9488 J. Am. Chem. Soc., Vol. 121, No. 41, 1999
Gill et al.
provide both nonpolar/hydrophobic and polar/hydrophilic func-
tionality are critical for achieving optimal lipase activation and
stabilization.9,10 Second, since commercial lipases are often
crude preparations with lipase contents as low as 0.1-5% w/w,
the immobilization protocol should enable the selective binding
of the enzyme from a complex protein mix. Also, lipase
immobilization efficiency, activation, and dispersion should be
maximized, and protein aggregation minimized,9,11 at the
elevated protein loads required for high catalytic density
immobilizates.
Despite the synthetic versatility of lipases and their heavy
commercial utilization, comparatively little attention has been
directed toward refining immobilization methods. Traditional
methods have largely relied upon adsorption onto hydrophobic
polymers,9,11 and only recently have more promising techniques
such as sol-gel entrapment and cross-linked crystals been
utilized.2,3 Although polymers such as polypropylene, alkyl-
agarose, polyacrylates and polystyrene can selectively bind and
activate lipases,9 enzyme stability can be compromised by
hydrophobic polymer surfaces,11 and enzyme activation and
adsorption capacity are typically limited by surface coverage
restrictions.9 Sol-gel entrapment offers mixed functionality
matrixes for efficient and stable activation2l-2q, but is limited
to low catalytic densities due to its poor discrimination toward
mixtures of proteins in crude lipases, enzyme precipitation in
sol-gel solutions at higher protein concentrations, and surface
rather than bulk matrix capture of lipase2l-2q. Cross-linked lipase
crystals can be prepared in a preactivated form, and display
excellent activity and stability, but to date this approach has
only been applied to two lipases.3a,-f,i,j
(2) (a) Avnir, D.; Braun, S. Biochemical Aspects of Sol-Gel Science and
Technology; Kluwer: Hingham, MA, 1996. (b) Avnir, D.; Braun, S.; Lev,
O.; Ottolenghi, M. Chem. Mater. 1994, 6, 1605. (c) Gill, I.; Ballesteros, A.
J. Am. Chem. Soc. 1998, in press. (d) Avnir, D.; Braun, S.; Lev, O.;
Ottolenghi, M. Sol-Gel Optics; Klein, L., Ed.; Kluwer Academic: Berlin,
1994; pp 539-582. (e) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem.
Mater. 1994, 6, 1605. (f) Armon, R.; Dosoretz, C.; Starosvetsky, J.;
Orshansky, F.; Saadi, I. J. Biotechnol. 1996, 51, 279. (g) Pope, E. J. A. J.
Sol-Gel Sci. Technol. 1995, 4, 225. (h) Campostrini, R.; Carturan, G.;
Caniato, R.; Piovan, A.; Filippini, R.; Innocenti, G.; Cappelletti, E. M. J.
Sol-Gel Sci. Technol. 1996, 7, 87. (i) Rietti-Shati, M.; Ronen, D.;
Mandelbaum, R. T. J. Sol-Gel Sci. Technol. 1996, 7, 77. (j) Braun, S.;
Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990,
10, 1. (k) Shabat, D.; Grynszpan, F.; Saphier, S.; Turniansky, A.; Avnir,
D.; Keinan, E. Chem. Mater. 1997, 9, 2258. (l) Reetz, M. T. AdV. Mater.
1997, 9, 943. (m) Reetz, M. T.; Zonta, A.; Simpelkamp, J. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 301. (n) Reetz, M. T.; Zonta, A.; Simpelkamp, J.
Biotechnol. Bioeng. 1996, 49, 527. (o) Reetz, M. T.; Zonta, A.; Simpelkamp,
J.; Ko¨nen, W. Chem. Commun. 1996, 1397. (p) Reetz, M. T.; Zonta, A.;
Simpelkamp, J.; Rufinska, A.; Tesche, B. J. Sol-Gel Sci. Technol. 1996,
7, 35. (q) Kuncova, G.; Miroslav, S. J. Sol-Gel Sci. Technol. 1997, 8,
667.
(3) (a) Lalonde, J. J. CHEMTECH 1997, 27, 38. (b) Zelinski, T.;
Waldmann, H. Angew. Chem., Int. Ed. Engl. 1997, 36, 722. (c) St. Clair,
N. L.; Navia, M. A. J. Am. Chem. Soc. 1992, 114, 7314. (d) Persichetti, R.
A.; St. Clair, N. L.; Griffith, J. P.; Navia, M. A.; Margolin, A. L. J. Am.
Chem. Soc. 1995, 117, 2732. (e) Lalonde, J. J.; Govardhan, C.; Khalaf, N.;
Martinez, A. G.; Visuri, K.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117,
6845. (f) Khalaf, N.; Govardhan, C. P.; Lalonde, J. J.; Persichetti, R. A.;
Wang, Y.-F.; Margolin, A. L. J. Am. Chem. Soc. 1996, 118, 5494. (g) Wang,
Y.-F.; Yakovlevsky, K.; Zhang, B.; Margolin, A. L. J. Org. Chem. 1997,
62, 3488. (h) Sobolov, S. B.; Draganoiu, M.; Bartoszko-Malik, A.;
Voivodov, K. I.; McKinney, F.; Kim, J.; Fry, A. J. J. Org. Chem. 1996,
61, 2125. (i) Margolin, A. L. Trends Biotechnol. 1996, 14, 223. (j)
Perischetti, R. A.; Lalonde, J. L.; Govardhan, C. P.; Khalaf, N. K.; Margolin,
A. L. Tetrahedron Lett. 1996, 37, 6507.
(4) (a) Yang, Z.; Mesiano, A.; Venkatasubramanian, S.; Gross, S. H.;
Harris, J. M.; Russell, A. J. J. Am. Chem. Soc. 1995, 117, 4843. (b) Wang,
P.; Sergeeva, M. V.; Lim, L.; Dordick, J. S. Nat. Biotechnol. 1997, 15,
789. (c) Panza, J. L.; LeJeune, K. E.; Venkatsubramanian, S.; Russell, A.
J. ACS Symposium Series 680; American Chemical Society: Washington,
DC, 1998; p 134. (d) LeJeune, K. E.; Panza, J. L.; Venkatsubramanian, S.;
Russell, A. J. ACS Polym. Prepr. 1997, 38, 563. (e) Yang, Z.; Williams,
D.; Russell, A. J. Biotechnol. Bioeng. 1995, 45, 10.
Results and Discussion
Preparation of Candida rugosa Lipase-PHOMS Adsor-
bates. While studying the encapsulation of proteins in matrices
derived from poly(hydroxymethylsiloxane) (PHOMS),12,13 we
discovered that this polymer was a very efficient adsorbent for
a variety of hydrophobic molecules, including proteins such as
lipases, phospholipases, papain, â-glucosidase, and thermolysin,
binding up to 26-34% of its weight of the enzymes from
aqueous solutions.14 Further investigation revealed some unusual
properties of PHOMS that made it a promising matrix for
immobilizing lipases: (i) PHOMS displays an exceptional
capacity for binding lipases, with maximal uptakes being in the
range of 28-41% w/w; (ii) PHOMS is available as a micro-
dispersed powder (2-13 µm) with a high surface area (451-
872 m2 g-1) and pore volume (0.38-1.23 mL g-1), which
undergoes considerable surface area/porous volume expansion
(5) (a) LeJeune, K. E.; Russell, A. J. Biotechnol. Bioeng. 1996, 51, 450.
(b) LeJeune, K. E.; Frazier, D. S.; Caranto, G. R.; Maxwell, D. M.; Amitai,
G.; Russell, A. J. Med. Def. Biosci. ReV., Proc. 1996, 1, 223. (c) Havens,
P. L.; Rase, H. F. Ind. Eng. Chem. Res. 1993, 32, 2254.
(6) (a) Wartchow, C. A.; Wang, P.; Bednarski, M. D.; Callstrom, M. R.
J. Org. Chem. 1995, 60, 2216. (b) Wang, P.; Hill, T. G.; Wartchow, C. A.;
Huston, M. E.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.; Callstrom,
M. R. J. Am. Chem. Soc. 1992, 114, 378. (c) Hill, T. G.; Wang, P.; Huston,
M. E.; Wartchow, C. A.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.;
Callstrom, M. R. Tetrahedron Lett. 1991, 32, 6823. (d) Wang, P.; Hill, T.
G.; Bednarski, M. D.; Callstrom, M. R. Tetrahedron Lett. 1991, 32, 6827.
(7) (a) Theil, F. Chem. ReV. 1995, 95, 2203. (b) Mori, K. Synlett 1995,
11, 1097. (c) Sih, C. J.; Gu, R.; Crich, J. Z.; Brieva, R. In Stereocontrolled
Organic Synthesis; Trost, B. M., Ed.; Blackwell: Oxford, 1994; p 399. (d)
Gill, I. In Synthesis in Lipid Chemistry; Tyman, J. H. P., Ed.; Royal Society
of Chemistry: Cambridge, 1996; p 175. (e) Gandhi, N. N. J. Am. Oil Chem.
Soc. 1997, 74, 621. (f) Akita, H. Biocatal. Biotransform. 1996, 13, 141.
(g) Patel, M. T.; Nagarajan, R.; Kilara, A. Chem. Eng. Commun. 1996,
152/153, 365. (h) Santaniello, E.; Ferraboschi, P.; Grisenti, P. Enzyme
Microb. Technol. 1993, 15, 367.
(8) (a) Ferrato, F.; Carriere, F.; Sarda, L.; Verger, R. Methods Enzymol.
1997, 286, 327. (b) Verger, R. Trends Biotechnol. 1997, 15, 32.
(9) (a) Bastida, A.; Sabuquillo, P.; Armisen, P.; Ferna´ndez-Lafuente, R.;
Huget, J.; Guisa´n, J. M. Biotechnol. Bioeng. 1998, 58, 486. (b) Bryjak, J.;
Bachmann, K.; Pawlow, B.; Maliszewska, I.; Trochimczuk, A.; Kolarz, B.
N. Chem. Eng. J. 1997, 65, 249. (c) Bosley, J. A.; Clayton, J. C. Biotechnol.
Bioeng. 1994, 43, 934. (d) Gitlesen, T.; Bauer, M.; Adlercreutz, P. Biochim.
Biophys. Acta 1997, 1345, 188. (e) Ruckenstein, E.; Wang, X. Biotechnol.
Bioeng. 1993, 42, 821. (f) Malcata, F. X.; Reyes, H. R.; Garcia, H. S.; Hill,
C. G., Jr.; Amundson, C. H. J. Am. Oil Chem. Soc. 1990, 67, 890.
(10) Investigations on the interactions of lipases with Langmuir-Blodgett
Layers (LBLs) and engineered polymers have demonstrated that hydrophobic
surfaces can initiate gross conformational disturbances in the adsorbed
species, leading to protein unfolding, and ultimately, diminished activity
and stability.11
(11) (a) Wannerberger, K.; Arnebrant, T. Langmuir 1997, 13, 3488. (b)
Wannerberger, K.; Arnebrant, T. J. Colloid Interface Sci. 1996, 177, 316.
(c) Schroen, C. G.; Stuart, M. A.; Van Der Voort Maarschalk, K. Langmuir,
1995, 11, 3068. (d) Duinhoven, S.; Poort, R.; Van Der Voet, G.; Agterof,
W. G. M.; Norde, W.; Lyklema, J. J. Colloid Interface Sci. 1995, 170,
351.
(12) Lu, S.; Melo, M. M.; Zhao, J.; Pearce, E. M.; Kwei, T. K.
Macromolecules 1995, 28, 4908.
(13) PHOMS can be prepared by oxidizing poly(hydrogenmethylsiloxane)
with dimethyl dioxirane (DMDO),12 or on the large scale more conveniently
by transfer dehydrogenation with acetone, using Zn(II)/Sn(II) as catalysts.
(14) The addition of PHOMS (20% w/w of a 50% w/w paste in 4:1
water-propan-2-ol) to aqueous protein solutions (10 mg mL-1 in 0.1 or
0.2 M metaphosphate buffer, pH 7, containing 20 mM calcium acetate, 5
°C), at an applied protein load of 10% w/w of PHOMS, gave the following
activity immobilizations: papain, 96%; thermolysin, >99%; almond â-D-
glucosidase, 98%; C. rugosa lipase-B, >99%; S. chromofuscus phospho-
lipase D, 98%. Adsorption took 10-30 min, and less than 3% leaching of
protein/activity was detected upon washing (3 × 10-fold volumes of buffer,
5 °C, 1 h).