Patent Application
20130224826
The invention relates to compositions and processes for stabilizing bioactive materials.
Bioactive macromolecules such as proteins, nucleic acids, and functional enzymes may be utilized in various aspects of biomedical and industrial applications. For example, nucleic acids may be utilized as genetic templates for polymerase chain reactions while proteins may be utilized in various detergent mixtures to enhance digestive cleaning efficiency of the detergent. Additionally, proteins such as digestive proteins or enzymes may be utilized to catalyze and decompose organic molecules. Digestive proteins may be utilized in organic media allowing various substrates to be utilized. Should the substrate be insoluble or only soluble in water, the maximum activity of the digestive proteins may not be achieved in an aqueous solution.
Although proteins such as digestive proteins or enzymes may be capable of decomposing and reacting with various organic molecules, they are generally not thermally stable at elevated temperatures. Additionally, such proteins or digestive enzymes are generally not stable in a non-aqueous organic solvent.
There is therefore a need in the art for a thermally stable bioactive composition that may be utilized in elevated temperatures and under dry conditions. There is also a need in the art for a bioactive composition that maintains a high catalytic activity in an organic solvent. There is additionally a need in the art for a thermally stable bioactive composition that maintains a high catalytic activity in organic solvent and a process for producing the bioactive composition.
In one aspect, there is disclosed a bioactive composition including a porous hydrogel matrix. At least one protein that is immobilized in the porous hydrogel matrix forming a hydrogel protein composite that is stable in an organic solvent.
In another aspect, there is disclosed a process for stabilizing a bioactive composition that includes the steps of: forming hydrogel matrix pores around protein molecules and reducing a water content within the hydrogel matrix pores forming a hydrogel protein composite that is stable in an organic solvent.
There is disclosed herein a bioactive composition that includes a porous hydrogel matrix and at least one protein immobilized in the porous hydrogel matrix that forms a hydrogel protein composite that is stable in organic solvent and at elevated temperatures. In one aspect, the hydrogel protein composite has a half-life that is at least 1000 times longer than the half-life of a free digestive protein counterpart in the organic solvent. Additionally, the hydrogel protein composite maintains an activity in the organic solvent. In one aspect, the hydrogel protein composite has an activity that is 1000 times greater than a free digestive protein counterpart in the organic solvent.
In addition to the stability of the hydrogel protein composite in an organic solvent, the hydrogel protein remains biologically active at elevated temperatures. In one aspect, the protein remains biologically active after exposure to a temperature of up to 100° Celsius.
Various proteins may be utilized in the bioactive composition disclosed herein. In one aspect, the protein may be selected from the group consisting of protease, amylase, cellulose, lipase, peroxidase, tyrosinase, glycosidase, nuclease, aldolase, phosphatase, sufatase, dehydrogenase, and lysozyme and combinations thereof. Additionally, bioactive agents may be utilized including antibodies, nucleic acids, fatty acids, hormones, vitamins, minerals, structural proteins, enzymes, and therapeutic agents including histamine blockers and heparin.
The bioactive composition includes a hydrogel matrix that has a water content of from 0 to 0.5 weight percent of the hydrogel matrix. In one aspect, the digestive protein is between 0.1 to 10 dry weight percent of the hydrogel matrix and in one aspect from 0.2 to 4.5 dry weight percent of the hydrogel matrix.
There is also disclosed herein a process for stabilizing a bioactive composition that includes forming a hydrogel matrix pore around a protein molecule and reducing a water content within the hydrogel matrix pores forming a hydrogel protein composite that is stable in organic solvent. In one aspect, the step of forming the hydrogel matrix pores around the protein molecules includes the step of dissolving a protein in deionized water at a desired concentration and dissolving a prepolymer in deionized water at a desired concentration and mixing the dissolved protein and dissolved prepolymer in a specified ratio. Next, polymerization is initiated of the prepolymer composition. In one aspect, the step of initiating the polymerization includes at least one step that may be selected from adding a cross linking agent, adding an initiator, adjusting a temperature of the mixture of the dissolved protein and the dissolved prepolymer. In one aspect, the ratio of dissolved prepolymer is from 20 to 28 percent by weight in relation to the total volume of the prepolymer.
The step of reducing a water content within the hydrogel matrix pores may include heating the hydrogel matrix to a temperature of from 20 to 100 degrees Celsius for a time period of from 24 hours to 7 days. Additionally, the step of reducing a water content within the hydrogel matrix may include heating the hydrogel matrix to a temperature of from 20 to 80 degrees Celsius for a time period of 24 hours followed by air drying at room temperature for a specified period such as 1 week.
Additionally, the step of reducing a water content within the hydrogel matrix pores may include heating the hydrogel matrix to a temperature of from 20 to 55 degrees Celsius for a time period of 24 hours followed by air drying at room temperature for a specified time period such as 1 week.
Further, the step of reducing a water content within the hydrogel matrix pores may reduce the pore volumes of from 15 to 21 percent compared to a wet gel volume. The step of reducing water content may reduce the pore volumes and allow a substrate to enter the pores allowing a reaction between the protein and a substrate. In this manner, the bioactive composition may be utilized in various reactions. In one aspect, the cross linking of the bioactive composition may be adjusted to again allow access of a substrate into the pores of the entrapped enzyme. The bioactive composition may have various shapes such as fibers, beads, filaments, or rods when used for various reactions.
In one aspect, the pretreatment of the bioactive composition may vary based on a transition temperature that would trigger a structural change of the enzyme being utilized. Referring to the table presented below, it can be seen that various enzymes have transition temperatures labeled TM in degrees Celsius. Listed in the table are various enzymes including glucose oxidase, peroxidase, α-chymotrypsin, thermolysin, α-amylase, and lipase. As can be seen in the table, the suggested pretreatment temperatures will vary based upon the transition temperature TM. As can be seen in the table, the suggested pretreatment temperature does not greatly exceed the transition temperature which would result in a large structural change of an enzyme.
Aspergillus niger
Bacillus thermopmfeolyticus
Bacillus amyloliquefaciens
Candida cylindracea
Various aspects of the present invention are illustrated by the following nonlimiting examples. The examples are for illustrative purposes and are not a limitation on the practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention.
Materials
Acylamide/Bis solution and NNN′N′-tetramethyleethylenediamine (TEMED) were the products of Bio-Rad Laboratories, Hercules, Calif., USA. D-(+)-glucose, glucose oxidase (GOx) from aspergillus niger (EC 1.1.3.4), peroxidase (HRP) from horseradish (EC 1.11.1.7), o-dianisidine, HPLC grade ethanol, methanol and chloroform, toluene, ammonium persulfate were obtained from Sigma Chemical Co., St. Louis, Mo., USA. Unless specially mentioned, all other reagents and solvents used in the experiments were of the highest grade commercially available.
Entrapment of Glucose Oxidase (GOx) into Polyacrylamide Hydrogel
The entrapment of GOx into polyacrylamide hydrogel was performed by the following procedure: 2 ml of 0.1M pH 7.0 sodium phosphate buffer containing 0.5-10 mg GO, were prepared and subsequently mixed with 6.8 ml of 30% acylamide/bis solution and 1.2 ml of DI H2O to make a 10 ml of solution with total monomers concentration of 20% (w/v) and cross-linker concentration of 5% (w/w). The polymerization using enclosures (8.3×7×0.075 cm3) to obtain hydrogel disks of predefined shape was initiated by adding 100 μl fresh prepared ammonium persulfate (10% w/v in DI water) and 4 μl of TEMED at room temperature. A period of at least 4 hours was needed for complete gelling to entrap the enzyme. The resulting hydrogel disks were removed from the glass enclosures and punched into small disks with a diameter of 16 mm for further tests.
Activity Assays for Native and Hydrogel-Entrapped GOx
A coupled-enzyme reaction using horseradish peroxidase and o-dianisidine was applied to determine the activity of GOx. For the native enzyme, the reaction mixture (1.1 ml) contained 0.1 mol glucose, 7 μg horseradish peroxidase, 0.17 mM o-dianisidine and 35 μl enzyme (0.4-0.8 U/ml) in 50 mM pH 5.1 sodium acetate buffer. The increase in absorbance at 500 nm at room temperature was recorded for activity calculation. The reaction with hydrogel-entrapped GOx was conducted in 20-ml glass vials.
To measure the activity of hydrogel-entrapped GOx, the dried hydrogel disc was immersed into DI water for at least 2 hours to reach the fully swollen state before activity test. The hydrogel disc was added to 20.7 ml of 0.1M glucose solution containing 0.14 mg horseradish peroxidase and 1.1 mg o-dianisidine. Aliquots of 1 ml each were taken periodically and recombined immediately after measuring the product concentration using UV absorbance at 500 nm.
Pretreatment (Drying) of Hydrogel-Entrapped Enzymes
For hydrogel-entrapped glucose oxidase, the effective pretreated temperature was found to be in the range from 20° C. to 80° C. Preferably, the fresh hydrogel discs were placed into a petri dish and incubated in the oven at 80° C. for 24 hours, followed with drying in the air at room temperature for 1 week allowing for complete desiccation. Finally the dry hydrogel discs were used for further testing and the activity was measured by hydrolysis in an aqueous solution as described above.
Thermal Stability of Pretreated Hydrogel-Entrapped GOx
GOx containing hydrogel discs were pretreated as stated above, placed on a glass plate and incubated in an oven at high temperature (80, 110, and 130° C.). At certain time periods hydrogel discs were withdrawn from the oven and residual catalytic activity was assessed using the assay procedure as described above.
Stability in Organic Solvents of Pretreated Hydrogel-Entrapped GOx
After pretreatment, the stability of hydrogel-entrapped enzymes was investigated in organic solvent such as polar type like acetone, methanol, ethanol or nonpolar type such as toluene. The pretreated hydrogel discs from the same batch were incubated in 10 ml of each solvent in screw-capped 20-mL vials. The native enzyme and non-pretreated hydrogel discs served as comparisons. The hydrogel discs were removed from the solvent at specific time periods for activity assay to determine the residual activity.
It was found that at room temperature the average lifetime of the pretreated hydrogel-entrapped-GOx in methanol was estimated as long as 5,650 hours whereas that of the native enzyme was less than 5 minutes and it was 50.3 hours for non pretreated wet hydrogel entrapped enzyme.
Stability of Dried Hydrogel-Entrapped GOx in Organic Solvent at High Temperature
After pretreatment, the stability of hydrogel-entrapped GOx was investigated under the concomitant impact of high temperature and a polar solvent. Typically, the dried hydrogel discs were incubated in 10 ml of pure ethanol at a fixed temperature of 74° C. Screw-capped 20-mL vials were used. At certain times the hydrogel discs were removed from ethanol solution to determine the residual activity.
As detailed in
Materials
Many of the same materials detailed in example 1 were used with the further use of, α-chymotrypsin (α-CT) from bovine pancreas (EC 3.4.21.1), n-acetyl-L-phenylalanine ethyl ester (APEE), dimethylsulfoxide (DMSO) and n-succinyl-ala-ala-pro-phe p-nitroanilide (SAAPPN) purchased from Sigma-Aldrich (St. Louis, Mo., USA). n-Propyl alcohol (n-PrOH, HPLC grade) were purchased from EM (Gibbstown, N.J.). All organic solvents were treated with 3 Å molecular sieve for at least 24 hours before being used. Unless specially mentioned, all other reagents and solvents used in the experiments were of the highest grade commercially available.
Entrapment of α-Chymotrypsin (α-CT) into Polyacrylamide Hydrogel
The entrapment of α-CT into polyacrylamide hydrogel was performed by the following procedure: 0.42 ml of 0.01 M pH 7.5 sodium acetate buffer containing 0.5-10 mg α-CT were prepared and subsequently mixed with 9.33 ml of 30% acylamide/bis solution and 0.25 ml of DI H2O to make a 10 ml of solution with total monomers concentration 28% w/v % and cross-linker concentration 5%. The polymerization using glass enclosures to obtain hydrogel disks of predefined shape (8.3×7×0.075 cm3) was initiated by adding 100 μl fresh prepared ammonium persulfate (10% w/v in DI water) and 4 μl of TEMED at room temperature. A period of at least 4 hours was needed for complete gelling to entrap enzyme. The resulting hydrogel disk was removed from the glass enclosures and punched into small discs with a diameter of 16 mm for further tests.
Aqueous Activity Assays for Native and Hydrogel-Entrapped α-CT
For native enzyme, 50 al of enzyme solution (1 mg/ml) were mixed with 2.44 ml of SAB and 13 μl of 160 mM SAAPPN stock solution in DMSO. The reaction rates were determined by monitoring the absorbance at 410 nm.
For hydrogel-entrapped α-CT, the dried gel disc was immersed into DI water for at least 2 hours to reach the fully swollen state before activity test. The activity was measured through the reaction in 20-ml vials with 4.975 ml of pH 7.5, 10 mM sodium acetate buffer with 5 mM calcium acetate and 25 μl of 160 mM SAAPPN stock. The reaction was initiated by the addition of hydrogel-entrapped enzyme with stirring at 200 rpm. Aliquots of 1 ml each were taken periodically and recombined immediately after measuring the product concentration using UV absorbance at 410 nm.
Pretreatment (Drying) of Hydrogel-Entrapped Enzymes
For hydrogel-entrapped α-chymotrypsin, the effective temperature for drying was found to be in the range from 20° C. to 55° C. Preferably, the fresh hydrogel discs were incubated in the oven at 55° C. for 24 hours followed by drying in the air at room temperature for 1 week allowing for complete desiccation. Finally the dry hydrogel discs were used for further testing and the activity was measured by hydrolysis in an aqueous as described above.
As can be seen in
Stability of Pretreated (Dry) Hydrogel-Entrapped α-CT in Methanol
After pretreatment, the stability of hydrogel-entrapped α-CT was investigated in methanol. A single pretreated hydrogel disc from the same batch was incubated in 10 ml of pure methanol in a 20-mL screw-capped vial. The native enzyme and non-pretreated hydrogel discs served as comparisons. The hydrogel discs were removed from the solvent at specific time to determine the residue activity (assay described above).
Gel-entrapped α-CT also showed greatly enhanced stability in organic solvent as detailed in
Transesterification Activity of Native and Pretreated Hydrogel α-CT
The transesterification activity of native α-CT in organic solvents was measured at room temperature in hexane or isooctane containing APEE (concentration ranged from 2.5 to 30 mM) and 0.5 M n-PrOH. Typically, 5 mg of native CT powder was added to 10 mL of reaction solution to initiate the reaction. During reaction at 200 rpm, aliquots of 200 μl from the reaction solution was periodically removed by filtration using a 0.22 μm PTFE syringe filter following centrifugation for 5 minutes at 13,000 rpm. A volume of 100 μl of supernatant was used for gas chromatograph analysis (GC method listed below).
For hydrogel-entrapped α-CT, one piece of dried hydrogel disc was added into 10 ml of hexane or isooctane containing APEE (concentration ranged from 2.5 to 30 mM) and 0.5 M n-PrOH. The reaction was shaken at 200 rpm, while aliquots of 200 μl were taken periodically and centrifuged for 5 minutes at 13,000 rpm. A volume of 100 μl of supernatant was used for gas chromatograph analysis.
The product concentration was monitored by using a gas chromatograph equipped with a FID detector and a RTX-5 capillary column (0.25 mm×0.25 μm×10 m, Shimadzu). A temperature gradient from 100 to 190° C. at a heating speed of 20° C./min, followed by 5-min retention at 190° C. was used. The injection temperature column was kept at 210° C. whereas the detector temperature was 280° C. The initial reaction rate of the formation of n-acetyl-L-phenylalanine propyl ester (APPE) was calculated. As shown in
Water Content Effects on the Activity
The activity of gel-confined α-CT was examined in organic solvent with varying water amount ranging from 0 to 1.5 v/v % in n-hexane. Native α-CT powder suspended in hexane with the same water content served as a control. As detailed in
Referring to
This application is a divisional of U.S. patent application Ser. No. 12/903,312, filed Oct. 13, 2010, the entire content of which is incorporated herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12903312 | Oct 2010 | US |
| Child | 13856244 | US |
