ENZYME FORMULATION AND METHOD FOR DEGRADATION

Abstract

An enzyme formulation includes an encapsulated fungal enzyme which is effective for degrading at least one material selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds. A degradation method includes treating one of the above-mentioned materials with an encapsulated fungal enzyme to degrade the material.

Claims

1-24. (canceled)

25. An enzyme formulation comprising a suite of enzymes comprising manganese peroxidase, lignin peroxidase, and laccase, ABTS, H.sub.2O.sub.2, and veratryl alcohol in a cross-linked hydrogel matrix.

26. The formulation of claim 1 in the form of beads and wherein the hydrogel matrix comprises manganese.

27. The formulation of claim 2 wherein the beads have a diameter within a range of from about 1.5 mm to about 5 mm.

28. The formulation of claim 1 wherein the cross-linked hydrogel comprises calcium alginate, manganese alginate, zirconium alginate, calcium poly(aspartate), manganese poly(aspartate) or zirconium poly(aspartate).

29. The formulation of claim 1 dispersed onto soil.

30. A method of making an enzyme formulation, comprising: providing a solution comprising: a suite of enzymes comprising manganese peroxidase, lignin peroxidase, and laccase, a cross-linked hydrogel, ABTS, and veratryl alcohol; and adding the solution dropwise to a bath of crosslinkers.

31. The method of claim 6 wherein the bath is a stirred bath comprising MnCl.sub.2, CaCl.sub.2 and BaCl.sub.2.

32. The method of claim 7 wherein the bath is a stirred bath comprising MnCl.sub.2.

33. A method of degrading a material, comprising: treating the material with the formulation of claim 1, wherein the material is selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds.

34. The method of claim 9 wherein the hydrocarbons include total petroleum hydrocarbons.

35. The method of claim 9 wherein the hydrocarbons include polycyclic aromatic hydrocarbons.

36. The method of claim 9 which includes remediating a soil contaminated with the material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1 illustrates the experimental setup for alginate encapsulation of enzyme.

[0079] FIG. 2 illustrates the reaction pathways of heme porphyrin with various reactants.

[0080] FIG. 3 illustrates the molecular structure of a tetrasaccharide monomer of the ionotropic alginate ion polymer.

EXPERIMENTATION

Laboratory Experiment Approach:

Dry Soil

[0081] Grow culture of P. chrysosporium and collect extracellular enzyme extract
Lyophilize enzyme extract and resuspend in 10 ML
Apply to soil microcosm and add hydrogen peroxide (reaction substrate)

pH 4.5, Temp 25 C

[0082]

TABLE-US-00002 Treatments Set Up Soil Enzyme Peroxide 1 ✓ 2 ✓ ✓ 3 ✓ ✓ ✓
20 g soil
2 mL purified enzyme at 20 U/mL
100 μL 10 mM hydrogen peroxide added every other day
TPH and PAH measured after 7 days and 14 days
All treatments prepared in duplicates

TABLE-US-00003 Results - Soil, Enzymes + H.sub.2O.sub.2, Day 7 Day 0 Day 7 Percent (mg/kg dry weight) (mg/kg dry weight) Loss TPH in Soil  C6-C12 ND >C12-C28 1,530 >C28-C35 581 Total C6-C35 2,110 PAHs in Soil Acenaphthene 0.231 Acenaphthylene 1.45 Benzo[a]anthracene 9.78 Chrysene 31.6 Phenanthrene 19.1 Fluoranthene 15.31

ADDITIONAL EXPERIMENTS

1. Selection of Fungal Strains for Production of High Concentrations of Enzymes: Manganese Peroxidase and Laccase.

[0083] Exp. 1. Phanerachaete chrysosporium 1309, Lenzites betulina 141, Trametes versicolor 159, Trametes cervina 33, Trametes ochraceae 1009, Trametes pubescens 11, Stereum hirsutum 42, Trametes zonatus 540, Trametes hirsuta 119, Phlebia radiata 312.
Composition of synthetic medium, (g/l): KH.sub.2PO.sub.4—1.0; MgSO.sub.4—0.5; CaCl.sub.2—0.1; FeSO.sub.4×7H.sub.2O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratryl alcohol—0.3, pH 5.0.

TABLE-US-00004 Laccase, U 1.sup.−1 pH Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 5.0 6.4 6.5 6.5 L. betulina 141 0 0 0 0 6.0 6.5 7.1 7.0 T. versicolor 159 2352 890 613 0 6.2 7.3 6.7 6.4 T. cervina 33 0 0 0 0 6.2 5.8 5.6 6.7 T. ochraceae 1009 121 252 231 111 6.0 5.9 6.1 6.0 T. pubescens 11 0 8 3 0 5.9 5.0 5.7 6.0 S. hirsutum 42 3 0 0 0 5.0 5.0 5.0 5.0 T. zonatus 540 4200 3276 2394 2100 5.8 6.2 6.8 6.6 T. hirsuta 119 256 143 336 806 4.8 4.3 6.0 5.5 P. radiata 312 17 8 10 15 6.1 5.8 5.8 5.3 MnP, U 1.sup.−1 (610 nm) MnP, U 1.sup.−1 (270 nm) Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 0 103 43 0 L. betulina 141 0 0 0 0 0 0 0 9 T. versicolor 159 77 890 16 0 99 60 21 0 T. cervina 33 59 0 559 254 21 125 168 236 T. ochraceae 1009 48 252 205 87 176 280 267 232 T. pubescens 11 0 8 0 0 0 0 0 0 S. hirsutum 42 0 0 0 0 0 9 9 13 T. zonatus 540 86 3276 144 100 146 112 99 86 T. hirsuta 119 0 143 0 0 13 9 9 17 P. radiata 312 0 8 0 0 0 9 0 0 LiP, U 1.sup.−1 Cultivation days 4 6 8 11 P. chrysosporium 1309 2 6 3 3 L. betulina 141 1 2 2 4 T. versicolor 159 9 4 2 2 T. cervina 33 1 1 3 6 T. ochraceae 1009 4 6 19 19 T. pubescens 11 2 1 8 2 S. hirsutum 42 0 0 1 1 T. zonatus 540 2 7 14 25 T. hirsuta 119 6 5 1 3 P. radiata 312 9 13 5 8
Composition of medium, (g/l): KH.sub.2PO.sub.4—1.0; MgSO.sub.4—0.5; CaCl.sub.2—0.1; FeSO.sub.4×7H.sub.2O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; MP—40.0. pH 5.0.

TABLE-US-00005 Laccase, U 1.sup.−1 pH Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 4.2 5.8 6.8 7.4 L. betulina 141 0 0 0 0 5.8 6.0 6.4 6.5 T. versicolor 159 106 143 235 0 5.1 6.2 6.1 6.0 T. cervina 33 0 0 0 0 5.2 5.4 6.2 6.3 T. ochraceae 1009 5544 5432 1596 722 3.2 5.0 5.2 5.6 T. pubescens 11 0 0 0 0 5.3 5.0 5.4 4.7 S. hirsutum 42 168 223 164 69 4.0 4.9 4.7 5.0 T. zonatus 540 8400 7896 5796 2520 3.8 5.2 5.2 5.7 T. hirsuta 119 1276 353 67 22 4.0 4.6 4.3 4.0 P. radiata 312 0 0 0 0 5.0 5.1 5.6 4.2 MnP, U 1.sup.−1 (610 nm) MnP, U 1.sup.−1 (270 nm) Cultivation days 4 6 8 11 4 6 8 11 P. chrysosporium 1309 0 0 0 0 0 0 0 0 L. betulina 141 0 0 0 0 17 0 0 13 T. versicolor 159 0 0 0 0 9 9 0 9 T. cervina 33 46 966 846 742 0 615 512 396 T. ochraceae 1009 100 164 171 104 374 318 310 387 T. pubescens 11 0 0 0 0 0 0 0 0 S. hirsutum 42 0 0 0 0 17 17 0 0 T. zonatus 540 129 103 97 99 129 215 159 172 T. hirsuta 119 0 0 0 0 30 9 26 0 P. radiata 312 0 0 0 0 0 0 0 4 LiP, U 1.sup.−1 Cultivation days 4 6 8 11 P. chrysosporium 1309 192 0 7 0 L. betulina 141 0 0 0 11 T. versicolor 159 0 15 37 15 T. cervina 33 0 0 0 0 T. ochraceae 1009 24 8 14 76 T. pubescens 11 0 0 0 3 S. hirsutum 42 0 2 1 38 T. zonatus 540 27 16 20 55 T. hirsuta 119 21 18 14 22 P. radiata 312 0 0 0 11
Exp. 2. Cerrena unicolor 300, Cerrena unicolor 301, Cerrena unicolor 302, Cerrena unicolor 303, Cerrena unicolor 305, Coriolopsis gallica 142, Merulius tremelosus 206, Pellinus tuberculosus 121, Pellinus tuberculosus 131, Cyatus striatus 978.
Composition of synthetic medium, (g/l): KH.sub.2PO.sub.4—1.0; MgSO.sub.4—0.5; CaCl.sub.2—0.1; FeSO.sub.4×7H.sub.2O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratrylalcohol—0.3, pH 5.0.

TABLE-US-00006 Laccase, U 1.sup.−1 pH Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 336 468 286 798 6.0 6.1 6.2 6.1 C. unicolor 301 77 134 69 185 5.0 5.2 5.3 5.8 C. unicolor 302 172 840 1260 7644 5.8 5.8 6.0 6.0 C. unicolor 303 76 151 133 407 5.3 5.8 5.4 5.8 C. unicolor 305 1025 420 176 210 5.5 5.5 5.5 5.7 C. gallica 142 105 332 470 504 5.3 5.7 5.6 5.7 M. tremelosus 206 605 504 181 66 4.9 4.3 4.5 4.5 P. tuberculosus 121 4 20 0 17 5.8 6.2 6.1 6.1 P. tuberculosus 131 0 0 2 8 6.0 6.2 6.0 6.0 C. striatus 978 4 0 0 20 6.1 6.1 6.1 5.8 MnP, U 1.sup.−1 (610 nm) MnP, U 1.sup.−1 (270 nm) Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 437 79 67 42 645 17 0 0 C. unicolor 301 156 221 552 81 206 482 507 155 C. unicolor 302 34 40 41 70 52 43 26 0 C. unicolor 303 394 734 874 55 507 1015 576 95 C. unicolor 305 206 225 101 49 284 507 215 146 C. gallica 142 29 59 22 3 26 17 34 30 M. tremelosus 206 4 0 0 0 0 0 9 0 P. tuberculosus 121 22 56 152 160 0 0 0 0 P. tuberculosus 131 119 201 308 128 0 0 0 0 C. striatus 978 44 5 0 0 0 0 0 0 LiP, U 1.sup.−1 Cultivation days 5 7 9 12 C. unicolor 300 1 2 3 0 C. unicolor 301 0 2 2 15 C. unicolor 302 4 3 2 0 C. unicolor 303 5 18 0 9 C. unicolor 305 1 4 1 30 C. gallica 142 9 12 12 3 M. tremelosus 206 0 0 0 0 P. tuberculosus 121 0 0 0 0 P. tuberculosus 131 0 0 0 0 C. striatus 978 0 0 4 0
Composition of medium, (g/l): KH.sub.2PO.sub.4—1.0; MgSO.sub.4—0.5; CaCl.sub.2—0.1; FeSO.sub.4×7H.sub.2O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; glycerol—10.0; MP—20.0. pH 5.0.

TABLE-US-00007 Laccase, U 1.sup.−1 pH Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 7392 6888 3654 9576 5.5 5.6 5.6 5.8 C. unicolor 301 4508 6552 2394 4620 4.7 4.8 5.0 5.0 C. unicolor 302 4620 7560 5516 12432 4.6 5.7 5.8 6.0 C. unicolor 303 2520 4340 2520 2016 4.3 5.0 5.0 5.2 C. unicolor 305 4620 5992 3276 5460 4.9 4.9 5.0 5.0 C. gallica 142 3528 2898 5292 2688 4.8 5.7 5.2 5.2 M. tremelosus 206 2982 3318 3570 3864 4.4 4.5 4.5 4.5 P. tuberculosus 121 13 42 34 121 4.7 5.6 5.5 5.7 P. tuberculosus 131 10 8 7 17 4.4 5.3 5,5 5.5 C. striatus 978 500 622 672 1596 4.7 4.6 4.6 4.6 MnP, U 1.sup.−1 (610 nm) MnP, U 1.sup.−1 (270 nm) Cultivation days 5 7 9 12 5 7 9 12 C. unicolor 300 423 92 30 14 980 120 34 0 C. unicolor 301 953 1122 760 808 2242 2761 705 1152 C. unicolor 302 16 24 13 24 0 17 9 0 C. unicolor 303 962 1072 313 22 2219 1376 237 56 C. unicolor 305 843 935 911 513 1213 1084 714 731 C. gallica 142 74 57 108 111 52 69 52 60 M. tremelosus 206 11 25 58 0 0 17 34 0 P. tuberculosus 121 701 898 591 124 0 22 0 0 P. tuberculosus 131 846 363 400 347 26 26 86 26 C. striatus 978 0 0 4 0 17 0 38 0 LiP, U 1.sup.−1 Cultivation days 5 7 9 12 C. unicolor 300 47 98 196 0 C. unicolor 301 1 37 67 110 C. unicolor 302 6 74 103 0 C. unicolor 303 24 35 59 5 C. unicolor 305 47 73 25 70 C. gallica 142 3 121 107 6 M. tremelosus 206 0 0 0 0 P. tuberculosus 121 0 0 0 0 P. tuberculosus 131 47 0 0 2 C. striatus 978 7 0 0 0

2. Alginate Encapsulation of Enzyme Cocktail

[0084] Mycorernedation via Encapsulation and Controlled Release of Ligninolytic Enzymes from Alginate microparticles

The goal of this work is to develop the use of alginate encapsulation approaches for ligninolytic enzymes for the stabilization and controlled release in soils contaminated with target hydrocarbons. The ideal result will be the identification of the materials and methods yielding alginate microparticles meeting the following:

[0085] Small enough size that they can be dispersed in aqueous medium and sprayed onto soil

[0086] High active until loading (Units/mass of dispersion)

[0087] Long term stability

[0088] Demonstrated ability to degrade hydrocarbons in contaminated soil.

Task 1—Investigate the Effect of 1VIn2+ on Enzyme Activity in Alginate Beads

[0089] We will test the effect of the inclusion of Mn2+ on the encapsulation of three ligninolytic enzymes: lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase. We will evaluate the capsules' size and enzyme loading. A promising formulation will be selected for investigation into methods to reduce the size.

Task 2—Investigate Methods of Reducing the Alginate Bead Size

[0090] Methods to be investigated are microemulsion and extrusion techniques. These methods will be evaluated based on particle size, dispersability, and potential sprayability. In addition, we will evaluate the enzyme loading in each microcapsule (units/mass capsule), The most promising method(s) will be chosen for testing long term stability and efficacy in contaminated soil.

Task 3—Stability and Efficacy

[0091] One or more promising methods will be chosen for final stability and efficacy tests. These will be tested against unencapsulated control enzymes. Results will be gauged on both the ability of the capsule to improve enzyme stability as well as ability to degrade the target hydrocarbons in contaminated soil.

TABLE-US-00008 TABLE 2 Experimental Test Matrix for Tasks 1 and 2. Alginate concentration will be constant at 20 mg/mL (2% w/v) based on prior results and published data. Enzyme loading will be chosen based on desired active units per mass of alginate. Processing Variables Compositional Variables Cross Method For Link Density/Amendment Extrusion, Excursion For Emulsion Run C.sub.BaCl2 C.sub.CaCl2 C.sub.MnCl2 Spray, Flow Rate Stir Rate C.sub.tween80 Task (#) (mM) (mM) (mg/mL) Emulsion (mL/hr) (RPM) (mg/mL) 1 1 0 10 100 Extrusion 10 2 1 0 100 Extrusion 10 2 3 TBD TBD 100 Extrusion 200 2 4 TBD TBD 100 Extrusion 400 2 41 TBD TBD 100 Extrusion 200 4

[0092] Encapsulation Experiments Continued

Background/Executive Summary

[0093] In the first round of alginate experiments, we saw that the conditions were not able to yield discrete alginate particles. The beads did not solidify and most of the collection bath became brown indicating that enzyme was not efficiently encapsulated. We hypothesized that the concentration of the crosslinking divalent ions were too low, and that this was resulting a weak encapsulating hydrogel matrix. To test this, we amended our test matrix to test three combinations of crosslinking divalent ions, each with higher concentrations of CaCl2 and BaCl2. The concentration of MnCl2 was kept constant at 100 mM since this is already high, and because the Mn2+ ion place a role in the enzyme activity in addition to crosslinking the alginate. The result confirmed our hypothesis and increasing the CaCl2 and BaCl2 concentrations yielded much more robust, and spherical beads. However, the collection bath still showed some brown color. We will run enzyme activity tests to quantify the units per bead. This will be done by dissolving a bead in 55 mM sodium citrate and running an assay on the solution. We will also run assays on the collection bath solutions.

Approach

Materials

[0094] A stock solution of Alginate in DI water was prepared at 40 mg/mL and dissolved by heating in an autoclave. Other stock solutions were prepared accordingly. ABTS (10 mg/mL), CaCl2 (200 mM), MnCl2 (200 mM) and BaCl2 (10 mM) in DI water. Enzyme (MnP from C, unicolor 300) was used as received. This was a vicious dark brown liquid with the following estimated enzyme concentrations: laccase (437 U/mL), MnPA270 (265 U/mL), yielding a total enzyme concentration of 840 U/mL.

Procedure

[0095] 1. To a 20 mL glass scintillation vial, add: [0096] a. 3.3 mL Alginate Stock (40 mg/mL; via 10 mL B-D syringe and 18 gauge hypodermic [0097] b. 2.9 mL enzyme (MnP from C unicolor 300; via 10 mL B-D syringe and 18 gauge hypodermic [0098] c. 0.132 mL of ABTS (10 mg/mL in DI water; via volumetric pipette) [0099] d. 0.289 mL of DI water (via volumetric pipette) [0100] 2. This resulted in pre-alginate solution with the following concentrations: [0101] a. 365 U/mL total enzyme (composed of the following enzymes) [0102] b. 190 U/mL Laccase [0103] c. 115 U/mL MnPA610 [0104] d. 60 U/mL MnPA610 [0105] e. 0.2 mg/mL ABTS [0106] f. 20 mg/mL Alginate [0107] 3. This solution was dispensed into collection baths with various concentrations of crosslinking ions (shown in Table 1 below), including MnCl2, CaCL2. For each run, 1 mL of pre-alginate solution was dispensed (at 10 mL/hr) through a 22 gauge stainless steel, blunt tipped needle into 50 mL of crosslinking solution in the collection bath. As the droplets hit the solution, they immediately solidified and sank to the bottom of the dish. The dish was rotated by hand to avoid accumulation of beads in one place in the dish. Note, that throughout the dispensing step, the collection bath gradually adopted a light brown color, indicating that some of the enzyme was diffusing from the beads into the collection bath. [0108] 4. After 1 mL was dispensed, the dish was left to sit for at least 30 minutes to allow the crosslinking to complete. Then the liquid was pipetted off the stored as the decantate. The dry beads were imaged using a camera (images were later analyzed for particle diameter using ImageJ software). These were then resuspended in 1 mL of DI water and stored in the refrigerator.

Results

[0109] The two of the runs (samples 3 and 4) yielded discrete beads that were able to be measured using ImageJ. If we assume that all of the enzyme in the pre-alginate solution was encapsulated in the beads (i.e. 100% encapsulation efficiency), and we estimate the bead volume from the measured diameters, we can estimate the enzyme concentration per bead (U/bead). This is shown in the Table below. Images and particle size distributions are shown in Figure below.

TABLE-US-00009 TABLE 1 Bead size and Estimated Enzyme loadings for successful runs (3 and 4). Bead Date Run C.sub.BaCl2 C.sub.CaCl2 C.sub.MnCl2 Diameter Enzyme Loading (U/bead) (y/m/d) (#) (mM) (mM) (mg/mL) (mm) Laccase MnP.sub.270 MnP.sub.610 2015 Mar. 18 1 10 100 0 NA NA NA NA 2015 Mar. 18 2 0 100 1 NA NA NA NA 2015 Mar. 23 3 50 100 2.5 2.8 ± 0.1 2.18 1.32 0.690 2015 Mar. 23 4 100 100 0 3.0 ± 0.1 2.69 1.63 0.848 2015 Mar. 23 5 0 100 5 NA NA NA NA

[0110] It looks like sample 3 had better encapsulation efficiency than 4. Although it could be improved. Most of the enzyme is being lost in the bath. There are three things we could try to improve the encapsulation efficiency. [0111] (1) Test higher concentrations of crosslinkers (CaCl2, and BaCl2) [0112] (2) Store the beads dry and then disperse them in water when we're ready to test [0113] (3) Reduce the amount of time that the beads are sitting in the bath before collection

[0114] Note: Encapsulation efficiency is based on enzyme activity assays. Thus, it is possible that there is some de-activation enzyme encapsulated that was not detected by the assay. This would mean that the actual encapsulation efficiency of the enzyme was higher by some unknown amount, and that this was effect by enzyme de-activation in the process.