7 6 5 4 3 2 1 0.0

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Glutaraldehyde Conc (%) Fig. 2. The activity of the immobilized BSH-SBA-15 sample as a function of the degree of cross-linking.

enough aldehyde groups on the surface of the support that were capable of bounding to BSH. Hence, it is clear that the glutaraldehyde in the immobilized enzyme play an important role in binding of the enzyme to the Am-SBA-15 support. 3.2. Structural and textural properties immobilized BSH enzyme Low-angle X-ray diffraction studies have confirmed the long range mesoscopic order of SBA-15 support is maintained in the BSH-SBA-15 sample (Fig. S2, Supporting information). A strong decrease in the intensity of the low-angle peak (10) is observed after BSH adsorption onto Am-SBA-15 at pH 6.5. Although this cannot be interpreted as a severe loss of structural order, yet it is likely that the larger contrast in density between the silica walls and the open pores relative to that between the silica walls and the BSH molecule is responsible for the observed decrease in intensity. In our earlier report [35], we found that ∼11.2% of amino groups are utilized for the immobilization and thereby reduces the surface area and the pore volume of SBA-15 material, conforming the success of grafting. The mesopores filling with enzyme molecules can be assessed by N2 adsorption. All the samples gave typical irreversible type IV adsorption isotherm with an H1 hysteresis loop (Fig. S3, Supporting information). Subsequently, when 4.1 mg of BSH was adsorbed on the Am-SBA-15 at a solution of pH 6.5, it further reduces the surface area from 605 to 188 m2 /g, which is ∼69% reduction in total surface area. The pore volume is reduced from 0.99 to 0.4 cm3 /g which is 60% reduction of the specific pore volume of the parent

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Am-SBA-15. This is consistent with the decrease in effective mesoporous diameter (from 6.6 to 6.5 nm), which indicates a considerable fraction of the enzyme is located inside the mesopores. The significant reduction in the mesopore volume points to significant pore blockage at the pore openings upon BSH adsorption. Because most of the pores (∼6.5 nm) are only a few A´˚ smaller or larger than the BSH molecules (5.0 nm) [39], it is reasonable to imagine that protein molecules (depending on their orientation at the pore opening) can partially enter the pores and block them at their openings. Due to the relatively larger size of the protein, nitrogen molecules may still adsorb into the channels where the BSH molecules have entered the pores. This significant reduction in the surface area is a result of the localized tight packing of the BSH molecules in the porous networks. The TEM image shown in Fig. 3 further justify that structural ordering and long range order is maintained in the BSH immobilized sample. Indeed, we observe the surface coverage after a reasonable amount of the BSH was immobilized on amine functionalized SBA-15. The BSH immobilized enzyme is bound to the surface or partially enter the pores of the SBA-15 particles and are not trapped within the pores of the SBA-15. This is understandable since the size of the BSH enzyme in this study is ca. 5 nm while the Am-SBA-15 pore diameter is estimated to be 6.5 nm (Fig. 3a and b). We have noticed that the immobilized BSH enzyme sample stored for six months at room temperature also show long-range order with hexagonal array of the cylindrical pores, which further confirms the stability of the material (Fig. 3c). This is fairly a good indication that structure of the SBA-15 matrix is retained after immobilization and subsequent enzyme treatment in aqueous medium. 3.3.

29 Si

CP-MAS NMR and FT-IR analysis

The solid-state 29 Si CP-MAS NMR spectra of two samples namely Am-SBA-15 and BSH-SBA-15 samples are displayed in Fig. 4 which further confirms the grafting of the modifying agent. The solid state 29 Si CP-MAS NMR spectrum of Am-SBA-15 sample shows the lines with chemical shift at −102 and −112 ppm which has been attributed to the presence of Q3 [Si(OSi)3 (OH)] and Q4 [Si(OSi)4 ] environment associated with the silica framework and T2 and T3 resonances between −50 and −69 ppm corresponding to two and three methoxy groups from the silane reacting with the surface. This suggests the organic moiety is covalently bound to the silica surface. The BSH immobilized samples also show similar behavior which indicates that the structure of the support is remained intact after immobilization and subsequent treatment conditions. FTIR spectra of SBA-15, Am-SBA-15 and BSH-SBA-15 samples are shown in Fig. S4 (Supporting information). The IR spectra of the Am-SBA-15 samples show the absorption band at 1468 and 1550 cm−1 attributable to C N stretching vibration. The appearance of these bands shows that the reaction is occurring between

Fig. 3. Transmission electron micrograph of (a, b) BSH-SBA-15 and (c) dry BSH-SBA-15 powder samples.

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Fig. 5. The effect of different pH on stability of the immobilized BSH-SBA-15 enzyme and compared to the free enzyme in solution.

Fig. 4.

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Si CP-MAS NMR spectra of (a) Am-SBA-15 and (b) BSH-SBA-15 samples.

3-amino propyltrimethoxysilane and silanol groups on the SBA15 silica surface. The IR spectra of BSH-SBA-15 sample exhibits a strong absorption band at 1635 and 1549 cm−1 which is attributed to C N stretching mode. On the other hand, the absence of C O and NH2 peaks and appearance of C N (azomethine) peak indicate that the expected imino compound was formed by condensation of terminal amine groups of the functionalized silica with the glutaraldehyde. The C N peak may also formed with the free aldehyde (C O group) of glutaraldehyde and the amino groups on the surface of the protein. 3.4. Biocatalytic activity measurement As mentioned before, the textural properties of mesoporous silica play an important role in loading and release kinetics of biologically active agents and it may also influence adsorption by promising host–guest interactions. It is observed that almost 57% of protein gets encapsulated in the silica matrix. Immobilized enzymes maintained good catalytic activity and exhibit 6.2 ␮mol/g/min of the specific activities than free enzymes (3.8 ␮mol/g/min) without significant leaching of enzyme over time, which is the additional advantage of immobilized enzyme being easy to recover and recycle. Particularly exciting observation is that the immobilized enzyme could be reused without significant loss in activity, which indicates potentially exciting industrial/biomedical application of this support. The cross-linking of protein also provides additional stabilization against unfolding and thus leads to both chemical and mechanical stability. These results clearly underline the remarkable reuse characteristics of the immobilized enzyme. 3.5. Effect of pH and temperature on the activity of the free and immobilized BSH The pH optima of bile salt hydrolase were shifted to lower pH after immobilization (Fig. S5, Supporting information). Shift in pH from 9.0 to 8.0 could be due to the loss of charges present on the surface of the molecule during treatment with glutaraldehyde. On the basis of the preceding arguments and the observation, we suggest that the immobilized enzyme experience a different (local) pH in pores of matrix than in the buffer solution and therefore shift in the pH optima was observed [35]. As shown in Fig.

S6 (Supporting information), the temperature profile for activity of the immobilized enzyme was shifted to higher temperature after immobilization compared to free enzyme. The immobilized enzyme retained (100%) residual activity than that of free enzyme (71%) at 60 ◦ C whereas on incubation at 70 ◦ C, it retained 82% of its activity compared to free enzyme (60%). Immobilized enzyme maintained good catalytic activity at high temperature range; this may be due to the stability of mesoporous materials, which prevents the denaturation of enzyme at higher temperature. Broadening of the temperature activity profile in the case of immobilized enzyme near its optimum activity indicates that the bound enzyme is stable over a wider temperature range as compared to the native enzyme. Fig. 5 shows the plot of the pH stability of free BSH enzyme in solution and immobilized BSH enzyme in the pH range 3–11. It is seen that the enzyme was stable at pH values from 3.0 to 11.0; at pH above 9.0 and below 4.0 it was rapidly inactivated. pH stability of immobilized enzyme showed highest enzymatic activity compared to the free BSH enzyme. At higher range of pH, free enzyme was less stable compared to immobilized enzyme. Enhancement in pH stability at the extreme side of pH may favor the use of immobilized enzyme for drug delivery purpose to with stand extreme pH conditions in the gastrointestinal conditions. This high lights the important role of support in enhancing and stabilizing the catalytic activity of the BSH in the immobilized state. We have investigated the thermostability of free and the immobilized BSH enzyme to determine whether the matrix could protect the immobilized enzyme from thermal denaturation. Thermal stability of free and the immobilized BSH enzyme were investigated over the temperature range 40–80 ◦ C. Maximum BSH activity for free enzyme was observed at 60 ◦ C, whereas for immobilized enzyme it was observed to be 70 ◦ C. The immobilized enzyme was stable at temperatures from 40 ◦ C to 80 ◦ C and retained (88%) residual activity than that of free enzyme (64%) after 1 h at 60 ◦ C whereas on incubation at 70 ◦ C, it retained 100% of its residual activity compared to free enzyme (40%) as shown in Fig. 6. The increase in the thermal stability of the immobilized enzyme may arise from the conformational integrity of the enzyme structure after binding to the support through amine and cysteine residues present in the enzymes. There is a correlation between protein stability and the number of arginine residues on protein surface. Thermophilic proteins have higher arginine content on the protein surface. The thermotolerance of enzymes increases because of stronger hydrogen bonding of the large guanidinium group of arginine with nearby polar groups. Enzymes find a more stable environment upon encapsulation in a silica host, because the

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Realative % of cholesterol

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0 Fig. 6. The effect of different temperature on stability of the immobilized BSH-SBA15 enzyme and compared to the free enzyme in solution.

siliceous framework creating a cage thus prevents leaching of proteins and protecting the enzyme from aggregation and unfolding. 3.6. Evaluation of kinetic parameters Michaelis–Menten Kinetics of BSH immobilized SBA-15 were compared with free enzyme in solution. A plot of reaction activity ‘V’ as a function of the substrate concentration [S] is shown in Fig. 7. As seen from the kinetic constants obtained from the nonlinear regression of Michaelis–Menten plots, the overall activity of the immobilized enzyme is slightly greater than that of enzyme in solution. The values of Km of the immobilized enzymes (7.65 ␮M) were higher than those of the free enzymes (2.43 ␮M), indicating the presence of partitioning and diffusional effects in the pores of the SBA-15 matrix. 3.7. Storage stability We examined the critical question of the storage and stabilities of the BSH immobilized SBA-15 catalysts. Furthermore, immobilized enzymes have a limited lifespan, and thus, deactivation is not always avoidable. Dry and wet storage of the immobilized BSH at room temperature over six months resulted in the marginal loss of the initial activity, demonstrating their working stability in aqueous and low-water media. During long term usage the enzyme support has to be stable. We have checked the biocatalytic activity of the BSH immobilized enzyme which has stored for 6 months 100

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Time, min Fig. 8. Co-precipitation of cholesterol over immobilized BSH-SBA-15 enzyme.

as a function of temperature and pH (Fig. S7, Supporting information). We want to emphasis on the point that the immobilized BSH enzyme still retains the activity even after 6 months of storage showing a great storage stability of the immobilized BSH on mesoporous SBA-15 silica sphere. This improved stability of immobilized enzyme would therefore lead to reduced conformational freedom with respect to change in environmental parameters such as pH variation and temperature alteration. It is also clear that the SBA-15 silica matrix exhibits as an excellent support for BSH enzymes that enhances biocatalytic activity as well as stability and provide high biocompatible environment for immobilized enzymes. High porosity and surface area is advantage of SBA-15 silica matrix, which allow one to immobilize higher amounts of enzymes without significantly reducing their accessibility. This silica lattice provides structural strength, durability and increases the tolerance of temperature and pH of the enzyme. 3.8. Co-precipitation of cholesterol In the present study, we have examined the effectiveness of BSH-SBA-15 enzyme for the co-precipitation of cholesterol. The biocatalyst was added to a cholesterol solution and the amount of cholesterol remaining is determined in order to study the activity of supported BSH enzyme. Co-precipitation of cholesterol was determined by the difference in the cholesterol level in the control which is incubated with and without the BSH immobilized enzyme. It can be inferred that there is a gradual decrease in the relative percentage of the cholesterol over a period of time on BSH-SBA-15 sample as shown in Fig. 8. The immobilized BSH sample has shown the interesting and superior activity for the co-precipitation of the Cholesterol in our preliminary investigation. Cholesterol co-precipitation experiment indicates that immobilized BSH enzyme may be the promising catalyst beneficial for bile deconjugation activity in vivo.

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4. Conclusions

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[S] Fig. 7. Michaelis–Menten plot for free and immobilized BSH-SBA-15 enzymes.

APTES functionalized mesoporous silica SBA-15, have been used for the immobilization of bile salt hydrolase for the first time. The immobilized sample ensures strong binding with negligible leaching from the SBA-15 matrix in addition to improving the thermal and pH stability compared to the soluble form of enzyme. Our studies indicate that the immobilized BSH enzyme is the promising catalyst for the co-precipitation of cholesterol and hence beneficial for reduction of serum cholesterol level. Thus the research on

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