Celite C22, CaCO3, Purolite 200 108/02/5, Cellulose AVICEL PH-101). The activities of the obtained preparates have been tested in esterification of lauric acid ...
Annals of West University of Timisoara Series Chemistry 17 (3) (2008) 61-66
DOUBLE IMMOBILIZATION OF PORCINE PANCREAS LIPASE C r ist in a Za rc u la, L iv ia Cor îc i, R am on a C r o it o ru , F. P ét er “Politehnica” University of Timişoara, Faculty of Industrial Chemistry and Environmental Engineering, C. Telbisz 6, Timişoara 300001, ROMANIA
SUMMARY Sol-gel encapsulation has proven to be a particularly easy and effective way to immobilize enzymes. An important extension of this method is the use of additional porous supports during the sol-gel process. In this work, double immobilization of porcine pancreas lipase (PPL) was investigated, using tetraethoxysilane and methyl- or phenyltriethoxysilane precursors and different porous solid supports (Celite 545, Celite 521, Celite C22, CaCO3, Purolite 200 108/02/5, Cellulose AVICEL PH-101). The activities of the obtained preparates have been tested in esterification of lauric acid with 1-octanol using n-hexane as reaction medium. The presence of methyl and phenyl nonhydrolizable groups in the sol-gel matrix gave recovery yield values of the total activity above 100%. Good esterification activities were obtained when NH3 (catalyst) and Celite 545 (solid support) were used in the immobilization protocol.
Keywords: Porcine pancreas lipase, Sol-gel entrapment, silane adsorption, enantioselectivity
precursors,
INTRODUCTION Bioencapsulation of enzymes into silica matrices prepared by sol-gel techniques has been extensively studied due to their well-known biochemical mechanisms, wide applications, commercial availability, and good stability [1, 2, 3]. An important extension of sol-gel method is the use of additional porous supports during the sol-gel process [4]. This type of “double immobilization” involves deposition of the sol-gel entrapped enzyme on the surface of a porous solid support which can lead to increased activities, whereas the aggregation during the immobilization process and
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diffusional drawback during enzyme utilization could be limited. Obviously, it is difficult to distribute the entrapped enzyme within the support pores whether the gelation process is too fast (seconds). Therefore, is more convenient to use tetraethoxysilane-based precursors that have slower gelation rates than tetramethoxy-silane derivatives [5]. In this work, double immobilization of porcine pancreas lipase (PPL) was investigated, using tetraethoxysilane and methyl- or phenyl-triethoxysilane precursors and different porous solid supports (Celite 545, Celite 521, Celite C22, CaCO3, Purolite 200 108/02/5, Cellulose AVICEL PH-101). Influence of immobilization conditions on the esterification activity and immobilization efficiency was studied. MATERIALS
AND
METHODS
Commercial porcine pancreas lipase (PPL) was purchased from Sigma Chemical Co.(USA). The silane precursors methyl- (MeTEOS) and phenyl-triethoxysilane (PhTEOS) were purchased from Aldrich and tetraethoxysilane (TEOS) from Fluka. Other materials used: tris-(hydroxymethyl)-aminoethan (Loba Chemie), HCl 1N (Chimopar), ethanol (99.2%, Chimopar), sodium fluoride (Fluka), Celite 545 (Merck), Celite 521 (Aldrich), Celite C22 (Loba Chemie), CaCO3 (Loba Chemie), Purolite 200 108/02/5 (Purolite), Cellulose AVICEL PH-101 (Aldrich), 1-octanol (95%, Fluka), lauric acid (Fluka), n-hexane (98%, Merck), n-hexadecane (99%, Merck), were of analytical grade and have been used as purchased. General procedure for immobilization by adsorption Method 1. 150 mg lipase (PPL) was suspended in 4 mL 0.1 M TRIS/HCl buffer solution, pH=8.0 and was added to 1 g (Celite 545, Celite 521, Celite C22, CaCO3, Purolite 200 108/02/5, Cellulose AVICEL PH-101). This mixture was stirred for 1 hour. 40 mL cold acetone (-15ºC) was added in droplets and the immobilized enzyme was filtered. The resulted powder was dried at room temperature. General procedure for immobilization by sol-gel entrapment and adsorption Method 2. The silane precursors in different molar ratio (total 3 mmoles) were mixed with 0.2 mL distilled water, 11 μL of 40mM HCl and in some cases 0.5 mL ethanol, to form a homogeneous sol. After 30 min stirring the enzyme solution (75 mg lipase PPL suspended in 1.1 mL 0.1 M TRIS/HCl buffer solution, pH=8.0) and 200 μL NaF or 100 μL NH3 was added. In the primer phase of gelation 0.5 g adsorbent (Celite 545, Celite 521, Celite C22, CaCO3, Purolite 200 108/02/5 or Cellulose AVICEL PH-101) was added and
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mixed. The resulting gel was kept at room temperature for 24 hours to complete polymerization. The bulk gel was washed with n-hexane (15 mL), filtered, and then dried 48 hours at room temperature. Method 3. The same procedure as described for Method 2 has been employed, excepting that 30 μL of 40mM HCl, without ethanol, were used for preparation of sol. General procedure for immobilization by adsorption and sol-gel entrapment Method 4. The silane precursors (total 3 mmoles) in different molar ratio were mixed with 0.2 mL distilled water, 30 μL of 40mM HCl to form a homogeneous sol. After 30 min stirring 1.1 mL 0.1 M TRIS/HCl buffer solution, pH=8.0 and 200 μL NaF or 100 μL NH3 were added. In the primer phase of gelation this solution was mixed with 0.5 g PPL adsorbed on Celite 521, Celite C22 or CaCO3. The mixture was kept at room temperature for 24 hours to complete the gelation process. General procedure for the esterification studies A 10 mL glass reactor was charged with lauric acid (0.2 mmole), 1-octanol (0.4 mmole), n-hexadecane (30 μL, internal standard for chromatographic analysis), n-hexane (2 mL, solvent) and free (5mg) or sol-gel immobilized PPL lipase (50mg). The mixture was incubated at 40ºC, with continuous stirring. Samples taken at different intervals were analyzed for esterification activity and activity recovery yields using a Dani 86.10 gas chromatograph equipped with flame ionization detector, and a 15 m x 0,32 mm BPX-5 capillary column (SGE Australia Pty. Ltd.). Esterification activity after 24h was calculated based on ester yields and it was expressed as micromoles of ester formed per hour per mg of immobilized enzyme. The activity recovery yields (%) of the enzymatic activity following immobilization were determined as the ratio of total activity of the immobilized enzyme and total activity of the free enzyme. RESULTS
AND
DISCUSSION
Double immobilization of porcine pancreas lipase (PPL) has been performed by three different methods, as described, and activities of the obtained preparates were tested in esterification of lauric acid with 1-octanol in n-hexane reaction medium. Adsorption of enzymes on porous inorganic supports is a widely used immobilization technique, but has the disadvantage that the enzyme-support bond is weak and the enzyme can be easily desorbed.
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As sol-gel immobilization resulted in preparates with high stability but lower esterification activity, we combined sol-gel entrapment with adsorption, to merge the advantages of both methods. We intended to obtain an immobilized enzyme with uniform distributed sol-gel network on the adsorbent surface, leading to increased preparate stability in comparison to the simple adsorption method. The investigated immobilization techniques differed by the precursors silane nature, catalyst nature and addition of ethanol in some cases to improve the system homogeneity. Using Celite C521 as solid adsorbent in the initial phase of gelation, the influence of immobilization conditions was investigated.
200
NH3
Recovered total activity (%)
240
180 160 140 120 100 80
450
Native enzyme
Recovered total activity (%)
220
Native enzyme Adsorption on Celite 521 NaF+EtOH NaF NH3+EtOH
260
400
NH3 catalyst NaF catalyst
350 300 250 200 150 100
60 50
40 20
0
0 MeTEOS:TEOS=1:1
PhTEOS:TEOS=1:1
Celite 545
Celite Celite 521 C22
CaCO3
Purolite
AVICEL
Figure 1. Influence of immobilization protocol
Figure 2. Influence of the type of adsorbent and
(silane precursor and catalyst nature, addition of
catalyst on the immobilization efficiency of double
ethanol) on catalytic efficiency of double
immobilized PPL (Method 2).
immobilized porcine pancreatic lipase (PPL).
As results from Figure 1, the highest recovery yields were obtained using NH3 as catalyst. A slight decrease of activity using MeTEOS/TEOS (1:1 molar ratio) as silane precursors and almost a 2.5-fold decrease for immobilizates from PhTEOS/TEOS (1:1 molar ratio) was observed by adding ethanol during the sol-gel process. In case of NaF catalyst, adding ethanol as additive led to complete activity loss. Testing different support materials for immobilizations by entrapment combined with adsorption, good recovery yields were obtained when NH3 was used as catalyst and Celite 521, Celite C22 and CaCO3 as adsorbents (Figure 2).
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Native enzyme o NaF, 25 C o NaF, 5 C o NH3, 25 C
150
o NH3, 5 C 100
50
160
Recovered total activity (%)
Recovered total activity (%)
200
140 120
Native enzyme NaF, entrapment+adsorption NaF, adsorption+entrapment NH3, entrapment+adsorption NH3, adsorption+entrapment
100 80 60 40 20 0
0 CeliteC22
CaCO3
Figure 3. Influence of the aging temperature on
Celite C22 CaCO3 Celite521 MeTEOS:TEOS=1:1molar ratio
Figure 4. Influence of the immobilization protocol
the immobilization efficiency of double immobilized
and catalyst nature on the immobilization
PPL (Method 4). MeTEOS and TEOS (1:1 molar
efficiency of double immobilized PPL (Method 2
ratio) were used as silane precursors, Celite C22
and Method 4).
and CaCO3 as adsorbents.
Presuming that deactivation could occur during gel maturation (24 hrs), we reduced the aging temperature from 25°C to 5°C. The recovery yield values showed a slight increase only when Lewis acid catalysis (NaF) was used, for both solid supports (Figure 3). It results that not the temperature caused lipase inactivation. Possible explanation could be steric hindrance or improper distribution of the enzyme on the outer surface of the support during gelation. Supposing that uniform distribution of the enzyme on the support surface could enhance the catalytic activity (as shown for simple adsorption), we also tested the possibility to immobilize the lipase first by adsorption, followed by blending with the solgel in initial gelation phase. It can be observed (Figure 4) that activities were lower as for sol-gel entrapped enzyme disposed on the adsorbent surface.
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CONCLUSIONS Immobilization of pancreatic lipase was performed by different methods: adsorption, sol-gel entrapment combined with adsorption and adsorption followed by solgel entrapment. Considering only adsorption, the best porous solid support proved to be Celite 545, but Celite C22 and CaCO3 gave high immobilization efficiencies, too. Using the combined immobilization method, in some cases even an enhancement of recovered total activity (values higher then 100%) was observed. Good results were obtained with TEOS and MeTEOS or PhTEOS as second precursor, NH3 as catalyst and Celite 521, Celite C22 and CaCO3 as adsorbents. Reducing the maturation temperature of gel from 25oC to 5oC (at same maturation time, 24 hrs), resulted in a slight increase of recovery yields only when Lewis acid catalysis was used. Thus, lipase inactivation during aging is not related to temperature. Inversion of the immobilization sequence by performing first the adsorption and subsequently the sol-gel entrapment did not provide better results compared to disposal of the sol-gel entrapped enzyme on the adsorbent surface. REFERENCES 1. 2. 3. 4. 5.
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Furukawa S., Onu T., Ijima H., Kawakami K., “Activation of protease by sol-gel entrapment into organically modified hybrid silicates“, Biotechnology Letters, 24 (2002), 13–16 Kandimalla V.B., Tripathi V. S., Ju H., “Immobilization of Biomolecules in Sol-Gels: Biological and Analytical Applications”, Critical Reviews in Analytical Chemistry, 36 (2006), 73–106 Péter F., Poppe L., Kiss C., Szõcs-Biró E., Preda G., Zarcula C., Olteanu A., Biocatal. Biotransform., 23 (2005), 251-260 Guisan J.M., “Immobilization of enzymes and cells”, Second edition, 2006, Humana Press Inc., Totowa, New Jersey. Péter F., Zarcula C., Kiss C., Csunderlik C., Conference of Chemical Engineering, Veszprém April 2007, p. 118-122
