Selective Precipitation of Proteins from Pancreatin Using Designed

May 11, 2007 - organic antisolvents in a drowning-out process. First, the solubility of pancreatin was measured in water- organic solutions when the o...
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Ind. Eng. Chem. Res. 2007, 46, 4289-4294

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SEPARATIONS Selective Precipitation of Proteins from Pancreatin Using Designed Antisolvents Ji-Hwan Hwang,† Hyun-Su Kim,‡ Jong-Min Kim,§ Sang-Mok Chang,§ In-Ho Kim,| and Woo-Sik Kim*,† Department of Chemical Engineering, Kyunghee UniVersity, Yongin 449-701, South Korea, Nensys Co., Ltd., Suwon 441-113, South Korea, Department of Chemical Engineering, Dong-A UniVersity, Busan 604-714, South Korea, and Department of Chemical Engineering, Choongnam National UniVersity, Daejeon, 305-764, South Korea

The feasibility of controlling the selective precipitation of pancreatic proteins was investigated using designed organic antisolvents in a drowning-out process. First, the solubility of pancreatin was measured in waterorganic solutions when the organic species and their composition in the solution were varied. Due to the hydrophilicity of the proteins, solubility was rapidly reduced when the organic species fraction in the solution was increased. This reduced solubility was further amplified with a less polar organic species. Plus, the pancreatin solubility as a predictor was found to agree well with the solution polarity as a single parameter, expressed in terms of the Hildebrand solubility parameter. The precipitation of pancreatin was then carried out with various antisolvents composed of alcoholic and nonalcoholic species. The precipitation was promoted along with an antisolvent of a lower polarity, which was also functionally related to the solution polarity. The amylase, lipase, and protease proteins contained in the pancreatin displayed different precipitation behaviors depending on the polarity of the antisolvent. Thus, it was possible to selectively separate the proteins and control their composition in the precipitated pancreatin. Introduction In the area of bioactive medicine, the selective precipitation of proteins has attracted much interest in relation to controlling the composition and purity of protein precipitates from mixtures. For example, pancreatin of protein mixture is normally obtained by extraction from a mammalian pancreas and characterized mainly by activities of amylase, lipase, and protease. For pharmaceutical uses as an anti-inflammatory, anticancer, and antipancreatitis agent, and digestive aid, the pancreatin should be purified and selectively designed by selective precipitation as a medical agent for a particular disease based on the composition of amylase, lipase, and protease in the precipitate. To assist with protein precipitation, the solubility of proteins has already been extensively investigated with regard to the molecular size, shape, electrostatic charge, isoelectric point (pI), and hydration of the protein and amino acid composition and sequence in the protein. Thus, since protein solubility is minimized near the isoelectric point of a solution, it increases as far as the solution pH deviates from the isoelectric point.1,2 Desnuelle3 reported that lipase (pI ) 5.0) could be isolated from amylase4 (pI ) 7.0) and proteases (pI > 8.3) through precipitation. In addition, the use of differential precipitation5 has been suggested for selective protein separation from a mixture via adjusting the solution conditions. Various models have already been developed to predict the solubility of a protein, including the use of different physico* To whom correspondence should be addressed. Tel.: +82-31-2012576. Fax +82-31-202-1946. E-mail: [email protected]. † Kyunghee University. ‡ Nensys Co., Ltd. § Dong-A University. | Choongnam National University.

chemical parameters of the solvent, such as the dielectric constant6,7 , dipole moment6,7 µ, empirical solvent polarity parameter7 ET, and hydrophobicity8,9 log P, all of which are commonly related to the polarity and hydrophobicity of the solvent. For example, in the study of lysozyme solubility in various organic solvents (more than 30 kinds of solvents) by Chin et al. , although the solubility related to the solvent polarity is implicitly suggested, the correlation between the solubility and solvent polarity is not developed. Here, many models of partition coefficient, dielectric constant, dipole moment, empirical solvent polarity parameter and Hildebrand solubility parameter are adopted to estimate the polarity of pure solvent.10,11 However, although such models are effectively used to estimate the solvent polarity and to predict the solubility, it is difficult to expand these models to multiple-component solvent systems due to the problems involved in determining the required parameter values in complex systems. For protein purification, precipitation is most widely adopted in unit operations due to the advantages of fast processing, high yield, low operating cost, and easy scale-up.12 According to previous reports, when performing the precipitation of proteins with different antisolvents, such as ethanol, methanol, dioxane, acetone, tetrahydrofurans, methylpentanediol, and polyalcohols, the amount and purity of protein precipitate were found to vary with the organic species of the antisolvent and the degree of the solubility drop.1,2,13,14 In these studies, the influence of the antisolvents on the precipitation was evaluated based only on a single organic species and the fraction in the solution, and not with a mixture of species. Plus, little has been reported on a quantitative description of the antisolvent influence of various organic species, which could help predict the precipitation.

10.1021/ie061280f CCC: $37.00 © 2007 American Chemical Society Published on Web 05/11/2007

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Shin et al.15,16 demonstrated that certain ionic surfactants of sodium [bis-2-ethylhexyl] sulfosuccinate (AOT) can be a good additive to improve the selectivity of lysozyme without deactivation for precipitation from a mixture with xylanase. However, in the case of lipase precipitation from pancreatin, severe deactivation of the protein results from the use of nonsteroidal surfactants as well as ionic surfactants of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB).17 Furthermore, such surfactants are not recommended for protein separation in industry due to their toxicity. Accordingly, the present study attempted to design antisolvents for the selective separation of amylase, lipase, and protease from pancreatic proteins using precipitation. Thus, many organic species of alcohol and nonalcohol were tested in singlecomponent and multicomponent systems, plus the influence of the antisolvent on the precipitation was described in terms of its polarity, as expressed by the Hildebrand parameter, thereby allowing the design of optimal antisolvents.

Figure 1. Solubility of pancreatic proteins in methanol-, ethanol-, n-propyl alcohol, and acetone-aqueous binary mixtures at 25 °C.

Materials and Methods Materials. Porcine pancreatin (9X, USP), supplied by Nensys Co., Ltd. (Suwon, South Korea), was used without further treatment as the raw protein mixture. The antisolvents used for the precipitation included methanol (MeOH), ethanol (EtOH), n-propyl alcohol (n-PrOH), isopropyl alcohol (i-PrOH), dimethylformamide (DMF), acetonitrile (AcN), dimethyl sulfoxide (DMSO), and acetone (AC), all purchased from Carlo-Erba Reagenti (ACS grade, Italy). Solubility. The solubility of the pancreatin was measured in various water-organic binary solutions with methanol, ethanol, n-propyl alcohol, and acetone, while the solution fractions were varied from 30 to 80 vol %. Here, the solubility was defined as the weight of total pancreatin proteins equilibrated in unit volume of solution, with the dimensions of milligram per milliliter [mg/mL]. An equilibrium of pancreatin proteins in the water-organic solution was achieved by dissolving the excess pancreatin proteins in 100 mL of the binary solutions with magnetic stirring for 1 day. The undissolved proteins were then removed by centrifugation and microfiltration. Finally, the equilibrium concentration of pancreatin in the various solutions was analyzed using the Miller-modified Lowry method18,19 with bovine serum albumin (Sigma-Aldrich, St. Louis, MO) as the standard. Precipitation. The precipitation (so-called drowning-out precipitation) of the pancreatic proteins was performed by adding various antisolvents (80 mL) to the pancreatin feed solution (20 mL) prepared by dissolving 45 g of pancreatin proteins into 1 L of distilled water. In the present study, the concentration of pancreatin feed solution was kept constant at 45 mg/mL, plus the volumetric ratio of the antisolvent to the pancreatin solution was fixed at 4:1. Thereby, it could be said that the amount of pancreatin proteins initially loaded into the 100 mL reactor was always constant at 900 mg. After 1 h of precipitation at 25 °C, the precipitated proteins were isolated by centrifugation (10 min, 3000g, 25 °C) and the pancreatin was estimated using the Miller-modified Lowry method.18,19 Analysis of Proteins. The protein profiles of the precipitates were obtained using an electrophoretic analysis,20 and the molecular weight distribution of the proteins analyzed using phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) as the standards (Bio-Rad, Hercules, CA). The amylolytic, lipolytic, and proteolytic activities of the precipitated precipitates were

Table 1. Solubility Parameter Values of Water and Neat Organic Solvents at 25 °C8 solvent

δ (MPa1/2)

deionized water (DW) methanol (MeOH) ethanol (EtOH) dimethylformamide (DMF) dimethyl sulfoxide (DMSO) n-propyl alcohol (n-PrOH) acetonitrile (AcN) isopropyl alcohol (i-PrOH) acetone (AC)

47.9 29.6 26.0 24.8 24.5 24.3 24.3 23.5 20.2

also measured to estimate the quantities of amylase, lipase, and protease, respectively, the three characteristic proteins of pharmacopeia pancreatin.21 Results and Discussion Solubility of Pancreatic Proteins. The solubility of the pancreatin was investigated in various water-organic solutions with different compositions, as shown in Figure 1. Since pancreatin is composed of many proteins, such as protease, lipase, amylase, and other unknown proteins, it is difficult to measure its precise equilibrium concentration (solubility) in pure water. Plus, a pancreatin concentration of more than 100 mg/ mL in water produces a paste. However, in the water-organic solutions with an organic fraction above 30%, an equilibrium concentration was clearly observed and dramatically reduced when the organic fraction was increased. Among the various water-organic binary solutions, methanol reduced the pancreatin solubility from 6.8 to 2.2 mg/mL (68% reduction of solubility) when the methanol fraction was increased from 30 to 80%, plus there was also a significant drop in the solubility when the binary solution included ethanol (95% reduction of solubility), n-propyl alcohol (almost 100% reduction of solubility), and acetone (almost 100% reduction of solubility). The reduced solubility in the water-alcoholic mixtures was due to the interaction of hydrophilic protein groups with nonpolar organic compounds. In addition, a bulk displacement of water and partial immobilization of the water molecules by the hydration of the nonpolar organic compounds occurred in these solutions,1 resulting in the solubility reduction of the proteins. The relationship between the pancreatin solubility and the solution was also predicted in terms of the polarity of the

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Figure 2. Correlation between solubility of pancreatic proteins and solubility parameter values of organic-aqueous binary mixtures. Solubility results from Figure 1 were replotted versus the solubility parameter values of the used binary mixtures in this work.

solution estimated using a solubility parameter model that was first proposed by Hildebrand.8 According to the model, the solubility parameter is directly dictated by the cohesive energy (Ecoh,i) composed of a linear combination of contributions from the dispersion interaction (Ed,i), polar interaction (Ep,i), and hydrogen bonding interaction (Eh,i), defined as

δi )

x

Ecoh,i ) Vi

x

Ed,i + Ep,i + Eh,i Vi

(1)

where δi is the solubility parameter [MPa1/2] and Vi is the molar volume of species i. Since the cohesion parameters are related to the corresponding interaction energies as

Ed,i + Ep,i + Eh,i ) δd,i2 + δp,i2 + δh,i2

(2)

the solubility parameter can be rearranged as

δi ) xδd,i2 + δp,i2 + δh,i2

(3)

The polarity of the water-organic solution (δm) was then estimated using a simple rule of mixing:

δm )

∑i xiδi

where xi is the volume fraction.

(4)

Figure 3. Correlation between solubility results in simple organic-aqueous binary mixtures and in extended ternary and quaternary ones. Each mixture composition and its symbol are depicted in Table 2, in detail.

As summarized in Table 1, the pure organic compounds of methanol, ethanol, n-propyl alcohol, and acetone exhibited relatively low polarities (solubility parameter of 29.6-20.0 MPa1/2) when compared with water (solubility parameter of 48 MPa1/2). From the experimental results (Figure 1), it appeared that the pancreatin solubility in the binary solutions dropped more significantly when the binary solution was prepared with less polar alcohols. Thus, when the pancreatin solubility was replotted with respect to the polarity of the solubility parameter of the binary solution, which ranged from 25 to 43 MPa1/2 for all the organic species and fractions in the binary solutions, a reasonably good correlation (r2 ) 0.9171) appeared, as shown in Figure 2. The reliability of the above correlation between the pancreatin solubility and the solution polarity was also examined when other organic compounds were used, such as isopropyl alcohol (i-PrOH), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetone (AC), and acetonitrile (AcN), to confirm whether the protein solubility was dictated by the polarity of the solution, regardless of the species and composition of the solution. In addition to using binary (water and one organic species) solutions, ternary (water and two organic species) and quaternary (water and three organic species) solutions were also investigated, resulting in variations of the solution polarity according to the species and composition of organic compounds in the solution, as summarized in Table 2. As such, the three kinds of solutions having the same polarity (28.0 MPa1/2) were prepared with 78% AC and 28% DW (binary solution), 50% DMF, 30% AC and 20% DW (ternary solution), and 30%

Table 2. Solubility Parameter Values and Their Symbols of the Binary, Ternary, and Quaternary Solvents Used in This Work at 25 °C

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Figure 4. Precipitates of pancreatic proteins by precipitation in organicaqueous binary mixtures at 25 °C.

Figure 5. Correlation between precipitates of pancreatic proteins and solubility parameter values of simple organic-aqueous binary mixture (open symbols) and extended ternary and quaternary ones (filled symbols).

DMSO, 30% i-PrOH, 20% AC, and 20% DW (quaternary solution). Then, the pancreatin solubilities in those solutions were compared with the solubility-polarity correlation obtained in Figure 2. Similar attempts were carried out at the solution polarities of 32.0, 36.0, and 40.0 MPa1/2. As shown in Figure 3, the pancreatin solubility in the solutions also matched the correlation (solid line) obtained from the solubility in the wateralcohol binary solutions (Figure 2). Therefore, these experimental results would seem to support the above hypothesis that the protein solubility is predetermined by the polarity of the solution, as a single parameter, regardless of the species and composition of the solution, which is very useful with regard to the design of antisolvents for the precipitation of proteins. Selective Precipitation of Pancreatic Proteins. When alcohols were added as antisolvents to reduce the equilibrium concentration of the pancreatin in the solution, the precipitation occurred quickly and approached equilibrium within 30 min. Samples of the pancreatin precipitate were then taken for at least 60 min after the precipitation for analysis. The pancreatin precipitation was promoted when the fraction of alcohol in the solution was increased, due to the reduced solubility, as shown in Figure 4. Also, more pancreatin precipitate was produced by the less polar antisolvents of acetone and n-propyl alcohol. Therefore, the experimental results revealed an analogy between the solubility of the pancreatin and the alcohol species and the

Figure 6. Differential precipitation profiles of pancreatic enzymes through drowning-out process based on the solubility parameter values of the solvents.

fraction in the solution, plus a correlation was observed between the pancreatin precipitate and the polarity of the solution, which varied according to the species and fraction of antisolvent in the precipitation. As shown in Figure 5, the pancreatin precipitate (y) was found to be linearly correlated with the polarity of the solution (x), in good agreement (r2 ) 0.9208) with the function y ) 2.719 0.061x. Thus, the polarity of the solution would seem to represent a single parameter that can predict the pancreatin precipitate in the precipitation. Consequently, the precipitation was enhanced when the solution polarity was reduced, based on the addition of a less polar antisolvent, and completed at a solution polarity of below 28 MPa1/2. Furthermore, the same description of the pancreatin precipitation in relation to the solution polarity was also confirmed when nonalcoholic organic compounds, such as dimethyl sulfoxide, dimethylformamide, and acetonitrile, were used as the antisolvents (filled symbols in Figure 5), showing an excellent agreement (r2 ) 0.98) with the function obtained above. The pharmaceutical properties of pancreatin are evaluated by the activity of the proteases, lipases, and amylases included in the pancreatin. For example, the pharmacopeias of the United States, Japan, Europe, and Korea require different criteria for the protease, lipase, and amylase activities in pancreatin.21-24 Thus, since it is difficult to satisfy the pharmacopeia criteria with a simple precipitation of pancreatin, selective precipitation has attracted much interest with regard to preparing pancreatin that satisfies the various pharmacopeias. The selectivity of the proteases, lipases, and amylases in the pancreatin precipitate were examined along with the polarity of the solution, as shown in Figure 6. In the pancreatin precipitated at a high solution polarity above 36.0 MPa1/2, little amylase was included. However, this sharply increased when the solution polarity was decreased to around 35.0 MPa1/2, and became slightly dependent on the solution polarity below 34.0 MPa1/2. In the case of the selective precipitation of lipase, this appeared to be independent of the solution polarity. To explain this result, it might be guessed that the sharp change of the protein precipitation along with the solution polarity was due to a sudden change of protein solubility related to the apparent hydrophilic and hydrophobic groups of amylase. As such, if the quantities of the apparent hydrophobic and hydrophilic groups of protein interacting with the solvent molecules (water + organic compounds) were somewhat

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Figure 7. Enzymatic activities of the recovered precipitates by drowningout process using the extended binary, ternary, and quaternary solvent mixtures. Each mixture composition and its symbol are depicted in Table 2, in detail. USP standard 1× unit designates that amylase, lipase, and protease are 25, 2, and 25 U/mg, respectively.

8.1-8.6),28 elastase (MW ∼ 26 kDa and pI ) 9.5),29,30 and trypsin (MW ∼ 24 kDa and pI ) 10.5),31 thereby including a broad range of molecular weights, isoelectric points, and hydrophilicity/hydropobicity; thus its precipitation behavior in relation to the solution polarity would be expected to be more complicated, as reflected in the gradual variation of the protease precipitation in relation to the solution polarity. It should be noted that a complete protein recovery was not achieved by the precipitation, as only 65% and 85% of the input lipase and amylase were precipitated out, respectively, even with the lowest solution polarity, while the protease precipitate did not exceed 95%. These results may have come from a denaturing of the proteins and protein peptidase by the protease during the precipitation. Figure 7 compares the activities of the amylase, lipase, and protease selectively precipitated in solutions with the same polarity, yet composed of different species of antisolvent. For example, acetone was used as the antisolvent for precipitation at a solution polarity of 28.0 MPa1/2, plus precipitation was also achieved at the same solution polarity of 28.0 MPa1/2 using an organic mixture of acetone and dimethylformamide as the antisolvent. Similar precipitations were then conducted at solution polarities of 32.0 and 36.0 MPa1/2. From the experimental results, it was interesting to find that the activities of the proteins precipitated at the same polarity with different antisolvents were identical within tolerance, implying that the selective precipitation of proteins was dictated by the polarity of the antisolvents, regardless of the species and composition. The above selective protein precipitation was also confirmed using SDS-PAGE, as shown in Figure 8. Thus, the two identical molecular weight profiles for the proteins precipitated using solutions with the same polarity, yet two different antisolvents, would seem to indicate that the protein composition of amylase, lipase, and protease was selectively designed according to the solution polarity of the antisolvents. Conclusions

Figure 8. Identical protein profiles of the recovered precipitates by drowning-out process based on the same solubility parameter values of the used solvent mixtures. Each mixture composition and its lane mark are described in Table 2, in detail. Lane “M” means the molecular weight standard.

balanced, a sudden change of the protein solubility might occur at the intermediate polarity of the solution (around 35.0 MPa1/2), and if the apparent balance of both groups in the protein were shifted to the hydrophobic one, it would occur at high polarity of solution. Thereby, it might seem that the lipase was somewhat hydrophobic so as to cause the sharp change of solubility at high solution polarity above 38 MPa1/2. Thus, the lipase appeared almost fully precipitated across the whole range of solution polarities used in the present experiment (23-38 MPa1/2). Meanwhile, the amylase would appear less hydrophobic than the lipase so that a sudden change in the solubility seemed to occur at an intermediate polarity of the solution (around 35 MPa1/2). In addition, since the precipitation occurred around pH 6.0, close to the isoelectric point of amylase (pI ) 7.0), this may also have contributed to the sensitivity of the amylase precipitation in relation to the solution polarity. The selective precipitation of protease gradually increased when the solution polarity was decreased through the addition of an organic antisolvent. Yet, protease can include a wide variety of enzymes active to peptidase, such as carboxypeptidase B (MW ∼ 34 kDa and pI ) 6.0),25,26 kallikrein (MW ∼ 35 kDa and pI ∼ 4.2),27 R-chymotrypin (MW ∼ 23 kDa and pI )

In the precipitation of pancreatin, the polarity of the antisolvent was identified as the most critical factor determining not only the amount and composition of the proteins precipitated, but also the solubility of the proteins. In water-organic solutions, the intervention of the organic species in the hydrophilic interaction between the proteins and water resulted in a significant decrease in the solubility of the pancreatin when the fraction of the organic species in the solution was increased. The relationship between the solubility of the pancreatin and the solution conditions was also examined when the polarity of the solution was varied using various alcoholic and nonalcoholic organic compounds. A good correlation was found between the solubility and the polarity, implying that the solution polarity can be considered an effective single parameter for predicting the solubility of pancreatin. Using the relationship between the solubility and the polarity, the precipitation of pancreatin was also predicted in terms of the Hildebrand solubility parameter, adopted as a quantitative scale of the polarity. Thus, a higher precipitation of pancreatin was expected when an antisolvent with a lower polarity was used, and this matched well with experimental results when various antisolvents composed of alcoholic and nonalcoholic organic compounds were used. In the precipitation, the amylase, lipase, and protease proteins contained in the pancreatin exhibited different behaviors based on unique solubility responses to the polarity of the antisolvent. The lipase was fully precipitated across the whole range of

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antisolvent polarities, although a sudden step change of solubility was observed in relation to the polarity. In contrast, the protease was gradually precipitated when the polarity of the antisolvent was decreased. It was also proven that the proteins that precipitated with the same antisolvent polarity were identical with regard to their molecular weight profiles and bioactivities, regardless of the organic species and composition of the antisolvent. Accordingly, these experimental results confirm the feasibility of designing optimal organic antisolvents for selective precipitation from pancreatin. Acknowledgment The authors are grateful for grants from the Korean Ministry of Health and Welfare (Project No. HMP-03-PJ1-PG1-CH140001). Literature Cited (1) Scopes, R. K. Protein purification; Springer-Verlag: New York, 1982. (2) Bollag, D. M.; Edelstein, S. J. Protein methods; Wiley-Liss: New York, 1994. (3) Desnuelle, P. The lipases. The enzymes; Boyer, P. D., Eds.; Academic Press: New York, 1972; Vol. VII, pp 575-616. (4) Cozzone, P.; Pasero, L.; Beaupoil, B.; Marchis-Mouren, G. Characterization of porcine pancreatic isoamylases. chemical and physical studies. Biochim. Biophys. Acta 1970, 207, 490. (5) Rothstein, F. Differential precipitation of proteins: science and technology. Bioprocess Technol. 1994, 18, 115. (6) Riddick, J. A.; Bunger, W. B. Organic SolVents. Physical Properties and Methods of Purification; Wiley: New York, 1970. (7) Reichardt, C. SolVents and SolVent Effects in Organic Chemistry; VCH: Weinheim, 1988. (8) Barton, A. F. M. CRC Handbook of Solubility Parameters and Other Cohesion Parameters; CRC Press: Boca Raton, FL, 1983. (9) Taylor, P. J. Hydrophobic properties of drugs. In QuantitatiVe drug design in ComprehensiVe Medicinal Chemistry; Ramsden, C. A., Eds.; Pergamon: Oxford, 1990; pp 241-294. (10) Chin, J. T.; Wheeler, S. L.; Klibanov, A. M. On protein solubility in organic solvents. Biotechnol. Bioeng. 1994, 44, 140. (11) Stevenson, C. L. Characterization of protein and peptide stability and solubility in non-aqueous solvents. Curr. Pharm. Biotechnol. 2000, 1, 165. (12) So¨hnel, O.; Garside, J. Precipitation; Butterworth-Heinemann: Oxford, 1992. (13) Cohn, E. J.; Hughes, W. L.; Weare, J. H. Preparation and properties of serum and plasma proteins. XIII. Crystallization of serum albumin from ethanol-water mixtures. J. Am. Chem. Soc. 1947, 69, 1753.

(14) McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor: New York, 1999. (15) Shin, Y. O.; Rodil, E.; Vera, J. H. Selective precipitation of lysozyme from egg white using AOT. J. Food Sci. 2003, 68, 595. (16) Shin, Y. O.; Wahnon, D.; Weber, M. E.; Vera, J. H. Selective precipitation and recovery of xylanase using surfactant and organic solvent. Biotechnol. Bioeng. 2004, 86, 698. (17) Gargouri, Y.; Julien, R.; Bois, A. G.; Verger, R.; Sarda, L. Studies on the detergent inhibition of pancreatic lipase activity. J. Lipid Res. 1983, 24, 1336. (18) Lowry, O. H.; Resebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951, 193, 265. (19) Miller, G. L. Protein determination for large numbers of samples. Anal. Chem. 1959, 31, 964. (20) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680. (21) The United States Pharmacopeial Convention, Inc. U. S. Pharmacopeia and National Formulary; The United States Pharmacopeial Convention, Inc.: Rockville, 2000. (22) The European Pharmacopeial Convention, Inc. The European Pharmacopoeia; European Directorate for the Quality of Medicines of the Council of Europe: Strasbourg, 2005. (23) The Japanese Pharmacopeial Convention, Inc. The Japanese Pharmacopoeia; The Society of Japanese Pharmacopoeia: Tokyo, 2001. (24) The Korean Pharmacopeial Convention, Inc. The Korean Pharmacopoeia; Yak-up shin-moon, Inc.: Seoul, 2002. (25) Smith, E. L.; Stockell, A. Amino acid composition of crystalline carboxypeptidase. J. Biol. Chem. 1954, 207, 501. (26) Folk, J. E.; Piez, K. A.; Carroll, W. R.; Gladner, J. A. Carboxypeptidase B. J. Biol. Chem. 1960, 235, 2272. (27) Raspi, G. Kallikrein and kallikrein-like proteinases: purification and determination by chromatographic and electrophoretic methods. J. Chromatogr. 1996, 684, 265. (28) Laskowski, M. Chymotrypsinogens and chymotrypsins. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; pp 8-26. (29) Lewis, U. J.; Williams, D. E.; Brink, N. G. Pancreatic elastase: purification, properties, and function. J. Biol. Chem. 1956, 222, 705. (30) Shotton, D. M.; Hartley, B. S. Evidence for amino acid sequence of porcine pancreatic elastase. Biochem. J. 1973, 131, 643. (31) Cunningham, L. W. Molecular-kinetic properties of crystalline diisopropyl phosphoryl trypsin. J. Biol. Chem. 1954, 211, 13.

ReceiVed for reView October 5, 2006 ReVised manuscript receiVed February 20, 2007 Accepted February 27, 2007 IE061280F