Source: http://www.google.com/patents/US20050029195?dq=Xerox+%2B+%22centroid
Timestamp: 2014-03-14 15:03:57
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Patent US20050029195 - Method of separating components in a sample using silane-treated silica ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention provides methods for separating one or more components of interest from a sample containing particulates and soluble materials. The method comprises the steps of: (a) filtering a sample through silica filter media whose surface silanol groups have reacted with one or more silanes,...http://www.google.com/patents/US20050029195?utm_source=gb-gplus-sharePatent US20050029195 - Method of separating components in a sample using silane-treated silica filter mediaAdvanced Patent SearchPublication numberUS20050029195 A1Publication typeApplicationApplication numberUS 10/830,935Publication dateFeb 10, 2005Filing dateApr 23, 2004Priority dateOct 1, 2002Also published asCN101421012A, EP1737551A2, EP1737551A4, US7264728, US7374684, US7850012, US20070267349, US20080185333, WO2005115580A2, WO2005115580A3Publication number10830935, 830935, US 2005/0029195 A1, US 2005/029195 A1, US 20050029195 A1, US 20050029195A1, US 2005029195 A1, US 2005029195A1, US-A1-20050029195, US-A1-2005029195, US2005/0029195A1, US2005/029195A1, US20050029195 A1, US20050029195A1, US2005029195 A1, US2005029195A1InventorsGary Gibson, Keith Hayes, Meng Heng, Csilla Kollar, Thomas Lane, Anthony Revis, Landon SteeleOriginal AssigneeGibson Gary L., Keith Hayes, Heng Meng H., Csilla Kollar, Lane Thomas H., Anthony Revis, Steele Landon M.Export CitationBiBTeX, EndNote, RefManReferenced by (9), Classifications (31), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetMethod of separating components in a sample using silane-treated silica filter mediaUS 20050029195 A1Abstract The present invention provides methods for separating one or more components of interest from a sample containing particulates and soluble materials. The method comprises the steps of: (a) filtering a sample through silica filter media whose surface silanol groups have reacted with one or more silanes, and (b) simultaneously capturing particulates and binding a soluble component to the silica filter media. The bound soluble component of interest is subsequently eluted from the silica filter media. In one embodiment of the invention, unwanted soluble materials are captured by the treated silica filter media and desired component of interest is recovered from the flow-through. In another embodiment of the invention, different components of interest are recovered from both the eluate and the flow-through. Preferred treated silica filter media are silane-treated rice hull ash or diatomaceous earth with functional quaternary ammonium group or functional sulphonate group. Particulates suitable for the present invention, for example, are microorganisms. Images(13) Claims(40)
Ligand densities are corrected for 0.43% C due to residual carbon on the original rice hull ash. Mixed silanes sample ligand density are based on first silane. Example 6 Surface Treated Rice Hull Ash for Protein Binding and Release Objective To test the binding and release of protein using surface treated rice hull ash (RHA). The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 6 summarizes the filter media samples and their surface treatments. TABLE 6 Sample Designation Treatment 6 3-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloride treated RHA 4 3-(trimethoxysilyl)propyloctadecyl- dimethylammonium chloride treated RHA Unground RHA Untreated from Producers FW12 Commercial diatomaceous earth (Eager Picher) HQ50 Commercial quaternary amine ion-exchange resin (PerSeptive BioSystems) Procedure 1. 2 g of each sample was measured into a 50-mL conical tube. 2. 25 mL of 25 mM Tris-HCL, pH 8.4 buffer was added. 3. Sample and buffer were mixed by inversion overnight. 4. Each of the wetted samples was transferred to a 15 mL conical tube, then centrifuged at 2500 g for 5 minutes and the supernatant was decanted. The resulting samples were used for binding test below. 5. Protein test solution description and preparation: Source: Micrococcus luteus particulate free concentrated broth recovered using the following steps: Fermentation broth was lysed using 200 ppm lysozyme (from chicken hen white). Lysed broth was flocculated using a poly-cationic polymer and filtered to remove particulates. Particulate broth was concentrated using an ultrafilter to dewater (Prep/Scale� TFF, Millipore). 6. The above solution was adjusted with 24 parts of 25 mM Tris-HCl pH 8.4 buffer. 7. 5 mL of protein test solution was added to each tube containing surface treated rice hull ash. 8. The tubes were mixed by inversion for 90 min. 9. The mixed tubes were centrifuged at 2500 g for 5 minutes and the supernatant was decanted. The fraction collected is referred to as �Flow Through or FT�. 10. 5 mL of 25 mM Tris-HCl pH 8.4 buffer was added to each of the tubes which were allowed to mix by inversion for 45 min. 11. The tubes were centrifuged at 2500 g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Wash�. 12. 5 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 2M NaCl) was added and mixed for 30 min. 13. 0.5 mL of 0.5M NaOH was added to each tube. 14. The tubes were mixed by inversion for 90 min. 15. The tubes were centrifuged at 2500 g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Eluate�. 16. All the fractions were analyzed by SDS-PAGE gel electrophoresis (procedure according to NuPAGE Electrophoresis System, U.S. Pat. No. 5,578,180, by NOVEX electrophoresis GmbH, Germany). Observations FIG. 1A (Binding and Analysis of Unbound Components) Unbound sample was detected by analysis of the Flow Through and the Eluate represents all or a portion of bound sample released in the elution process. FW12 (commercial diatomaceous earth) did not bind any protein from the feed (lane #3 versus lane #2). The slightly lower intensity for all the bands is due to the dilution by the solution used to pre-wet the test sample. Untreated RHA selectively bound a protein band above 6 kd and below 14.4 kd (lane #4 versus lane #2). The slightly lower intensity for all the bands is due to the dilution by the solution used to pre-wet the test sample. HQ50 (commercial quaternary amine ion-exchange resin) bound most of the proteins from the test solution except below 14.4 kds (lane #5 versus lane #2) Treated RHA Sample 4 selectively bound near and above 97 kd region, between 55.4 and 36.5 kd, near 21 kd and 14.4 kd proteins. Note that the bands below 14.4 kd were not captured, as in the case with HQ50. The overall protein band intensity appears lower than the untreated rice hull ash and FW12, which suggests greater binding by treated RHA. Treated RHA Sample 6 demonstrated similar protein binding selectivity as sample 4 but appears to have lower binding capacity. Note that the bands below 14.4 kd were not captured, as in the case with HQ50. The overall protein band intensity appears lower than the untreated rice hull ash and FW12. FIG. 1B (Release and Analysis of Bound Components) FW12 eluate contains trace amount of proteins which are most likely from the physically trapped/carried over liquid (lane #2). The protein, below 14.4 and above 6 kd bands, bound to untreated RHA were released (lane #3) All the proteins captured by HQ50 were released (lane #4) Eluate from sample 4 contains protein bands above 116 kd, near and below 55 Kd and near 6 kd. The above 36.5 kd band appears to remain bound (lane #5). Eluate from sample 6 contain mostly above 116 kd and near 55 kd bands. Others that were bound either remain bound or are too low to be detected by the analysis (lane #6). Untreated diatomaceous earth did not exhibit protein-binding capability. The untreated rice hull ash demonstrated some protein binding capability. The two treated rice hull ashes, sample 4 and 6, demonstrate protein-binding capability. Example 7 Surface Treated Rice Hull Ash for Protein Binding and Release Objective To test the binding and release of protein using additional surface treated rice hull ash. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 7 summarizes the filter media samples and their surface treatments. TABLE 7 Sample Desig- nation Treatment 7 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 8 3-(trimethoxysilyl)-2-(p,m-chloromethyl)-phenylethane treated 9 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 10 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 11 N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treated 12 N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride treated Procedure Same as in Example 6. Observations Sample 7 Protein Binding and Release (FIG. 2A) Selectively bound all MW bands below 55 kd except near 21.5 kd (lane 2 versus lane 3). The wash has similar profile compared to flow through. The eluate has a very light band near 55 kd, and not many other bands. The other bands appear to be tightly bound and were not eluted under conditions used. Sample 8 Protein Binding and Release (FIG. 2A) Similar observations as above, Sample 7. Sample 9 Protein Binding and Release (FIG. 2B) Alnost all the proteins were bound except for the band below 14 kd (lane 1 versus lane 2) No protein bands were detected in the wash fraction. Eluate fraction contained mostly near 55 kd band, other bands remain bound. This demonstrated selective release for the near 55 kd band resulting in a protein purity >90+% based on band intensity. Sample 10 Protein Binding and Release (FIG. 2B) Similar observations as the sample 9 above Sample 11 Protein Binding and Release (FIG. 2C) Almost all, except some low MW bands, were bound. This demonstrated selective protein binding. (lane 1 versus lane 3) No protein bands were detected in the wash fraction. Most of the bands bound were eluted under conditions used (lane 5). Appears to have relatively high binding capacity compared to other surface treated rice hull ashes. Sample 12 Protein Binding and Release (FIG. 2C) Similar observations as the sample 9 above Conclusions Unique protein binding and release were observed for surface treated rice hull ashes. Selective binding was observed (Sample 7 and Sample 8). Selective release (sample 9 and sample 10) resulted in >90% protein purity fractions. Example 8 Surface Treated Rice Hull Ash for Protein Binding and Release Objective To test the binding and release of protein using additional surface treated rice hull ash. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 8 summarizes the filter media samples and their surface treatments. TABLE 8 Treated Rice Hull Ash Identification Surface Treatment 14 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 13 3-(N-styrylmethyl-2-aminoethylamino)- propyltrimethoxysilane hydrochloride treated 17 3-aminopropyltrimethoxysilane treated 18 3-aminopropyltrimethoxysilane treated 19 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated 20 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated surface Procedure 1. 2 g of each surface treated rice hull ash was weighed into a 50 mL conical tube and 40 mL equilibration buffer (25 mM Tris-HCl pH 8.4) was added. The tubes were mixed by inversion for 30 min. 2. The tubes were centrifuged at 2500�g for 5 minutes and the supernatant was decanted. 3. Protein test solution source: Micrococcus luteus particulate free concentrated broth was prepared as in Example I followed by partial digestion using 10 ppm protease. 4. The above solution was adjusted with 24 parts of 25 mM Tris-HCl pH 8.4 buffer. 5. 20 mL of protein test solution was added to each prepared surface treated rice hull ash. 6. The samples were mixed by inversion for 30 min. 7. The samples were centrifuged at 2500�g for 5 minutes and the supernatant was decanted. The fraction collected is referred to as �Flow Through or FT� 8. 20 mL of 25 mM Tris-HCl pH 8.4 buffer was added to the samples which were allowed to mix by inversion for 15 min. 9. The samples were centrifuged at 2500�g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Wash�. 10. 20 mL elution buffer (25 mM Tris-HCl pH 8.4 containing 1M NaCl) was added to each sample and the samples were mixed by inversion for 30 min. 11. The samples were centrifuged at 2500�g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Eluate #1� 12. Steps 9 and 10 were repeated using 10 mL of the same elution buffer+50 mM NaOH. The fraction collected is referred to as �Eluate #2�. 13. All of the fractions were analyzed by SDS-PAGE gel electrophoresis. Observations Sample 14 Protein Binding and Release (FIG. 3A) The flow through fraction has relatively low protein band intensity, which indicates sample 14 has relatively good binding capacity (lane #6 versus lane #1) The band below 14.4 kd remains in the flow through, which indicates selective binding. The bound proteins were partially eluted by 1M NaCl. Addition of NaOH to the 1M NaCl containing buffer further eluted the bound proteins. Sample 13 Protein Binding and Release (FIG. 3B) All the feed proteins were bound (lane #3 versus lane #2) Only a small amount of bound protein was eluted at 1M NaCl (lane #5), which suggests that the binding may not be ion exchange. Addition of caustic to the 1M NaCl elution buffer successfully eluted bound protein. The behavior was similar to sample 14. Sample 17 Protein Binding and Release (FIG. 3C) Relatively good binding as shown in lane #7 flow through fraction. Selectively did not bind the below 14 kd protein. Required high NaCl/NaOH for elution. Sample 18 Protein Binding and Release (FIG. 3C) Relatively good binding as shown in lane #1 flow through fraction. Selectively did not bind the below 14 kd protein. Required high NaCl/NaOH for elution. The results are similar to those of sample 17. Sample 19 Protein Binding and Release (FIG. 3D) Relatively good binding, as shown in the flow through fraction on lane #7 having low protein bands. Selectively did not bind the below 14 kd band. Most of the bound proteins were eluted at 1M NaCl. The addition of NaOH to 1M NaCl containing buffer further eluted the near 55 kd bands. Sample 20 Protein Binding and Release (FIG. 3E) Relatively good binding as indicated by the low protein bands in the flow through fraction (lane #3). Some leakage during wash (lane #4). Selectively did not bind the below 14 kd band (lane #3). The bound proteins were eluted mostly at 1M NaCl (lane #5). The results are similar to those of sample 19. Conclusions For the above surface treated rice hull ash samples tested, three general binding/release behaviors were observed when the samples were tested under conditions suitable for binding based on anion exchange and release by high salt and/or high pH: Relatively good binding, elute with NaCl/NaOH: Sample 14 (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated) Sample 13 (3-(N-styrylmethyl-2-aminoethylamino)-propyltrimethoxysilane hydrochloride treated) Sample 17 (3-aminopropyltrimethoxysilane treated) Sample 18 (3-aminopropyltrimethoxysilane treated) Relatively good binding, elute with NaCl: Sample 19 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated) Sample 20 (N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated) The binding/release test was designed to test for anion exchange behavior. The observations are consistent with the RHA surface modifications. The responses of sample 14 and sample 13 are consistent with a combination of ion exchange and hydrophobic characteristics. Sample 17 and sample 18 also demonstrated a mixture of behaviors. Sample 19 and sample 20 have typical characteristics similar to anion-exchange behavior in terms of both binding and release. Example 9 Surface Treated Rice Hull Ash for Protein Binding and Release (Cation Exchange) Objective To test the binding and release of protein using surface treated rice hull ash. The protein solution is particulate free, derived from Aspergillus niger fermentation. Table 9 summarizes the samples designation and their surface treatments. TABLE 9 Treated Rice Hull Ash Identification Surface 41 1st step 3-glycidoxypropyltrimethoxysilane and 2nd step Na2S2O5 treatment Unground Untreated RHA from Producers Porous HS50 Commercial �SH cation exchange resin (PerSeptive BioSystems, Farmington, MA) Procedure 1. 2 g of each surface treated rice hull ash were placed into a 50 mL conical tube and 40 mL equilibration buffer (100 mM Sodium Acetate, pH 4.0) was added. The tubes were mixed by inversion for 30 min. 2. The tubes were centrifuged at 2500�g for 5 minutes and the supernatant was decanted. 3. Protein test solution description and preparation: a. Source: Aspergillus niger particulate free concentrated broth recovered using the following steps: i. The fermentation broth was filtered to remove cell. ii. Ultrafilter (dewater (Prep/Scale� TFF, Millipore) cell free broth to dewater. b. The above solution was adjusted with 14 parts of 100 mM Sodium Acetate, pH 4.0 buffer. 4. 20 mL of protein test solution was added to each prepared surface treated rice hull ash. 5. The samples were mixed by inversion for 70 min. 6. The samples were centrifuged at 2500�g for 5 minutes and the supernatant was decanted. The fraction collected is referred to as �Flow Through or FT�. 7. 20 mL of 100 mM Sodium Acetate pH 4.0 buffer was added to each sample, and the samples were allowed to mix by inversion for 15 min. 8. The samples were centrifuged at 2500�g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Wash #1�. 9. Steps 7 & 8 were repeated and the fraction collected is referred to as �Wash #2� 10. 20 mL elution buffer (100 mM Sodium Acetate pH 4.0 buffer containing 1M NaCl) was added and the samples were mixed by inversion for 60 min. 11. The samples were centrifuged at 2500 g for 5 min and the supernatant was decanted. The fraction collected is referred to as �Eluate #1� 12. Repeated step 9 and 10 using 10 mL of the same elution buffer+50 mM NaOH. The fraction collected is referred to as �Eluate #2�. 13. All the fractions were analyzed by SDS-PAGE gel electrophoresis. Observations Sample 41 Protein Binding and Release (FIG. 4A) Selectively binds near 97 kd and below 3 kd bands. There were relatively low to no protein bands detected in the �Washes #1 and #2�, respectively (see lane #3 and lane #4, respectively), which implies that the binding was specific/strong. The bound proteins were eluted in 1M NaCl containing buffer. Untreated RHA Protein Binding and Release (FIG. 3A) The near 97 kd and below 3 kd bands were not present in the flow through. However, no proteins were eluted in either Eluate #1 or Eluate #2. Porous HS50: Protein Binding and Release (FIG. 4B) Selectively binds near 97 kd and below 3 kd bands. There were relatively low to no protein bands detected in the �Washes #1 and #2�, respectively (see lane #3 and lane #4, respectively), which suggests that the binding was specific/strong. The bound proteins were eluted in 1M NaCl containing buffer. Conclusion The surface treated rice hull ash sample 41 has very similar binding and release characteristics to the positive control. Example 10 Surface Treated Silica Filter Media for Protein Binding and Release (Ion Exchange) Objective To test the binding and release of protein using surface treated silica filter media. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Table 10 summarizes the samples designation and their surface treatments. TABLE 10 Sample Identification Description Sample 42 1st step 3-aminopropyltrimethoxysilane and 2nd step Glycidyltrimethylammonium chloride treatment Sample 40 1st step N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and 2nd step Glycidyltrimethylammonium chloride treatment Sample 34 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celite 545 Sample 29 N-(2-aminoethyl)-3-aminopropyltrimethoxysilane treated Celpure P1000 (commercial diatomaceous earth) AgriSilicas Untreated RHA Celite 512 Untreated commercial diatomaceous earth (World Minerals) Procedure Same as in Example 8 for all samples except sample 29, sample 30 and CelPure P100, which have the following variations: The protein test solution was diluted by 100� (versus 25�). Steps 4 and 5 were repeated and the wash fraction collected is referred to as �Wash #2�. Observations Under the test conditions used, the amount of protein test solution was in excess. As a result, all the flow through fractions had similar protein band patterns compared to the feed test solution. No attempt was made to qualitatively describe the protein binding capability of each silica filter media sample tested. The following observations are based on the eluate fractions only. Sample 42 (FIG. 5A) Most of the bound proteins were eluted at 1M NaCl (lane #5). Sample 40 (FIG. 5B) Most of the bound proteins were eluted at 1M NaCl (lane #5). Sample 34 (FIG. 5C) No significant amount of protein was eluted at 1M NaCl. Small amount of proteins were eluted subsequently using high pH. Sample 29 (FIG. 5D) Both eluate fractions contain proteins, and the compositions seem similar in these fractions (lane #5 for 1M NaCl eluate and lane #6 for high pH eluate). Untreated AgriSilica RHA (FIG. 5B) The eluted fractions contain proteins, especially at MW lower than 14.4 kd. Celite 512 (FIG. 5C) The fraction eluted at 1M contains proteins near 97 kd, near and below 55 kd and especially between 14.4 kd and 6 kd (lane #10). Conclusion Samples 40 and 42 (surface treated rice hull ash) and samples 29 and 34 (surface treated diatomaceous) demonstrate protein-binding capability over the corresponding untreated counterparts. Example 11 Surface Treated Rice Hull Ash for Dynamic Protein Binding and Release (Ion Exchange) Objective To test the dynamic binding and release of protein using surface treated rice hull ash sample 9. The experiment design is based on ion exchange. The protein solution is particulate free, derived from Micrococcus luteus fermentation. Procedure 1. 6 g of sample 9 was placed into a 50 mL conical tube. 2. 50 mL equilibration buffer (25 mM Tris-HCl pH 8.4) was added and the sample was mixed by inversion for 30 min. 3. The samples was centrifuged at 2500 g for 5 minutes and the supernatant was decanted. 4. 30 mL of equilibration buffer was added and the sample was mixed well by inversions. 5. The sample was poured into a gravity flow column. 6. The surface-treated rice hull ash was allowed to settle and pack to a 10 mL volume. 7. The pre-filter was placed onto the packed bed. 8. 20 ml of equilibration buffer was added. 9. 25 mL of protein test solution was added (prepared the same way as in Example 6) 10. Flow through fractions were collected in 15 mL conical tubes. 11. 30 mL of equilibration buffer was added, and the �wash� was collected in 15 ml conical tubes. 12. The following steps were used sequentially for election and collection of multiple elutes as shown in Table 11: a. 0.2M NaCl in equilibration buffer was added. b. 2M NaCl in equilibration buffer was added. c. 0.1M NaOH was added. 13. All the fractions were analyzed by SDS-PAGE gel electrophoresis. Observations (FIG. 6) The amount of solution loaded was higher than the capacity, hence significant breakthrough in the FT fractions (lanes 3, 4 and 5) The surface treated rice hull ash, sample 9, had good flow property. All the steps performed above were easily accomplished by gravity flow. FIG. 6 shows that at the at 10 mL load, the feed solution appears to breakthrough the 10 mL packed sample 9. Under the binding conditions tested, sample 9 appears to selectively bind the near 96 kd, near 55 kd, the two bands below the 55 kd, bands near and between the 14.4 kd and 6 kd. The following were observed with the three elution steps: Three bands (near 97 kd, near 55 kd, and below 14.4 kd) were eluted at 0.2M. At 2M NaCl, near 97 kd and near 55 kd bands were eluted. Under 0.1M NaOH, near 55 kd, below 31 kd bands and near 14.4 kd proteins were eluted. Table 11 shows a summary of fractions collected for the binding test. TABLE 11 Volume Fraction (mL) Feed 25 mL loaded FT#1 10.5 mL FT#2 (after 10 mL feed was loaded) 10 mL FT#3 (after 14.5 mL was loaded) 4.5 mL FT#4 (after 25 mL feed was loaded) 10 mL Wash 30 mL Eluate #1 (0.2 M NaCl) 10 mL Eluate #2 (2 M NaCl) 10 mL Eluate #3 (1st 0.1 M NaOH fraction, very dark) 4.5 mL Eluate #4 (2nd 0.1 M NaOH) 3.5 mL Conclusions This example demonstrates that surface-treated rice hull ash can be used in a packed bed chromatography mode for protein binding and release and as a filter aid with gravity flow alone. The binding and release characteristics are similar to those of batch mode. The example also illustrates that selective elution can be achieved by using different elution buffers. Example 12 Surface-Treated Rice Hull Ash for Simultaneous Particulate Capture and Soluble Capture/Release Objective To test the characteristics of surface-treated rice hull ash for simultaneous particulate filtration, and protein binding and release. The surface treated rice hull ash was designated sample 19, which was demonstrated to have anion exchange characteristics (see Example 8). The untreated rice hull ash was also tested in parallel. Buffers Equilibration Buffer: 25 mM Tris-HCl, pH 8.4. Elution Buffer: 25 mM Tris-HCl, 1M NaCl, pH 8.4; 1M NaOH; 1M HCl Test Solution Flocculated Micrococcus luteus fermentation broth referred to as �feed� was prepared according to the following: After harvest, the broth was lyzed using 100 ppm lysozyme (chicken egg white). The lysed broth was flocculated using poly-cationic polymer. The flocculated sample was diluted with 1 part equilibration buffer before testing. Procedure 1. Surface-treated rice hull ash preparation: 5 g of untreated RHA was placed into each of the two 50 mL conical tubes. 40 mL equilibration buffer was added and the tubes were mixed by inversion for 30 min. 2. The tubes were centrifuged at 2500�g for 5 minutes and decanted in step #1 for the untreated rice hull ash. 3. 50 mL of the prepared test solution �feed� was added to each prepared rice hull ash. 4. The tubes were mixed by inversion for 30 min at room temperature. 5. A 1 mL small sample was centrifuged using a bench top centrifuge (4 min) and the supernatant was collected (referred to as �Bench FT�). 6. 0.45 μm 250 mL-Nalgen unit was prepared for filtration: The unit was connected to a house vacuum outlet. The other prepared rice hull ash was suspended in 50 mL of equilibration buffer. The suspension was poured into the filter unit, and (house) vacuum was applied to form a pre-coat (cake). The filtrate reservoir was emptied. The reservoir was reconnected for the filtration test. 7. The protein solution with rice hull ash ad-mix from step 4 was poured into the prepared filtration unit and vacuum was reapplied to start filtration. The collected filtrate sample is referred to as �FT Filtrate�. 8. The vacuum was discontinued and 50 mL of Equilibration Buffer was added and mixed by stirring. The vacuum was reapplied to start filtration. The filtrate sample was collected and referred to as �Wash�. 9. Step 8 was repeated with 50 mL of Elution Buffer and mixed for 15 min before vacuum was reapplied to start filtration. The filtrate sample was collected and is referred to as �Eluate�. 10. All the fractions were analyzed by 4-12% Tris-Bis SDS-PAGE gel electrophoresis with MES running buffer (see separate Excel file for procedure). 11. Steps 1-10 were repeated with the untreated rice hull ash. Observations/Comments 1. The surface-treated rice hull ash, sample 19, appears to have slightly thinner cake thickness than the untreated rice hull ash. 2. All the fractions collected (FT filtrate, wash and eluate) from both rice hull ashes were clear, free of particulate. 3. The surface-treated rice hull ash, sample 19, has a particulate filtration rate comparable to the filtration rate of the untreated rice hull ash: Sample 19: 12.8 mL/min Untreated RHA: 14.0 mL/min 4. Sample 19 demonstrates good capture and release over untreated RHA: Untreated RHA (FIG. 7A) The �FT filtrate� (lane #4) has very similar profile as the feed (lane #2). All the bands are slightly lighter than the feed, which is an artifact of dilution from the buffer used to condition the rice hull ash. The protein solution physically trapped within the rice hull ash was displaced and this is represented by the �wash� fraction (contains very faint protein bands, see lane #5) There was only trace amount of protein in the Eluate (lane #6). 5. Sample 19 (FIG. 7B) Demonstrates good binding and recovery of the bound protein. The �FT filtrate� fraction has very low to no protein (lane #4). The �Bench FT� supernatant (lane #3) has slight protein bands when compare to the �FT Filtrate�, which indicates that proteins were captured as they passed through the cake. The wash has low to no protein bands (lane #5). The Eluate has similar band patterns but slightly less intense than the feed (lane #6). Conclusion The surface-treated rice hull ash simultaneously captured soluble proteins of interest by ion exchange and separated particulates from the feed protein solution. The captured proteins can be subsequently extracted from the surface treated rice hull ash by elution with a high-salt buffer. The results demonstrate that surface-treated rice hull ash can be used to separate a particulate-containing protein solution into three streams: particulates trapped in surface-treated rice hull ash pre-coat and body feed, non-protein components bound to surface treated rice hull ash, and protein components bound to and eluted off the surface treated rice hull ash. Example 13 Surface Treated Rice Hull Ash for Simultaneous Particulate Capture and Soluble Capture/Release Objective To repeat Example 12 using a Aspergillus niger broth using the same surface-treated rice hull ash (sample 19) and untreated rice hull ash. Test Solution Aspergillus niger fermentation was diluted with 4 parts of DI water and pH was adjusted to 8.06 using NaOH. Procedure Same as in the Example 12. Test solution volume was 100 mL. Observations/Comments The surface treated rice hull ash, sample 19, has a comparable particulate filtration rate to the untreated rice hull ash. All the fractions collected (FT filtrate, wash and eluate) from both rice hull ashes were clear, free of particulate. Under the conditions tested, the amount of test solution used was in excess of the binding capacity. As a result, the flow through fractions (both �bench FT� and �Filtrate FT�) for both sample 19 and untreated RHA were not significantly different from the feed solution. See FIG. 8, lanes #2, 3 and 4 versus lane #1 for untreated RHA and lanes #7, 8 and 9 versus lane #1 for sample 19. The following observations confirmed that sample 19 has protein-binding capability over the untreated RHA (see FIG. 8): Untreated RHA Wash (lane #5) contains more protein than the sample 19 (lane #10). The eluted fraction from sample 19 (lane #11) shows higher protein band intensity than the eluted fraction from untreated RHA (lane #6). Conclusion This example demonstrates that the surface-treated rice hull ash simultaneously captured soluble proteins of interest by ion exchange and separated particulates from the Aspergillus niger derived feed protein solution. The captured proteins can be subsequently extracted from the surface treated rice hull ash by elution with high salt buffer. The results demonstrate that surface-treated rice hull ash can be used to separate a particulate containing protein solution into three streams: particulates trapped in surface treated rice hull ash pre-coat and body feed, non-protein components bound to surface treated rice hull ash, and protein components bound to and eluted off the surface treated rice hull ash. Example 14 Protein Binding Test Materials MilliQ H2O Protein solution (filtered catalase DFC) 50 mL Oak Ridge tubes Sorval RC 5B Plus centrifuge with Sorval SA600 rotor BCA Protein assay kit (Pierce) Compat-Able Protein Assay Preparation Reagent Set (Pierce) Pre-Diluted Protein Assay Standards, bovine serum albumin fraction V set (Pierce) 5 μm syringe filter (Sartoris, Minisart, #17594) Procedures 1. 1 g of each silane treated rice hull ash Sample #54-67 was added into a 50 mL Oak Ridge tube. 2. 20 mL MilliQ H2O was added to each tube. 3. The contents of each tube was mixed by turning end-over-end at 8 rpm for 10 minutes at room temperature. 4. Each tube was centrifuged at 16,000 rpm, 15� C. for 15 minutes. 5. The supernatant was carefully removed using plastic transfer pipettes. 6. 1 part protein solution was diluted with 24 parts MilliQ H2O (Feed). 7. 10 mL Feed was added to each tube (gave 10% w/v solid). 8. Each tube was incubated at room temperature for 2 hours, turning end-over-end at 8 rpm. 9. Each tube was centrifuged at 16,000 rpm, 15� C. for 30 minutes. 10. Each supernatant was filtered through a 0.45 μm syringe filters. 11. The protein concentrations of Feed and filtered supernatants (Step 10) were measured by BCA assay using microtiterplate protocol. The results are shown as Table 12. TABLE 12 Protein concentration ug/ml Sample # Silane Type Original 160.66 Untreated RHA 98.75 54 trimethoxysilylpropyl-ethylenediamine, triacetic acid, trisodium 120.09 salt 55 N-(triethoxysilylpropyl)-O-polyethylene oxide urethane 136.10 56 Bis-(2-hydroxyethyl)-3-aminopropyltriethoxysilane 112.02 57 ((chloromethyl)phenylethyl)trimethoxysilane 103.21 58 N-(3-triethoxysilylpropyl)-gluconamide 81.02 59 3-mercaptopropyltriethoxysilane 73.43 60 N-(triethoxysilylpropyl)-4-hydroxybutyramide 98.83 61 3-(triethoxysilyl)propylsuccinic anhydride 73.49 62 Tris(3-trimethoxysilylpropyl)isocyanurate 81.41 63 2-Hydroxy-4-(3-triethoxysilylpropoxy)-diphenylketone 49.57 64 Ureidopropyltrimethoxysilane 100.04 65 3-isocyanatopropyltriethoxysilane 76.12 66 N-(3-trimethoxysilylpropyl)pyrrole 59.32 67 Bis[(3-methyldimethoxysilyl)propyl]-polypropylene oxide 110.24 Results The protein concentrations of the Feed materials were decreased after the Feed materials were mixed with sample numbers 54-67, centrifuged, and filtered. The results indicate that silane-treated silica sample numbers 54-67 and untreated RHA all bound proteins from the Feed protein solution. Example 15 Test of Antimicrobial Activity (Bacillus subtilis) Microorganism tested: Bacillus subtilis Filter media tested: filter media samples 43, 44, 4 and FW12 (untreated diatomaceous earth) Protocol: Bacillus subtilis fermentation broth was diluted in sterile PBS to �104 CFU/mL (1 OD≈5*108 CFU/mL was used to estimate CFU/mL in fermentation broth) Use 0.5 g filter media/5 mL liquid (10% solid) 1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted broth sample were plated on LA plates to determine actual CFU/mL used. Plates were incubated over night at 34� C. 2. Filter media and diluted bacterial sample (or PBS control) were mixed in a sterile 125 mL baffled flask for 2� hours at 30� C., 200 rpm. 3. Liquid part of the treated samples (2) were plated on LA plates (5 plates for each sample, one plate for control) and incubated overnight at 34� C. 4. The plates were counted for bacteria. Results: The results are summarized in Table 13. By mixing the bacteria with filter media samples 4 and 44, the CFUs were reduced, which indicates that filter media samples 4 and 44 had antimicrobial activity and killed the bacteria by contacting. TABLE 13 Sample CFU/mL Diluted broth - start 6.53*103 � 2.47*103 Sample 43 + bacteria - mixing 1.04*104 � 1.50*103 Sample 44 + bacteria - mixing 1.30*102 � 3.00*101 Sample 4 + bacteria - mixing TFTC FW12 + bacteria - mixing 5.90*104 � 8.00*103 Diluted broth sample - mixing 1.05*103 � 5.00*101 Notes and Abbreviations: PBS: Phosphate buffered saline (prevents cells from lysing due to osmotic chock) CFU: colony forming units (a measure of viable cells) TFTC: Too Few To Count The CFU/mL are reported as: Average�Difference (number of plates) [the difference is between the average and the observations farthest from the average]. Only plates with between 20-300 colonies were counted. Example 16 Test of Antimicrobial Activity (Bacillus subtilis) Microorganism tested: Bacillus subtilis Filter Media tested: filter media samples 1, 4, 6, 44, and 45. Protocol: Bacillus subtilis fermentation broth was diluted in sterile PBS to �104 CFU/mL. 0.5 g filter media/5 mL liquid (10% solid) was used. 1. Serial dilutions (made in sterile 0.9% w/v NaCl) of the diluted broth sample were plated on LA plates to determine actual CFU/mL used. Plates were incubated over night at 34� C. 2. Filter media and diluted bacterial sample (15 mL liquid) were mixed in a sterile 250 mL baffled flask. 2 flasks were used for each filter media. (A flask with PBS instead of bacterial sample was included for the following filter media: Samples 1, 6 and 45) 3. The above was mixed for 2 hours at 30� C., 250 rpm. 4. Treated samples (the liquid part) were plated on LA plates (4 or 5 plates for each sample). Plates were incubated overnight at 34� C. 5. The plates were counted for bacteria. Results: The results are summarized in Table 14. By mixing the bacteria with filter media samples 1, 4, 6, 44, and 45, the CFUs were significantly reduced. TABLE 14 Sample CFU/mL Diluted broth - start 3.45*104 � 4.50*103 Diluted broth - mixing 1.72*104 � 1.55*103 Sample 1 A TFTC B TFTC Sample 4 A TFTC B TFTC Sample 6 A TFTC B 1.00*102 � 0.00*100 Sample 44 A 3.10*102 � 9.00*101 B 6.00*102 Sample 45 A TFTC B TFTC Example 17 Test of Antimicrobial Activity and Filtration (Lactobacillus brevis) Microorganism tested: Lactobacillus brevis Filter media tested: Samples 4, 43, 45 & FW12. Used 0.5 g filter media/5 mL culture (10% solid). Protocol: 1. A Lactobacillus brevis overnight culture was diluted to �105 CFU/mL (based on 1 OD600≈2.7*108 CFU/mL) in two steps�the first dilution was made in sterile Lactobacillus MRS broth, the second in sterile PBS. 2. Serial dilutions (in 0.9% w/v NaCl) of the culture were made (second dilution). 3. Diluted samples were plated on Lactobacillus MRS broth plates, to determine actual starting CFU/mL. 4. Filter media and diluted bacterial sample (10 mL liquid) were mixed in a sterile 125 mL baffled flask, sealed with PARAFILM�, for 2 hours 15 minutes at room temperature on an 20 orbit shaker (�60 rpm). 5. Serial dilutions (in 0.9% w/v NaCl) were made of treated sample and plated on Lactobacillus MRS broth plates. 6. Selected samples/dilutions of samples 4, 43 and 45 were filtered through a 5 μm filter. 7. The filtered samples were plated on Lactobacillus brevis broth plates, and incubated in a candle jar at 30� C. for 2 days. 8. The plates were counted. Results: The results are summarized in Table 15. CFUs were reduced by mixing Samples 4, 43, and 45 with bacteria. CFUs were further reduced by filtering the mixture through a 5 μm filter. TABLE 15 Sample CFU/mL Lactobacillus brevis culture - start 1.05*105 � 2.50*103 Lactobacillus brevis culture - mixing 1.23*105 � 2.50*103 Sample 4 (mixing) 3.22*104 � 4.77*103 Sample 43 (mixing) 3.43*104 � 5.67*103 Sample 45 (mixing) 5.55*102 � 4.50*101 FW12 (DE) 8.60*104 � 4.75*103 Filtered Sample 4 TFTC Filtered Sample 43 TFTC Filtered Sample 45 TFTC Example 18 Test of Antimicrobial Activity (E. coli) Microorganism tested: E. coli (MG1655) Filter media tested: FW12, samples 43, 1, 4, 6, 44 and 45. Protocol: 0.5 g Filter Media/5 mL Feed (=10% solid). 1. An E. coli culture (not yet in stationary phase) was diluted to �105 CFU/mL (based on 1 OD600≈5*108 CFU/mL) in two steps�the first dilution was made in sterile LB media, the second in sterile PBS (this was the Feed). 2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made. 3. 100 μL of the diluted feed samples were plated on LA plates, to determine the actual starting CFU/mL. 4. Filter media and 10 mL feed were mixed in a sterile 125 mL baffled flask for 2 hours at 25� C., 200 rpm (� inch stroke). 5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made and 100 μl of each was plated on LA plates, and incubated overnight at 30� C. 6. Plates were counted. Results: The results are summarized in Table 16. TABLE 16 Sample CFU/mL MG1655 - start 6.80*104 � 4.00*103 MG1655 - mixing 5.35*105 � 2.50*104 diatomaceous earth 2.28*105 � 1.72*105 Sample 43 9.05*103 � 5.50*102 Sample 1 1.28*103 � 2.45*102 Sample 4 1.73*104 � 2.03*103 Sample 6 TFTC Sample 44 2.70*103 � 1.23*102 Sample 45 5.20*103 � 2.00*102 Example 19 Test of Antimicrobial Activity and Filtration (Lactobacillus brevis) Microorganism tested: Lactobacillus brevis type strain (ATCC#14869) Filter media tested: Samples 43, 4, and 44 Protocol: 0.5 g Filter media/5 mL Feed (=10% solid) 1. A Lactobacillus brevis culture was diluted to �105 CFU/mL (based on 1 OD600≈2.7*108 CFU/mL) in two steps�the first dilution was made in sterile Lactobacillus MRS broth, the second in sterile PBS (this was the Feed). 2. Serial dilutions (in 0.9% w/v NaCl) of the Feed were made. 3. 100 μL of the diluted samples were plated on Lactobacillus MRS broth plates, to determine the actual starting CFU/mL. 4. Filter media and 5 mL Feed were mixed in a sterile 15 mL conical tube for 2 hours at 25� C., 250 rpm (� inch stroke). 5. Serial dilutions (in 0.9% w/v NaCl) of mixed samples were made and plated on Lactobacillus MRS broth plates (100 μl each). 6. All samples were filtered through 5 μm syringe filter. 7. Serial dilutions (in 0.9% w/v NaCl) of filtered samples were made and plated on Lactobacillus MRS broth plates. 8. Plates were counted in a candle jar at 30� C. for 2 days. 9. Plates were counted. Results: The results are summarized in Table 17. CFUs were reduced by mixing Samples 4, 43, and 44 with bacteria. CFUs were further reduced by filtering the mixture through a 5 μm filter. TABLE 17 Sample CFU/mL CFU/mL (filtered) ATCC#14869 - start 2.83*104 � 4.67*103 ATCC#14869 - mixing 4.00*104 � 2.00*103 1.27*104 � 5.80*102 Sample 43 4.55*103 � 3.50*102 2.40*103 � 2.00*102 Sample 4 1.95*102 � 5.00*100 TFTC Sample 44 8.10*102 � 1.40*102 5.50*101 � 5.00*100 Example 20 Test of Antimicrobial Activity (Lactobacillus brevis) Microorganism tested: Lactobacillus brevis Filter media tested: Samples 48, 50, 51, and 52. Protocol: 1. Lactobacillus brevis (gram positive) culture was streaked on MRS agar and incubated anaerobically at 26� C. until growth was sufficient. 2. Working inoculum was prepared by diluting colonies from the MRS plates into 0.1% peptone, targeting 5�104 cfu/mL. 3. 0.5 g filter media was added to 10 mL inoculum in a 30 mL glass tube (5%). 4. The glass tube was sealed and incubated at room temperature for 30 minutes with mixing (8 inversions/minute). 5. Serial dilutions of 1:10 were prepared in 0.9% NaCl and plated with MRS agar, using the pour plate method to enumerate bacterial population. 6. Plates were incubated at 26� C., anaerobically (GasPak), until growth was sufficient to count. 7. Plates that had 20-200 colonies were counted. The Results are summarized in Table 18. Example 21 Test of Antimicrobial Activity (Acetobacter pasteurianus (gram negative)) Microorganism tested: Acetobacter pasteurianus (gram negative) Filter media tested: Samples 48, 50, 51, and 52. Protocol: 1. Acetobacter pasteurianus (gram negative) culture was streaked onto MRS agar and incubated aerobically at 27� C. until growth was sufficient. 2. Culture was stocked by adding 1 mL loop of agar plate colonies to 99 mL of MRS broth and incubated at 27� C. 3. Working inoculum was made by diluting an aliquot of the MRS stock culture into either phosphate buffered saline (PBS) or 0.1% peptone. 4. 0.5 g of filter media was added to 10 mL inoculum in a 30 mL glass tube. 5. The glass tube was sealed and incubated at room temperature for 30 minutes with mixing (8 inversions/minute). 6. Serial dilutions of 1:10 were performed in 0.1% peptone and plated with MRS agar, using the pour plate method to enumerate bacterial population. 7. Plates were counted at 27� C., aerobically, until growth was sufficient to count. 8. Plates that had 20-200 colonies were counted. The Results are summarized in Table 18. Example 22 Test of Antimicrobial Activity (Saccharomyces diastaticus (yeast)) Microorganism tested: Saccharomyces diastaticus (yeast) Filter media tested: Samples 48, 50, and 51. Protocol: 1. Saccharomyces diastaticus (yeast) culture was streaked onto YM agar and incubated aerobically at 30� C. until growth was sufficient. 2. Working inoculum was prepared by diluting colonies from the YM plates into phosphate buffered saline (PBS), targeting 3�104 cfu/mL. 3. 0.5 g to filter media was added to 10 mL inoculum in a 30 mL glass tube. 4. The glass tube was sealed and incubated at room temperature for 30 minutes with mixing (8 inversions/minute). 5. Serial dilutions of 1:10 were performed in 0.9% NaCl and plated with MRS agar, using the pour plate method to enumerate bacterial population. 6. Plates were incubated at 30� C., aerobically, until growth was sufficient to count. 7. Plates that had 20-200 colonies were counted. The Results are summarized in Table 18. TABLE 18 Lactobacillus Acetobacter Saccharomyces Brevis, grams pasteurinus, distaticus, Sample positive (+) gram negative (−) yeast No. Treatment Silica Type % Reduction % Reduction % Reduction 48 3-(N-styrylmethyl-2- RiceSil 100 100% 18% 41% aminoethylamino)- propyltrimethoxy- silane hydrochloride 51 3-trihydroxysilylpropyl- RiceSil 100 20% 10% 33% methyl phosphonate, sodium salt 50 N-(2-Aminoethyl)-3- RiceSil 100 90% 20% 3% aminopropyltrimethoxy- silane Glycidyltrimethyl- ammonium chloride 52 N- RiceSil 100 100% 90% Octadecyldimethyl(3- Trimethoxysilylpropyl) ammonium chloride Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications could be made without departing from the scope of the invention. Referenced byCiting PatentFiling datePublication dateApplicantTitleUS7514010Jul 9, 2007Apr 7, 2009Salmon Daniel JWater filtering method and apparatusUS7531632Feb 16, 2006May 12, 2009Gtc Biotherapeutics, Inc.Clarification of transgenic milk using depth filtrationUS7828967Dec 14, 2006Nov 9, 2010Shaw Mark DWater purification systemUS8029677Sep 21, 2010Oct 4, 2011Shaw Mark DWater purification methodUS20100249279 *Mar 19, 2010Sep 30, 2010Taiyo Ink Mfg. Co., Ltd.,Thermally curable resin composition and cured product thereofUS20110077393 *Aug 9, 2010Mar 31, 2011University Of Louisville Research FoundationMethods of synthesis and purification by use of a solid supportWO2007106078A2 *Mar 8, 2006Sep 20, 2007Gtc Biotherapeutics IncClarification of transgenic milk using depth filtrationWO2008073499A1 *Dec 13, 2007Jun 19, 2008Tad J HeymanWater purification systemWO2009094299A1 *Jan 16, 2009Jul 30, 2009Jie LuFreshwater diatomaceous earth products containing reduced soluble metal levels, processes for reducing soluble metal levels in freshwater diatomaceous earth products, and methods of using the same* Cited by examinerClassifications U.S. Classification210/656, 422/70, 436/161, 210/198.2International ClassificationB01J20/286, B01D15/08, B01J20/10, B01D15/36, B01D15/38, B01J20/32Cooperative ClassificationB01J20/3259, B01J20/14, B01J20/3204, B01J20/3257, B01D15/3804, B01J20/3242, B01J20/286, B01J2220/4887, B01J20/3244, B01D15/361, B01J20/103European ClassificationB01J20/32F8B4J4, B01J20/32F8B4J, B01J20/32B4, B01J20/32F8, B01J20/32F8B, B01J20/14, B01J20/286, B01J20/10B, B01D15/36B, B01D15/38ALegal EventsDateCodeEventDescriptionFeb 10, 2011FPAYFee paymentYear of fee payment: 4Jun 7, 2010ASAssignmentFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:DANISCO US, INC.;REEL/FRAME:24492/316Owner name: DOW CORNING CORPORATION,MICHIGANEffective date: 20100603Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:DANISCO US, INC.;REEL/FRAME:024492/0316Owner name: DOW CORNING CORPORATION, MICHIGANMar 1, 2010ASAssignmentOwner name: DANISCO US INC.,CALIFORNIAFree format text: CHANGE OF NAME;ASSIGNOR:GENENCOR INTERNATIONAL, INC.;US-ASSIGNMENT DATABASE UPDATED:20100302;REEL/FRAME:24006/253Effective date: 20070215Free format text: CHANGE OF NAME;ASSIGNOR:GENENCOR INTERNATIONAL, INC.;REEL/FRAME:024006/0253Owner name: DANISCO US INC., CALIFORNIASep 29, 2004ASAssignmentOwner name: DOW CORNING CORPORATION, MICHIGANFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GIBSON, GARY L;KOLLAR, CSILLA;LANE, THOMAS H;AND OTHERS;REEL/FRAME:015197/0513;SIGNING DATES FROM 20040730 TO 20040924Owner name: GENENCOR INTERNATIONAL, INC., CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HENG, MENG H;STEELE, LANDON M;REEL/FRAME:015197/0192Effective date: 20040907RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google