EP1402011A2 - Proteine in einer porösen matrix - Google Patents

Proteine in einer porösen matrix

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Publication number
EP1402011A2
EP1402011A2 EP02706428A EP02706428A EP1402011A2 EP 1402011 A2 EP1402011 A2 EP 1402011A2 EP 02706428 A EP02706428 A EP 02706428A EP 02706428 A EP02706428 A EP 02706428A EP 1402011 A2 EP1402011 A2 EP 1402011A2
Authority
EP
European Patent Office
Prior art keywords
protein
matrix material
oph
porous matrix
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02706428A
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English (en)
French (fr)
Inventor
Eric J Ackerman
Jun Liu
Lei Chenghong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Battelle Memorial Institute Inc
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Battelle Memorial Institute Inc
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Filing date
Publication date
Priority claimed from US09/791,138 external-priority patent/US7846684B2/en
Application filed by Battelle Memorial Institute Inc filed Critical Battelle Memorial Institute Inc
Publication of EP1402011A2 publication Critical patent/EP1402011A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to proteins in porous supports, methods of supporting proteins, and methods of using supported proteins.
  • the invention also provides an improved method for making organophosphorous hydrolase (“OPH").
  • OPH organophosphorous hydrolase
  • Chemical weapons i.e. nerve gases, especially sarin and VX
  • organophosphorous pesticides e.g. parathion, paraoxon and acephate
  • the OPH enzyme offers tlie potential to inactivate chemical weapons or organophosphorous pesticide without the need for complex and expensive incineration facilities.
  • tlie invention provides improved protein systems that can better meet tlie challenges described above. Although tlie invention generally applies to immobilized enzyme systems, etc., in some specific examples, tlie invention also provides an improved method for making OPH and systems containing active OPH.
  • One concept of the invention is H e engineering of support structures that match protein sizes to support structure pore sizes. It has been surprisingly found that well-matched sizes can produce protein systems having desirable qualities such as high activity, enhanced stability, and a relatively high density of active protein. Coupling of proteins in pores that are either too small or too large results in inferior properties. Other factors, such as surface area, pore density, pore uniformity and distribution, protein population within a support, and type and density of cross- linking sites may also be utilized to control the characteristics of the protein system.
  • the invention provides a protein system for use in facilitating chemical reactions.
  • Tl e system includes a porous matrix material that has pores within a solid matrix.
  • the protein system comprises: a porous matrix material having a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in tlie range of 50 to 400 A, and a chemically-active protein bonded to the matrix material.
  • "Bonded” refers to covalent, ionic and/or electrostatic attachment to tl e matrix material.
  • the protein is covalently bonded to H e matrix through coupling groups.
  • the protein system comprises: a porous matrix material being sized such that the protein system comprises 0.01 to 1 mmol of protein per gram of matrix material and wherein the protein in the protein system exhibits an activity of at least 65% that of the activity of the protein in the active state.
  • the invention further provides a protein system comprising a porous matrix material containing an entrapped protein wherein the protein system is characterized by having at least 0.01 mmol of protein per gram of matrix material and an activity that is at least 2 times greater than tlie activity of a protein system that has been formed under identical conditions except on a normal silica matrix material.
  • "Nonnal silica” is uncoated silica bulk material witli a pore size of 300 A with a 12 micrometer bead size; if available the normal silica should be purchased from PolyLC Inc., Columbia Maryland, USA, item #BMSI 1203.
  • tl e invention provides a protein system comprising a porous matrix material containing an entrapped protein wherein the protein system is characterized by having at least 0.01 mmol of protein per gram of matrix material and enhanced stability as defined by the stability testing procedures described herein.
  • the invention also provides a method of forming a protein system comprising H e steps of: providing a porous matrix material having a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in the range of 50 to 400 A, and reacting tl e porous matrix material witli a protein so that Hie protein chemically bonds to Hie porous matrix material.
  • this method comprises a step of reacting the porous matrix material witli a cross- linking agent to form a porous matrix material having cross-linking agents covalently bound to the surface, and reacting tlie porous matrix material having cross-linking agents covalently bound to the surface with a protein so that the protein chemically bonds to the porous matrix material.
  • tlie invention provides methods in which a protein system is made by adding a protein to a porous matrix material without a cross-linking agent. It has been found that stable and active protein systems can be obtained in which the protein is entrapped by non- covalent bonding.
  • a protein system is formed by: providing a porous matrix material having a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in tlie range of 50 to 400 A, wherein the porous matrix material has a functionahzed surface, and adding a protein so that the protein is entrapped by non-covalent bonding in the porous matrix material.
  • the invention also provides a method of making OPH.
  • a host cell is transfected with a vector comprising a sequence encoding OPH, the sequence being operably linked to a T7 expression control sequence.
  • the transfected host cell is cultured under conditions permitting expression under the control of the expression control sequence.
  • the OPH is purified from tl e cell or tlie medium of tlie cell.
  • the protein system is engineered to match the size of the individual protein with the size of the individual pores, in preferred embodiments, the volume of the individual protein occupies between 5 and 40 % of the average volume of each pore.
  • the invention also includes methods of using these systems in facilitating chemical processes (i.e., processes of making chemicals) such as hydrolysis, oxidation, hydrogenation, and proteolysis.
  • chemical processes i.e., processes of making chemicals
  • the invention also encompasses the use of active enzymes in porous supports in filtration equipment for individual soldiers, pesticide workers, vehicles, aircrafts, ships and buildings such as civilian and military defense shelters, to perform detoxifications.
  • Various embodiments of the present invention can provide numerous advantages including: high protein activities on a porous support; stability under a variety of conditions; high densities of active protein; capability in industrial-scale applications; and providing environmentally safe methods of destroying chemical weapons and organophosphorous pesticides, and avoid the dangers inherent in burning these materials.
  • advantages can be envisioned in view of tl e following descriptions and examples.
  • FIG. 1 is a conceptionalized, cross-sectional representation of an enzyme disposed in a porous substrate.
  • FIG. 2 is a ribbon diagram for OPH plasmid.
  • FIG. 3 is the relevant DNA sequence from the constructs BamH I to Bgl II site that encompasses the region immediately preceding the T7 promoter to just beyond the OPH stop codon, SEQ ID: 2.
  • FIG. 4 is the OPH amino acid sequence, SEQ ID : 1.
  • FIG. 5 shows a comparison of Normal Silica and NH 2 -SAMMS for OPH immobilization.
  • FIG. 6 shows an ionic strength effect on OPH entrapped in HOOC-SAMMS.
  • FIG. 7 shows the effect of pH on OPH entrapment in HOOC-SAMMS.
  • FIG. 8 shows immobilization efficiency of OPH in HOOC-SAMMS at different pH.
  • FIG. 9 shows an ionic strength effect on OPH immobilization efficiency in HOOC-SAMMS.
  • FIG. 10 illustrates stability of OPH immobilized inNH 2 -SAMMS.
  • FIG. 11 shows a comparison of Normal Silica and NH 2 -SAMMS for Glucose Oxidase (GOD) immobilization.
  • FIG. 12 illustrates a coverage effect of a functional group of NH 2 -SAMMS on GOD entrapment.
  • FIG. 13 shows the effect of pH on GOD entrapment in NH 2 -SAMMS.
  • FIG. 14 shows immobilization efficiency of GOD inNH 2 -SAMMS at different pH.
  • FIG. 15 illustrates the stability of GOD immobilized in NH 2 -SAMMS.
  • FIG. 16 shows a comparison of Normal Silica andNH 2 -SAMMS for GI immobilization.
  • FIG. 17 shows a comparison of GI immobilization and pore size effect of NH 2 -SAMMS.
  • FIG. 18 shows immobilization efficiency of GI in NH 2 -SAMMS.
  • FIG. 1 A conceptual illustration of one embodiment of the protein system 2 of the present invention is shown in Fig. 1.
  • a matrix material 4 has pores 6 containing protein 8.
  • the protein 8 is connected to Hie matrix via connecting moieties 10.
  • Many variations of this structure are possible.
  • the porous matrix material preferably has a pore volume wherein at least 90% of the pore volume is composed of pores having sizes in tlie range of 50 to 400 A, more preferably, 100 to 200, and still more preferably 100 to 120 A.
  • pore size distribution is measured by N 2 adsorption using techniques that are well-known in the art. For materials with especially large pores, N 2 adsorption may need to be supplemented by mercury porisimetry or microscopy to get an accurate pore size distribution.
  • pore size refers to pore diameter.
  • tlie protein system the pore size distribution is to be measured without protein in tlie matrix - for measurement purposes, protein can be removed from the matrix by proteases or other appropriate means. For measurements on protein systems, the coupling agents remain bound to tlie matrix during measurement of pore size distribution.
  • Hie pore size distribution of the porous matrix material is measured without coupling agents.
  • the composition of the matrix material can vary, but is preferably an inorganic-oxide-containing material.
  • Inorganic oxide based materials (such as silica-based materials) offer advantages over many organic supports - these advantages can include mechanical strength and chemical and thermal stability.
  • the protein system comprises a coupling agent disposed between the inorganic porous matrix and the protein.
  • the unreacted (that is, before reacting with a protein) inorganic oxide support typically has surface hydroxyl groups.
  • these surface hydroxyls are reacted witli relatively low molecular weight organic compounds to form a functionahzed monolayer.
  • Treatment with the appropriate coupling agent can produce selected functionahzing moieties on the surface of the porous support.
  • Preferred coupling moieties are mercapto (-SH), amino (-NH 2 ), carboxyl (-COOH), hydroxyl (-OH), and azido (-N 3 ).
  • a particularly preferred embodiment utilizes the functionahzed mesoporous support described by Feng et al. in "Functionahzed Monolayers on Ordered Mesoporous Supports," Science, vol. 276, 923-926 (1997).
  • the surface hydroxyls can be reacted with mercaptopropyltrimethoxysilane, (MeO) 3 Si(CH 2 ) 3 SH, to form a functionahzed surface with terminal mercapto groups.
  • Functionahzed surfaces are superior to the nonfunctionalized surfaces because t ey provide better and more controllable chemical environments and bonding to proteins.
  • tlie surface of the porous matrix material is functionahzed
  • the degree of functionalization effects the activity level of the bonded protein.
  • surface coverage is at least 2%, more preferably between about 20 and 70%, more preferably, between 20 and 50 %. Too many coupling moieties can reduce activity while too few reduces covalent attachment of the protein to the matrix and can reduce tlie stability of tlie protein system. It has been found, especially for embodiments in which a coupling agent isn't used, that numerous factors can effect the porous matrix's ability to entrap proteins.
  • a non-exhaustive list of these factors can include: the degree of functionalization, Hie type of functionahzing moieties (for example, amino, carboxyl, etc.), pH, ionic strength, buffering, and pore size.
  • electrostatic interactions can be very important for controlling entrapment of the protein.
  • carboxyls in tl e pores of tlie matrix material can interact with aminos on tlie surface of the protein, and vice versa.
  • hydrophilic/hydrophobic interactions can be controlled to tailor a matrix material for entrapping proteins; for example, hydrocarbon chains in the pores of the matrix material can interact with hydrophobic regions on proteins. Identifying the conditions for entrapping a selected protein can be determined through routine experimentation in view of the descriptions set forth herein.
  • Proteins are polymeric organic compounds comprising more than about 100 amino acid residues, and typically having molecular weights in the range of about 8,000 to about 300,000 daltons.
  • chemically active proteins that is, those proteins that are capable of facilitating a chemical process such as hydrolysis, oxidation, reduction, oxygen transport, optical inversion, dehydrogenation, elimination, etc.
  • enzymes that is, those proteins that catalyze chemical reactions.
  • organophosphorus hydrolase OHPH
  • One particularly preferred protein is organophosphorus hydrolase (OPH) which is known and has been reported in the literature, see, for example, Muchandani et al., "Biosensor for direct determination of organophosphate nerve agents. Potentiometric enzyme electrode," Biosensors & Bioelectronics, 14, 77-85 (1999).
  • a protein can be comprised of amino acids that are all connected through covalent bonds. Proteins can also be comprised of subunits that are held together by non-covalent interactions. For example, hemoglobin is a protein that is comprised of four subunits. Proteins can also include other components such as metal atoms, porphyrin rings, and other manmade or naturally occurring modifications.
  • OPH is a dimeric enzyme that has a diameter of about 45 to 80A with a volume of about 1.95 x 10 5 A 3 . Thus, if a protein system were designed such that OPH occupied 10% of the average pore volume, the matrix would have an average pore volume of about 1.95 x io 6 A 3 .
  • Protein size in the present invention is defined in the conventional sense based on the radius of gyration in tlie non-denatured state, hi Hie protein systems of the present invention, a preferred type of proteins are enzymes having volumes in the range of 0.5 x 10 5 A 3 to 3 x 10 5 A 3 , because proteins within this size (volume) range are especially advantageous in tlie porous matrices of the protein systems of tlie present invention.
  • the protein in the matrix can be compared to the protein in the "active state.”
  • the definition of activity (or "unit activity") for an immobilized protein is the same as the accepted definition for the non-immobilized protein.
  • Activity units are defined in terms of the quantity of protein required to produce a product from a known or characterized substrate in certain buffer conditions at a certain temperature for a specified time. For many enzymes and classes of enzymes, there are commonly accepted activity units. One source of commonly accepted activity units is the Worthington Enzyme Manual (available from tlie
  • the protein of the invention is at least 50% of the activity in the active state, more preferably at least 75%. We have found that activity of the OPH-containing protein systems have excellent activity. In some preferred embodiments, proteins, in systems of the present invention, have activities of 65 to 95 %.
  • tihe protein system of the present invention can also be defined in comparison to an equal mass of a system prepared and tested under the same conditions, including tlie same functionalization conditions, except using a normal silica as the porous matrix material; preferably the protein system has at least 2 times (2X), more preferably at least 5X, still more preferably at least 10X, and still more preferably at least 50X greater activity as compared to a system that is prepared identically except having a normal silica in the porous matrix material.
  • the invention can also be defined as being able to maintain activity under the same conditions and to about Hie same or greater extent as described in tlie examples.
  • a nonlimiting list of proteins that may be employed in various embodiments of the invention is as follows: Adenosine aminohydrolase, Adenosine deaminase, Alcohol oxidase, Alcohol dehydrogenase, Amino acid oxidases, Amino acid deaminases, Amino acid decarboxylases, ATP kinase, ATP phophatase, Bilirubin oxidase, Catalase, Chymotrypsin, Creatine phosphokinase, Catecholase, Cholesterol oxidase, Chitinase, Chitodextrinase, Chitosanase, Cholesterol dehydrogenase, Cholesterol esterase, Choline oxidase, Choline phophatase, Citrase, Cocaine esterase, Cytochrome C, Cytochrome C oxidase, Cytochrome C reductase, Cytochrome
  • the invention can be defined as including any of the proteins listed above (either exclusively, substantially, or including).
  • the invention can be defined wherein the protein is exclusively (i.e., consists of), or substantially (i.e., consists essentially of), or includes (i.e., comprising) OPH (or, alternatively, any of the other proteins from the above list).
  • the coupling agent can be reacted via hydroxyl moieties on tlie support with amines on Hie protein (see, for example, U.S. Patent No. 5,077,210 which is incorporated herein by reference).
  • These connecting moieties are preferably organic moieties having a chain length of 2 to 20 atoms, more preferably 4 to 10 atoms.
  • each protein is bound to the matrix via at least one coupling moiety, more preferably via 2 to 10 moieties.
  • the number of moieties bound to each protein can be detennined by appropriate analytical techniques, for example, by cleaving off the bound proteins and analyzing Hie cleaved molecules by mass spectrometry.
  • a coupling agent can have a siloxane (-Si(OR) x ) terminal group that forms oxo bonds to the surface, a flexible organic chain (e.g., (CH 2 ) n ), and a thiol (-SH) terminal group that bonds with a protein.
  • a siloxane (-Si(OR) x ) terminal group that forms oxo bonds to the surface
  • a flexible organic chain e.g., (CH 2 ) n
  • -SH thiol
  • Measurable properties that can define various embodiments of H e invention include: pore size, pore volume, pore size distribution, surface area, activity, stability, density of protein in support, density of system, and strength of system. It has been discovered that superior properties can be obtained by engineering supports with pore sizes (or pore volumes) that correspond to protein sizes (or protein volumes). Preferably the volume of a protein is between 5 and 40 % of the average pore volume (where, for purposes of this metric, the average pore volume is based only on those pores in tlie size range of 50 to 40 ⁇ ), more preferably the volume of a protein is between 10 and 25 % of the average pore volume. This size matching of protein to pore size can produce surprising improvements in activity and stability.
  • Protein volume can be measured by biophysical methods such as analytical ultracentrifiigation or x-ray crystallography.
  • the activity, measured per protein molecule is at least 60% of the protein's activity in the active state.
  • the system comprises less than 40 volume % protein; more preferably 5 to 40 volume % protein; still more preferably 10 to 25 volume % protein.
  • the system possesses stability such that when exposed to 8M urea (as set forth in the examples), tlie protein in the protein system is at least twice as stable as the free protein, in some embodiments about 3 to about 5 times more stable.
  • the protein in tlie protein system is at least twice as stable (as measured by the rate of denaturing), more preferably at least 3 times as stable, as tlie free protein when placed in solution under any of the conditions that are set forth in tlie Examples.
  • tlie present invention is high surface area, as measured by N 2 adsorption, of tlie protein system.
  • surface area is measured on the functionahzed surface for protein systems and on the unfunctionalized matrix material for the invention defined by methods and systems made by these methods.
  • Surface area is preferably at least 700 m 2 /g; more preferably at least 900 m 2 /g.
  • the upper limit of surface area may be limited by the upper limit of the mesoporous matrix materials of the type described by Feng et al. and similar materials.
  • Another advantage of the present invention is that it can produce a relatively dense protein system.
  • the protein in the system has a density of at least 0.01 mmol/g; more preferably a density of 0.1 to 1 mmol/g.
  • the inventive systems can be characterized by exhibiting any one of its properties or several of its properties in various combinations.
  • the protein system exhibits an activity of 65 to 95% that of the active protein and has a density of 0.1 to 1 mmol g.
  • the porous matrix material including entrapped protein
  • a microchannel is a channel that has at least one dimension of 2 mm or less, preferably 1 mm or less.
  • Microchannels can offer advantages such as enhanced heat and/or mass transfer and compactness of design. Testing with OPH cross-linked into SAMMS disposed in a 0.25 mm wide channel has demonstrated low pressure drop (flow under gravity or peristaltic pumping or less than 1 bar) and fast reaction times.
  • the invention includes apparatus and methods in which a porous matrix material, including entrapped protein, is disposed within a microchannel. hi the methods, for example, a reactant can be passed through the microchannel to form products.
  • Proteins can be prepared by known procedures and, in preferred embodiments, do not need special procedures before reaction with coupling agent(s) to bond to the support. Preferably, prior to bonding within the matrix, the protein should be about 95% pure in an aqueous solution that stabilizes activity, and the buffer should not hinder the coupling chemistry.
  • a host cell is transfected with a vector comprising a sequence encoding OPH, the sequence being operably linked to a T7 expression control sequence.
  • the transfected host cell is cultured under conditions permitting expression under the control of tlie expression control sequence.
  • the OPH is purified from the cell or the medium of Hie cell.
  • the vector is provided with the sequence encoding OPH operably linked to the T7 expression control sequence.
  • tlie OPH has an activity of about 13,000 units/mg.
  • tlie vector is a plasmid.
  • the host cell can be a prokaryotic cell, eukaryotic cell, or yeast cell.
  • the prokaryotic cell is preferably a bacterium, more preferably tlie bacterium is Escherichia coli.
  • the yeast cell is preferably Pichiapastoris.
  • the matrix is preferably a mesoporous oxide material made from soluble precursors.
  • Examples of preferred syntheses are provided in U.S. Patents Nos. 5,645,891 and 5,922,299 and U.S. patent application ser. no. 09/020,028, all three of which are incorporated herein as if reproduced in full below, Liu et al., "Molecular Assembly in Ordered Mesoporosity: A New Class of Highly Functional Nanoscale Materials," J. Phys. Chem., 104, 8328-8339 (Aug. 2000), and the Feng et al. article referenced above.
  • a typical synthesis for a matrix material was reported by Feng et al., Science, 276, p923 (1997).
  • a CTAC/OH solution was prepared by contacting a CTAC solution with a strongly basic ion exchange resin (DOWEX-1 , 0.2 g resin per gram of 29% CTAC solution). 13 g of colloidal silica, 51 g of tetramethylammonium silicate and 28 g of mesitylene were added to each 100 g of CTAH/OH solution. The mixture was sealed in a teflonTM-lined vessel and heated at 105 °C for 1 week. The product was recovered by suction filtration, dried at ambient temperature, and calcined at 540°C for 12 hours in air.
  • DOWEX-1 strongly basic ion exchange resin
  • the surface of the resulting mesoporous material was fiinctionalized by a variety methods.
  • the surface can be functionahzed with thiol groups by reaction with tris(methoxy)mercaptopropylsilane.
  • the resulting functionahzed matrix is called a "SAMMS.”
  • the percent surface coverage was estimated based on (i) the surface area of the support, (ii) the weight change after the functionahzed monolayer was attached, and (3) the ideal loading density that could be achieved on flat surfaces.
  • the percent surface coverage can be verified by electron energy-dispersive spectroscopy (EDS).
  • the support is preheated with a coupling agent, such as bis [2- (sulfosucch ⁇ imidoxycarbonyloxy)ethyl]sulfone (BSOCOES). Excess coupling agent can be washed out. A protein is subsequently reacted with the coupling-agent-treated surface.
  • a coupling agent such as bis [2- (sulfosucch ⁇ imidoxycarbonyloxy)ethyl]sulfone (BSOCOES).
  • BSOCOES bis [2- (sulfosucch ⁇ imidoxycarbonyloxy)ethyl]sulfone
  • protein can first be reacted with the coupling agent, and subsequently reacted with the surface of Hie matrix. Excess protein can be washed out and recovered.
  • the functionahzed porous matrix is first reacted with a cross- linking agent.
  • a cross- linking agent For example, an amino-derivatized SAMMS is reacted with sulfo-BSOCOES or glutaric dialdehyde. If present, it is desirable that excess cross-linking agent is removed. Then, a protein is added. Preferably, the protein is in a low concentration solution. This procedure reduces or eliminates the possibility intermolecular cross-linking of the protein by the cross- linking agent.
  • a porous matrix material (preferably a functionahzed porous matrix material) is combined witli a protein, without die addition of a cross-linking agent.
  • a protein system can be prepared in which the protein is noncovalently bonded to a porous matrix.
  • This method is preferably conducted by tailoring electrostatic interactions such that tlie porous matrix creates a complementary environment for the selected protein (for example, amino groups in the pores of the matrix match up witli carboxyl groups on Hie protein). Factors that may be considered in tailoring the electrostatic interactions have been discussed above.
  • Protein systems as described herein, can be made using any of the methods described herein, and these methods are also part of the present invention.
  • fermentor media Purification and Yeast extract
  • pETl laTM, pETl 5bTM Components of fermentor media (Peptone and Yeast extract) were obtained from Gibco BRL.
  • expression vectors pETl laTM, pETl 5bTM
  • Primers for PCR were ordered from Genosys Inc.
  • Bulk chromatography media for the protein purification was obtained from Perseptive Biosystems (HSTM and HQTM).
  • Polypropyl ATM columns as well as nonderivatized silica resin for comparison of OPH linking obtained from Poly LC Inc., Columbia, MD.
  • Cross-linking reagents for enzyme immobilization were purchased from Pierce Chemical Company, Rockford, IL.
  • CTAC cetyltrimetliylammonium chloride OPH, organophosphorous hydrolase,
  • SAMMS self-assembled monolayers on mesoporous silica
  • Sulfo-BSOCOES bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone
  • DI deionized
  • CPTS monolayer's 3-cyanopropyltrimethoxysilane
  • the treated mesoporous silica was washed with toluene to remove any unreacted silanes.
  • the air-dried CPTS-SAMMS materials were then treated with 50% H 2 S0 4 solution and refluxed for 3 hours. After washed with DI water extensively, the white sample was dried under vacuum at 70°C overnight.
  • APTS aminopropyl
  • MPTS mercaptopropyl
  • OPH sequence was cloned by PCR using pCMS75 plasmid in E.coli FM5 (Amgen Inc.) which had been obtained from American Type Culture Collection, Rockville, MD (ATCC® #67778).
  • OPH sequence was subcloned using Ndel, BamHI restriction sites into pETl la. The resulting
  • OPH sequence encoded the mature portion of OPH enzyme, i.e., without N-terminal 29 amino acid signal sequence, so that the length of the sequence is 1010 bp, which corresponds to 337 amino acids in total or 36,419 Da in MW.
  • the confirmation of tlie correct product of cloning was made by PCR and restriction enzyme digest.
  • FIG. 2 A plasmid diagram for OPH is illustrated in Fig. 2.
  • the organophosphorous hydrolase was purified from E.coli expression system using oph-pETl la plasmid and BL21(DE3) pLyssTM, Novagen Inc., as a host strain. (N-terminal His-
  • Oxygen level was maintained at 35% using DO-agitation-oxygen triple cascade, setting up agitation range 200 rpm minimum to 800 rpm maximum.
  • Initial glucose concentration in the media was 10 g/1, glucose level was monitored during the run using regular glucose strips and kept to be not less than 2 g/1.
  • Thirty-one (31) ml of 40 % glucose were added to bring the glucose level to 5 g/1.
  • the temperature was lowered to 28 °C, the mixture was induced with 0.25 mM IPTG and another 31 ml of 40 % glucose were added.
  • the bacterial cells (60 g) were suspended in 420 ml of lysis buffer A, containing 100 mM HEPES pH 8.5, 50 uM CoCl 2 , 1 mM DTT, antiprotease cocktail (pepstatin, leupeptin and aprotinin), and cells were lysed using French pressure cell 2 times.
  • Soluble protein supernatant obtained by 100,000 x g centrifugation for 1 hr (AvantiTM, Bechman), was loaded on 500 ml HQTM anion-exchange column (Perseptive Biosystems) equilibrated in buffer A, with substitution of lmM DTT for 5 mM beta-mercaptoethanol (2-me).
  • the column flow rate was 25 ml/min. Flow-through containing OPH was collected, pH was adjusted to 7.5 using 1 M MES, pH 5.5, and applied on a 250 ml HS cation exchange column (Perspective Biosystems), equilibrated in 0.1 M HEPES, pH 7.5, 50 uM CoCl 2j 5 mM 2-me. The column flow rate was 25 ml/min. Flow-through of HS column was retained, enough dry (NH 4 )S0 4 was added to make final conductivity of the sample to be equal to conductivity of 1 M (NH 4 )S0 4 solution (i.e., 105 mS /ml), using a conductivity meter Orion 126, Cell 012210.
  • a sample was loaded on a 180 ml Polypropyl ATM (PolyLC, Inc.) column, equilibrated in 1 M (NH 4 )S0 4 , 0.1 M HEPES pH 7.5, 5 mM 2-me.
  • the column flow rate was 10 ml/min.
  • the buffer contained beta-mercaptoethanol, the competitive inhibitor of OPH activity, and no CoCl 2 , the other the buffer contained 50 ⁇ M CoCl 2 and no 2-me.
  • SAMMS derivatized with SH-, COO-, NH 2 - active groups, 5 % and 20 % coating (5 % and 20 % of all available silane groups get modified or derivatized with active groups).
  • Characteristics of the media 250 A 12 - 15 um beads, surface area around 450 m 2 /g
  • Poly LC Silica Purchased uncoated, derivatized in PNNL witli NH 2 -, COO- groups, 20
  • cross-linking agents were tested, among them 2 were found to be especially efficient: Sulfo-BSOCOES, bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone, and DTSSP, dithiobis(sulfosuccinimidylpropionate).
  • Sulfo-BSOCOES bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone
  • DTSSP dithiobis(sulfosuccinimidylpropionate).
  • the advantages of these 2 cross-linking agents are as follows: i. Both cross-linking agents have spacer arms (12 A in length for DTSSP and 13 A for BSOCOES). A spacer arm is beneficial to avoid steric hindrance, ii. Both of them are water soluble due to the sulfo- functioning group. iii. Pierce (Rockford, IL) recommended PBS as the linking buffer, pH 7.5
  • This pH is favorable for OPH because OPH tends to aggregate and lose the metal from active center at an acidic pH, i.e., pH lower than 6.5.
  • a higher pH has another advantage: The hydrolysis of NHS-esters proceeds faster at a higher pH. Therefore when we used a higher pH, we obtained a higher molar ratio of cross-linking agent per protein, iv. Both produce stable covalent amide bond, which in case of DTSSP is cleavable with tliiols (DTT, mercaptoethanol, etc.). This feature could be useful for certain applications.
  • a nitrogen glove box filled with nitrogen was used for taking aliquots of Hie content out of the tubes containing Sulfo-BSOCOES.
  • OPH was coupled to PolyLC at 20 % and 100 % coating surface using Sulfo-BSOCOES and DTSSP, with lOx, 25x, and 50x molar excess of cross-linking agent per protein.
  • the procedures were similar to Example 1 to 6, except for step 2: 1000 mg of PolyLC were used per 5 ml H 2 0 and made ⁇ 50 % slurries (v/v).
  • the estimated amount of OPH linked to the resin is listed in Table II.
  • inventive compositions are capable of higher density loading than witli conventional silica (PolyLC).
  • preferred embodiments of the invention can be characterized by loading densities.
  • tlie protein system has density that is 2 to about 7 times higher (in mg/g) than PolyLC with the same coating %, more preferably about 5 to about 7 times higher.
  • the densities are measured at a 20% coating.
  • the immobilized enzyme was far more stable than the free protein.
  • preferred embodiments of the invention can be characterized by their stability to denaturing agents.
  • the protein system has a stability, in 8M urea, that is at least twice as stable as the free protein, and in some embodiments, about 3 to about 5 times more stable.
  • OPH-SAMMS has approximately
  • Mass in tlie table refers to mass of OPH.
  • OPH-SAMMS and soluble OPH were subjected to lyophihzation conditions (IM MES, pH 5.0) and it was found that OPH-SAMMS retained 50% of its activity after 1 and 24 hours, while the soluble OPH retained only 15% of its activity after 1 and 24 hours.
  • the estimated protein amount of OPH covalently linked was 11.00 mg/g SAMMS by Pierce BCA Assay Kit (Pierce, 23227) wiih the initial specific activity 11386.07 Units per mg linked OPH.
  • Example 15 (1) 10.17 mg GOD (Sigma G-7016) was dissolved in 5 ml of pH7.5, 20 mM
  • Phosphate/0.15 MNaCl as the GOD stock.
  • the enzyme stock contained 395.05 units (1.05 mg OPH protein amount) per ml.
  • the estimated protem amount of GOD covalently linked was 14.85 mg/g SAMMS, witli the initial specific activity 114.31 Units per mg linked GOD.
  • GOD GOD
  • the enzyme stock contained 372.25 units (1.08 mg OPH protein amount) per ml.
  • the washing solution was pH 7.0 instead of pH 7.5 used in Example 15.
  • the estimated protem amount of GOD covalently linked was 81.64 mg g SAMMS, witli the initial specific activity 72.34 Units per mg linked GOD.
  • GI purified from Sfreptomyces rubiginosus, was obtained from Hampton Research, Inc. It was dialyzed in pH7.5, 20 mM Phosphate/0.15 M NaCl/1 mM MgS0 4 , aliquoted in 1 ml, stored at -80°C and thawed in a refrigerator before use.
  • the GI stock contained 180.22 units (4.11 mg GI protein amount) per ml.
  • Example 15 Similar to Example 15, but used the GI stock instead of the GOD stock. And the incubation was carried out at 40°C. (3) The washing solution was pH7.5, 20 mM Phosphate/0.15 M NaCl 1 - 20 mM
  • GI Activity definition at the present condition 1 unit is 1 micromole D-Fructose converted to D-Glucose in one minute at pH 7.5, at 60°C, at 1.923 M D-Fructose concentration. The activity was measured in a general procedure as the following: 5-20 ⁇ l of the GI-SAMMS suspension was added with 0.5 ml 2M of D-Fructose
  • the estimated protein amount of GI covalently linked was 48.47 mg/g SAMMS, witli the initial specific activity 53.40 Units per mg linked GOD.
  • Example 18 (1) Similar to Example 17, but used 200 - 400 ⁇ l of 5% Glutaric Dialdehyde (Aldrich, 34085-5) in pH 7.5, 20 mM Phosphate/0.15 M NaCl instead of the sulfo-BSOCOES solution as tlie covalent linker source. (2) The estimated protein amount of GI covalently linked was 30.45 mg/g SAMMS, witli the initial specific activity 39.19 Units per mg linked GOD.
  • Example 20 1.17 mg GOD (Sigma G-7016) was dissolved in 5 ml of pH7.5, 20 mM
  • the enzyme stock contained 395.05 units (1.05 mg OPH protein amount) per ml.
  • the enzyme stock contained 395.05 units (1.05 mg OPH protein amount) per ml.
  • (3) GOD activity was measured using Sigma standard method (revised on 08/30/96).
  • the estimated protein amount of GOD entrapped was 13.35 mg/g SAMMS, with the initial specific activity 99.33 Units per mg entrapped GOD.
  • the washing solution was pH 7.0 instead of pH 7.5 used in Protocol 7.
  • the estimated protein amount of GOD entrapped was 14.59 mg/g SAMMS, with the initial specific activity 136.28 Units per mg entrapped GOD.
  • GI purified from Sfreptomyces rubiginosus, was obtained from Hampton Research, Inc. It was dialyzed in pH7.5, 20 mM Phosphate/0.15 M NaCl/1 mM MgS04, aliquoted in 1 ml, stored at -80°C and thawed in a refrigerator before use.
  • Tlie GI stock contained 180.22 units (4.11 mg GI protein amount) per ml.
  • Example 20 Similar to Example 20, but used the GI stock instead of the GOD stock. And the incubation was carried out at 40°C. (3) The washing solution was pH7.5, 20 mM Phosphate/0.15 MNaCl/1 - 20 mM
  • the estimated protein amount of GI entrapped was 94.94 mg/g SAMMS, with the initial specific activity 43.26 Units per mg entrapped GOD.
  • Example 23 (1) Purified OPH, aliquoted in 2 ml, stored at -80°C, was thawed, dialyzed from pH 8.5,
  • the enzyme stock contained 4830.00 units (0.39 mg OPH protein amount) per ml.
  • a aliquot of 82.45 mg of 300 A 2% HOOC-SAMMS in 50 ml tube was added with 12 ml of tlie OPH stock, shaking at speed 350 RPM on Innova 4330 refrigerated incubator shaker at 25°C for 2 h 47 min. Then, the resulting suspension was aliquoted in 0.4 ml.
  • tlie enzyme incubation solution was separated and the resulting deposit was washed by n x 400 ⁇ l pH 7.5, 20 mM HEPES (n>10). In between, it was centrifuged at 14,000 RPM for
  • the washing solution was pH 7.0, 20 mM Phosphate buffer.
  • GOD activity was measured using Sigma standard method (revised on 08/30/96).
  • the estimated protein amount of GOD entrapped was 107.94 mg/g SAMMS, with the initial specific activity 76.22 Units per mg entrapped GOD.
  • GI purified from Strptomyces rubiginosus, was obtained from Hampton Research, Inc. It was dialyzed in pH7.5, 20 mM Phosphate/0.15 M NaCl/1 mM MgS04, aliquoted in 1 ml, stored at -80°C. Six aliquots were thawed in a refrigerator before use and diluted together by 22.5 ml of pH 7.0, 20 mM Sodium 1 mM MgS0 4 as the GI stock. The GI stock contained 42.10 units (0.86 mg GI protein amount) per ml.
  • Example 2 Similar to Example 24, but used the GI stock and 100.98 mg of 300 A 20% NH 2 - SAMMS. And the incubation was carried out at 40°C.
  • the washing solution was pH7.5, 20 mM Phosphate/1 - 20 mM MgS04.
  • the estimated protein amount of GI entrapped was 77.26 mg/g SAMMS, with the initial specific activity 45.06 Units per mg entrapped GI.
  • Nonnal Silica (Item#: BMSI 1203), which is commercially available from PolyLC, Inc, was functionahzed in a similar way for SAMMS and used to replace tl e SAMMS in the protocols mentioned above under the same experimental conditions for comparison.
  • BMSI 1203 Nonnal Silica
  • washing solutions were used to replace tlie washing solution in the protocols mentioned above under tlie same experimental conditions for investigating tlie effects of the buffer composition, pH, stability and ion strength.
  • 300 A 20% NH 2 -SAMMS means a pore size of 300 A mesoporous silica with a 20% coverage of NH 2 - functional monolayer derivatized from tris-(methoxy)aminopropylsilane
  • 300 A 2% HOOC-SAMMS means the same mesoporous silica with a 2% coverage of HOOC- functional monolayer from tris-(methoxy)carboxyletl ⁇ ylsilane.
  • Fig. 5 shows comparative data for OPH immobilized on 5 different matrices.
  • the three examples using normal silica had activities of 48 units per mg silica while the OPH immobilized on SAMMS demonstrated activities of 156 and 125. See Examples 14 and 19.
  • the protein systems made from normal silica demonstrated significantly lower activities.
  • Type of functional group and degree of functionalization were found to be important factors in forming protein systems.
  • example 19 activity 156) was repeated with 20 % functionalization, instead of 2%, (on either 100 A, 170 A or the 300 A SAMMS), activities of less than 1 were obtained.
  • Example 14 activity 125 was repeated with 20 % functionalization, instead of 2%, (on either 100 A, 170 A or the 300 A SAMMS), activities of less than 20 were obtained.
  • a 300 A 20% HOOC-SAMMS treated with OPH demonstrated an activity of 97 while a 300 A 20% NH 2 -SAMMS similarly treated with OPH had an activity less than 1.
  • Figs. 6 and 9 show that activity is sensitive to buffer concentration (ionic strength) both for proteins entrapped at varying buffer concentrations (Fig. 6) and specific activity, relative to a solution of OPH at pH 7.2, of entrapped proteins at varying buffer concentrations (Fig. 9). As shown in Figs.
  • a sample prepared at pH 7.0 contained more OPH than samples prepared at higher pH, while tlie specific activity of Hie protein system appeared to be optimized near pH 7.5.
  • the specific activity in Fig. 8 is the specific activity of the immobilized OPH divided by the specific activity of a reference solution of OPH at pH 7.2. Whether held by non-covalent bonds or by covalent bonding, entrapped OPH demonstrated significantly better stability as compared to OPH in solution. As shown in Fig. 10, entrapped protein showed a loss of less than about 30% activity when refrigerated at 4 °C for 110 days. Some activity was recovered, presumably through renaturing, after 40 additional days at room temperature.
  • Fig. 11 shows comparative data for GOD immobilized on 6 different matrices.
  • the three examples using normal silica had activities of 0.2, 0.3 and 0.4 while the GOD immobilized on SAMMS demonstrated activities of 2.0 and 5.9. See Examples 20 and 15.
  • the protein systems made from nonnal silica demonstrated significantly lower activities.
  • the unfunctionalized SAMMS, labeled "300 A Meso-Silica," (i.e., 0% NH 2 ) treated under the same conditions showed an activity of only 0.3.
  • a GOD protein system prepared from 100 A 20% NH 2 -SAMMS showed the same activity as 300 A 20% NH 2 -SAMMS, but a GOD protein system prepared from 100 A 20% NH 2 -SAMMS witli a covalent cross-linker showed an activity of 4.0 as compared to tlie 5.9 activity of the analogously-prepared protein covalently cross-linked in 300 A 20% NH 2 -SAMMS.
  • a porous matrix with a 20% amino surface coverage performed significantly better than a 2% surface coverage.
  • the presence of 0.15 M sodium chloride caused a substantial decrease in activity of GOD in the NH 2 -SAMMS system.
  • tl e non-covalently bonded protein has superior stability relative to the covalently cross-linked system when tested according to tlie procedures described herein.
  • Fig. 16 shows comparative data for GI immobilized on 8 different matrices.
  • Tlie four examples prepared under analogous conditions using nonnal silica had activities of 0.0, 0.0, 0.3 and 0.0 while tlie GI immobilized on SAMMS demonstrated activities of 4.1, 1.2 and 2.6, respectively. See Examples 22, 17 and 18.
  • the unfunctionalized SAMMS labeled "300 A Meso-Silica," (i.e., 0% NH 2 ) treated under the same conditions showed an activity of 0.0.
  • a significantly larger effect was seen based on pore size (Fig. 17), which was not unexpected in view of GI's larger size.
  • Fig. 17 pore size
  • GI in NH 2 -SAMMS was found to be nearly unaffected by the presence of 0.15 M sodium chloride.
  • 300 A 2% NH 2 -SAMMS and 300 A 20% HOOC-SAMMS were found to have little or no activity.
  • GI is a robust enzyme and entrapment in SAMMS showed relatively little stability enliancement for the time period and conditions tested.

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