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Louis DeFilippi, Ph.D. Publications and Patents

REVIEWED PUBLICATIONS

Kabak, J., DeFilippi, L., Engel R. and Tropp, B., "Synthesis of the Phosphonic Acid Isostere of Glycerol 3-Phosphate," J. Medicinal Chem. 15, p. 1074, 1972. (Note my name is misspelled as "DeFilippe" in the original publication... a good reason to give galley proofs to all of the authors!)
In this publication we describe the synthesis of the dilithium salt of 3,4-dihydroxybutyl-1-phosphonic acid (see Fig 1).

          O-Li⁺         OH
           |                  |
     O=P-CH₂CH₂CHCH₂OH
   |
          O-Li⁺

Fig 1. 3,4-dihydroxybutyl-1-phosphonic acid, dilithium salt

This compound has been of interest since its structure is equivalent to glycerol 3-phosphate, except that the O in the phospho-ester linkage found in glycerol 3-phosphate (bolded in the next box),

|
=P-O-CH₂-
|

is replaced by a much more stable methylene, -CH₂-, linkage (bolded in the next box),

|
=P-CH₂-CH₂-
|

Enzymatic cleavage of the latter linkage does not readily occur. The compound has found use in studies relating to the Escherichia coli glycerol 3-phosphate transport system.

DeFilippi, L. J. and Hultquist, D. E., "A Microscale Isolation of Hemins from Hemeproteins by Use of Polyacrylamide Gel Electrophoresis," Arch. Biochem. Biophys. 170, pp. 670-675, 1975.
Until this work, there was no good method for the microscale isolation of hemins from hemeproteins (hemoproteins). We took advantage of our observations that the bovine erythrocyte green heme protein (found later to possess the same protein moiety as the NADPH-dependent methylene blue reductase, see below) would lose its prosthetic group during disc-gel electrophoresis, and that the addition of cyanide shifted the equilibrium towards the dissociation of the heme from the apoprotein. Under neutral or alkaline conditions hemin (see Fig 2) is negatively charged and is bound to the apoprotein portion of the parent hemoprotein through a number of forces. Cyanide acts to weaken these forces and aids in the displacement of hemin from the apoprotein. Urea, in combination with cyanide, was also found to further assist displacement of hemins from certain hemoproteins.
Hemin is relatively small, when compared to the apoprotein. During an electrophoretic separation the hemin tends to migrate more quickly to the anode than does the apoprotein and travels with what would have been the dye front. These properties are the basis for the electrophoretic separation of hemin or other hemes from their respective apoproteins. We did not attempt to isolate the apoprotein, but we suspect it would be straightforward.

Fig 2. Hemin (Credit to Sigma Chemical for figure)

Fig 3. Hemin, ball and stick model. The black balls are carbon, the blue balls nitrogen, the red-brown balls oxygen and the central light-green ball, iron. Hydrogen atoms are not shown. Carboxylate groups are in the upper left and lower left. Note rotation of the structure by approximately 120 degrees counterclockwise, relative to Fig 2. (Credit to Scott Prahl, Oregon Medical Laser Center, http://www-class.unl.edu/chem/Chem192H/WWW_Pgs/Lesa/ for figure).

Dean, R. T., DeFilippi, L. J. and Hultquist, D. E., "Esterification of Hemins with Trimethyloxonium Tetrafluoroborate," Anal. Biochem. 76, pp. 1-8, 1976.
        As can be seen in Figs. 2 & 3, hemin (protohemin IX) contains two free carboxylic acid groups. It is often desirable to methylate these groups before analysis or other chemical reactions. The problem was that the methylation conditions then in existence were harsh, being performed under highly acidic conditions. Since it was one of the major goals in the laboratory at the time to determine the structure of the prosthetic group of the erythrocyte green hemoprotein, and it appeared that the prosthetic group was acid labile, we needed to develop new techniques.
         Protohemin IX contains two potentially reactive vinyl groups. There are other hemins that contain reactive groups, such as hydroxyls, that are potentially methylatable. Since we desired to work with acid-labile hemins, and hemins that may have other reactive substituents, we needed to develop a method that worked under neutral to alkaline conditions to selectively methylate the carboxylate group, yet leave hydroxyl groups untouched. In specific, we wished to use this technique for structural determination of both heme a and the prosthetic groups of the erythrocyte green hemoproteins.
        We initially used benzilic acid as a model compound. We found that carboxylate group of the sodium salt of benzilic acid could be readily methylated (see Fig. 4) under aqueous conditions that left the hydroxyl group untouched. After washing and drying the product, the yield was 82.4% of theoretical.
        We were subsequently successful in applying the synthetic method to the methylation of a variety of hemins, including protohemin IX, hemato hemin and heme a. As with benzilic acid, the carboxylate groups were methylated while the hydroxyl groups were left untouched. Vinyl groups were similarly not altered.

         f                                              f
            |                                           |
     f-C-COOH +   Me₃OBF₄ Ž f-C-COOCH₃
             |                                                |
         OH                                           OH

Fig. 4. Methylation of 8.77 mMol benzilic acid in 110 mL H₂O and 96.7 mMol Na₂CO₃, using a 10 X molar excess of Me₃OBF₄

DeFilippi, L. J. and Hultquist D. E., "High-Pressure Liquid Chromatography and Field Desorption Mass Spectroscopy of Heme a, Heme a Dimethyl Ester and Acetyl Heme a Dimethyl Ester," Biochim. et Biophys. Acta 498, pp. 395-402, 1977.
(Notes from the author: Back when it was first used for analysis, HPLC was called High-Pressure Liquid Chromatography. It is now usually called High-Performance Liquid Chromatography. Also, the published abstract describes the base peak m/e of 582. This is a typo. It should read 852). The present study was initiated when we realized that characterization of heme a might be furthered by combining three new (at the time!) techniques, esterification of hemes at neutrality with oxonium salts (see Dean, DeFilippi, and Hultquist, above), purification of hemes by high pressure liquid chromatography, and molecular weight determination by field desorption mass spectral analysis (see Evans, N. et al., J. Chromatogr. 115, pp. 325-333, 1975).

The above structure, with the formyl group substituted for the circled methyl group, and the hydroxyethylfarnesyl group, as shown, is the chemical structure of heme a. (Credit to Anthony Crofts, at the University of Illinois at Urbana-Champaign, for figure. Mail to: a-crofts@uiuc.edu and view web page at: http://arc-gen1.life.uiuc.edu/Bioph354/Cyt_ox.html)

Weiss, L., Wolff, J., Knowles, F. C., DeFilippi, L. J. and Gibson, Q. H., "Synthesis and Characterization of N-(2,4-Diphosphobenzyl)-1-Amino-5-Naphthalenesulfonic Acid, a New Fluorescent Analogue of Diphosphoglyceric Acid," J. Biol. Chem. 253 (7), pp. 2380-2385, 1978.
At the time this work was performed, it was already well established that anions have a marked effect on ligand binding by hemoglobin. In particular, the polyanions diphosphoglycerate and inositol hexaphosphate have been shown to markedly decrease the affinity of hemoglobin for oxygen when added to solutions in media of low ionic strength. We employed this fluorescent compound to study the R to T transition of human hemoglobin.

DeFilippi, L. J. and Hultquist, D. E., "The Green Hemoproteins of Bovine Erythrocytes, I. Purification and Characterization," J. Biol. Chem. 253 (9), pp. 2946-2953, 1978.
        In this publication we describe the purification and characterization of the bovine green hemoproteins (two forms) that were initially discovered in human erythrocytes.
        The two green hemoproteins, hitherto undetected in bovine tissues, were isolated from the supernatant fraction of bovine erythrocyte hemolysates. The two hemoproteins were co-purified by a procedure which entailed hypotonic lysis, storage at -70^oC, removal of stromata through pH adjustment, and chromatography on DEAE-cellulose, Ambelite CG-50, and Bio-Gel P-60 gel exclusion chromatography. This procedure also yields superoxide dismutase and glutathione peroxidase. The green hemoprotein fraction was then separated on DEAE-Sephadex into two forms (I and II). Both forms appear homogeneous by electrophoresis on polyacrylamide gel electrophoresis (PAGE) at pH 6.9 and 7.6, on polyacrylamide gels at 8.6 in the presence of sodium dodecyl sulfate (SDS), and on cellulose acetate strips at pH 8.6. Heterogeneity was seen upon isoelectric focusing. The heterogeneity of form I on gel electrophoresis at pH 8.9 appears to result from protein degradation and dissociation into apoprotein and free hemin.
        Antibodies were raised against the hemoproteins in rabbits. Immunological studies gave no indication of impurities. Moreover, by immunological techniques, the two forms are indistinguishable and show no structural relationship to hemoglobin.
        The two forms exhibit identical molecular weights of 23,000 by SDS-PAGE and 27,000 by gel exclusion chromatography and both therefore exist as monomers in solution.
        The reduced pyridine hemochromes of the denatured hemoproteins show absorption maxima for form I at 434, 544 and 580.5 nm, and for form II at 431, 538, and 573 nm, demonstrating the heme of form I possesses substituents with greater electron-withdrawing power than the substituents on the heme of form II. These spectra show that the two hemoproteins are similar to two hemoproteins detected in human erythrocytes. The hemin prosthetic groups of the two proteins have been hitherto undetected in bovine tissues.

DeFilippi, L. J. and Hultquist, D. E., "The Green Hemoproteins of Bovine Erythrocytes, II. Spectral, Ligand-Binding, and Electrochemical Properties," J. Biol. Chem. 253 (9), pp. 2954-2962, 1978.
        In this publication we describe many of the physical characteristics of the green hemoproteins (two forms) that were purified from the cytosolic fraction of bovine erythrocytes.

A brief summary of subsequent work on the
Erythrocyte Green Hemoproteins

In a continuation of the work on the bovine erythrocyte green hemoproteins (now, once again renamed, to bovine erythrocyte green heme binding protein, or GHBP) by Quandt and coworkers in the laboratory of Donald Hultquist at the University of Michigan, it was found that the protein components of GHBP was identical to erythrocyte flavin reductase (1). Further work by Xu and coworkers demonstrated that this protein is the cytosolic NADPH-dependent reductase that catalyzes the reduction of methylene blue and flavins and, in the presence of redox couplers, catalyzes the reduction of methemoglobin (2). This is the same NADPH-dependent reductase (variously referred to as NADPH-dependent methemoglobin reductase, NADPH dehydrogenase, or NADPH diaphorase), shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, that was the basis for the use of methylene blue or riboflavin as therapeutic agents in the treatment of congenital and toxic methemoglobinemia. The flow of reducing equivalents (electrons) is:

NADPH Ž Methylene blue Ž Methemoblobin

Xu et al. concluded that this reductase is a binding protein with affinity for hemes, porphyrins, and fatty acids. "By identifying the binding properties of the reductase, demonstrating that the protein is not unique to erythroid cells, determining that it is present at a relatively high intracellular concentrations, and remembering that the reductase activity has a very low turnover number, we bring into questions long-held beliefs as to the catalytic function of the protein. The protein may participate in transport, storage, or scavenging of heme, roles that have been suggested for intracellular heme-binding proteins. Ongoing studies aim to establish whether the physiological significance of the protein relates to these heme-binding properties or, alternatively, whether the role of the protein is to (I) bind fatty acids, (ii) catalyze heme-regulated reduction reactions, or (iii) protect cells from oxidative damage by catalyzing the reduction of the higher oxidations states of hemeproteins." (2).

We will try (some time in the near future) to put together a summary page tying together work on the NADPH-dependent methemoglobin reductase = Erythrocyte Green Hemoprotein.

(1) Quandt, Kim S., Xu, Feng, Chen, Ping and Hultquist, Donald E., Biochem. Biophys. Res. Comm. 178 (1), pp 315-321, 1991.

(2) Xu, Feng, Quandt, Kim S., and Hultquist, Donald E., Proc. Natl. Acad. Sci. 89, pp. 2130-2134, 1992.

DeFilippi, L. J., Toler, L. S. and Hultquist, D. E., "Reactivities of Hydroxylamine and Sodium Bisulfite with Carbonyl-Containing Haems and with the Prosthetic Group of the Erythrocyte Green Haemoproteins," Biochem J. 179, pp. 151-160, 1979.
       The reaction of hydroxylamine, NH₂OH, with carbonyl groups has classically been used as a test for the detection of carbonyl-containing porphyrins and, less frequently, carbonyl-containing hemes (haems in the UK). The reaction is conveniently followed by observations made in the near-UV-visible absorption spectrum. The magnitude of the blue-shift of the UV/VIS peaks that accompanies oxime formation is much larger for formyl (-CHO)-containing tetrapyrroles than for acetyl (-COCH₃)-containing tetrapyrroles.
        The bottom line here is that when we attempted to repeat the reactions reported in the literature we found that they actually did not occur as reported and had led previous investigators to draw wrong conclusions.
      This paper describes the spectral alterations that are observed upon reaction of carbonyl-containing hemes with NH₂OH and sodium bisulfite (NaHSO₃), the structural conclusions that may be drawn from these alterations, the kinetics of reaction of heme a with NH₂OH (it reacts much more rapidly under alkaline or acidic conditions than neutral conditions, with a second order rate constant of 50 mM^-1 in an aqueous solution of 20% pyridine, v/v, and 0.5 M NaOH), and equilibrium values for the adduct of heme a and NaHSO₃ (dissociation constant of 0.97 M). We studied the reaction (or lack of reaction) of deutroheme IX, heme a, spirographis heme, isospirographis heme, 2,4-diacetyldeuteroheme, protoheme IX, and the prosthetic group of the bovine erythrocyte green hemeprotein with the above reagents. The formyl-containing hemes reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced hemochrome spectrum.

Abstract and introduction as published:

ABSTRACT

The reactivities of alkaline NH₂OH and neutral NaHSO₃ with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3 nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH₂OH and neutral NaHSO₃. The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH₂OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH₂OH, but not with neutral NaHSO₃. These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH₂OH or NaHSO₃, but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem. 251, 3927-3932] may have been premature. These studies also provide evidence that the a (alpha)-hydroxyfarnesylethyl side chain of haem a affects the a-band maximum, but not the b-(beta-) or Soret bands of the reduced pyridine haemochrome spectrum of haem a.

INTRODUCTION

The reaction of NH₂OH with carbonyl groups to give the corresponding oxime derivative has classically been used as a test for the detection of carbonyl-containing porphyrins and, less frequently, carbonyl-containing haems (Rawlinson & Hale, 1949; Lemberg & Falk, 1951; Oliver & Rawlinson, 1955; Connelly et al., 1958; Parker, 1959; Morrison et al., 1960; Clezy & Barrett, 1961; Clezy et al., 1964). The magnitude of the blue-shift of the near-U.V.-visible absorption spectrum that accompanies oxime formation is much larger for formyl-containing tetrapyrroles than for acetyl-containing tetrapyrroles. Oxime formation results in a 17-22 nm shift of the a-peak of the reduced pyridine haemochrome of haems with a formyl group in conjugation with the tetrapyrrole nucleus, but gives only a 1-2 nm shift with haems containing a conjugated acetyl group (Lemberg & Falk, 1951). This difference has been used to distinguish between formyl and acetyl substitution on the periphery of the porphyrin nucleus.

A blue-shift of a porphyrin spectrum on reaction with NaHSO₃ has likewise been cited as evidence for the presence of a formyl group in conjugation with a tetrapyrrole nucleus, since the acetyl-containing porphyrins, cryptoporphyrins p, are reported not to undergo bisulphite-adduct formation (Clezy et al., 1964).

The unique prosthetic group of a human erythrocyte green haemoprotein (Hultquist et al., 1976) was found to undergo reactions with NH₂OH and NaHSO₃ under the conditions that have been used with other haems; characterization of this prosthetic group and its derivatives distinguished it from all other naturally occurring prosthetic groups and suggested that it is a complex haem containing both a formyl group and polar acetylatable functional groups. Similarly, we have studied two bovine erythrocyte green haemoproteins and shown that these proteins differ in terms of the spectral and chemical properties of their prosthetic groups (DeFilippi & Hultquist, 1978a,b).

In attempting to identify unambiguously the side chains of these haems we further studied the reactions of NH₂OH and NaHSO₃ with model compounds. We discovered that haem a, which has been shown to possess a formyl group as one of its substituents (Lemberg & Falk, 1951; Lemberg, 1953; Connelly et al., 1958; Caughey et al., 1975), does not rapidly undergo oxime formation under the neutral or mildly alkaline conditions at which the reaction was believed to occur. However, we found that the reaction proceeds rapidly in the strongly alkaline conditions used in pyridine haemochrome formation, a procedure that was believed to be simply a process to assess visually the extent of the reaction. Moreover, we realized that the reaction of formyl-containing haems with NaHS0₃ has received relatively little attention in the literature (Orii & Washio, 1977; Kitagawa et al., 1977). Reactivity of porphyrins (but not haems) with HS0₃^- was apparently first described by Parker (1959) as yielding "alteration" in the absorption spectrum of cryptoporphyrin a in dilute cold pyridine; reference was made to unpublished work by R. Lemberg.

In the present paper we report the reactivities of haems with alkaline NH₂OH and neutral NaHSO₃. These reactivities constitute a rapid and straightforward means of differentiating among formyl, acetyl and other electron-withdrawing side chains of haems and have allowed us to reexamine the question of whether a formyl group is present on the prosthetic groups of the erythrocyte green haemoproteins.

Hydroxylamine = NH₂OH
Sodium bisulfite = NaHSO₃

    British to American translation!
heme = haem
    thus
diacetyldeuterohaem = diacetyldeuteroheme
protohaem = protoheme
haemochrome = hemochrome
    and
bisulphite = bisulfite

Click here for further information.

See the following paper for an application of the chemistry of fluorescent hydroxylamine to proteomics studies:

H. Fai Poon, Laila Abdullah, Jon Reed, Sarah M Doore, Cyndi Laird, Venkat Mathura, Michael Mullan and Fiona Crawford, "Improving image analysis in 2DGE-based redox proteomics by labeling protein carbonyl with fluorescent hydroxylamine" Biol. Proced. Online 2007;9:65-72.

DeFilippi, L. J., Ballou, D. P. and Hultquist, D. E., "Reaction of Bovine Erythrocyte Green Hemoprotein with Oxygen and Hydrogen Peroxide," J. Biol. Chem. 254 (15), pp. 6917-6923, 1979.

Xu, F., DeFilippi, L. J., Ballou, D. P., and Hultquist, D. E., "Peroxide-dependent Formation and Bleaching of the Higher Oxidation States of Bovine Erythrocyte Green Hemeprotein" Arch. Biochem. Biophys. 301 (15), pp. 184-189, 1993.

Turner, R.J., Aikens, J., Royer S., DeFilippi, L. Yap, A., Holzle, D, Somers, N, Fotheringham, I.G., D-Amino Acid Tolerant Hosts for D-Hydantoinase Whole Cell Biocatalysts Engineering in Life Sciences. 4 (6), pp. 517-520, 2004. http://www3.interscience.wiley.com/cgi-bin/abstract/109746241/ABSTRACT

ABSTRACT

Whole cell biocatalysts which enable the concerted use of D-hydantoinase, D-carbamoylase, and racemase enzymes are valuable for the production of D-amino acids. However, Escherichia coli host strains used for this purpose efficiently degrade D-amino acids. This work demonstrates that D-amino acid degradation occurs largely through the concerted action of D-amino acid dehydrogenase, encoded by the dadA gene, and D-serine dehydratase, encoded by the dsdA gene. Deletion mutants of E. coli which lack these activities were constructed and compared against wild type strains in D-amino acid degradation. An E. coli dadA mutant reduced the degradation of D-methionine by one third, D-phenylalanine by two-thirds, and D-2-aminobutyric acid nearly completely. Even though the dadA mutant had no effect on D-serine degradation, a dadA dsdA double mutant of E. coli additionally reduced degradation of D-serine, as well as D-phenylalanine, almost entirely. These strains are appropriate hosts for whole cell biosynthesis of D-amino acids using general approaches such as the hydantoinase system.

ABSTRACTS, PROCEEDINGS AND PUBLISHED LECTURES

DeFilippi, L. J. and Hultquist, D. E., "Isolation of Hemes from Hemeproteins Using Polyacrylamide Gel Electrophoresis," Federation Proceedings 34 (6), p. 602, 1975.

DeFilippi, L. J. and Ballou, D., "Mechanism of Dithionite- and H₂O₂-Induced Bleaching of The Erythrocyte Formylhemin-Containing Protein," Federation Proceedings 35 (6), p. 1393, 1976.

DeFilippi, L. J. and Gibson, Q. H., "Problems in Defining the Hemoglobin T-State," Federation Proceedings 37 (6), p. 1672, 1978.

DeFilippi, L. J., "High Surface Area Immobilized Enzyme Electrode," Federation Proceedings 45 (6), p. 1945, 1986.

DeFilippi, L. J. and Lupton, F. S., "Bioremediation of Soluble Cr(VI) Using Anaerobic Sulfate Reducing Bacteria," Proceedings of R&D 92 National Research & Development Conference on the Control of Hazardous Materials, San Francisco CA, pp. 138-141, February 4-6, 1992.

DeFilippi, L. J. and Lupton, F. S., "Bioremediation of Hexavalent Chromium Using Marine Sulfate Reducing Bacteria" (Invited Lecture, NY/NJ Port Authority, no published abstract), May 4-5, 1992.

DeFilippi, L. J. and Lupton, F. S., "Bioremediation of Chromium (VI) Contaminated Solid Residues Using Sulfate Reducing Bacteria" Emerging Technologies in Hazardous Waste Management V, American Chemical Society Division of Industrial and Engineering Chemistry Special Symposium, pp. 117-120, September 21-23, 1992, Atlanta, GA.

DeFilippi, L. J., "Bioremediation of Chromium (VI) Contaminated Solid Residues Using Sulfate Reducing Bacteria" U.S. Environmental Protection Agency Forum on Innovative Hazardous Waste Treatment Technologies: Domestic and International, Nov. 17-19, 1992, San Francisco, CA

DeFilippi, L. J., Sanyal, S. and Love, T. P., "Performance Improvement of a Fixed-Film Biological Reactor by the Use of Mixed Packing Media" Emerging Technologies in Hazardous Waste Management V, American Chemical Society Division of Industrial and Engineering Chemistry Special Symposium, pp. 130-132, September 27-29, 1993, Atlanta, GA.
This article is based upon the Sanyal et al. patent referenced below.

Click on thumbnail to view.

The above image depicts the performance enhancement observed when mixed media is substituted for all plastic media in a microbiological fixed film reactor. The plot depicts ppm phenol on the y axis, vs. sampling date on the X axis. Operating conditions are:

HRT: 13.2 hrs.

Temperature: Ambient (around 22 deg. C).

Eff-1: Effluent from packed bed of all-plastic media (tripack)

Eff-2: Effluent from mixed bed composed of 15 ppi PUF and tripack

Inf: Influent concentration of phenol. Note that the influent concentration is 10X higher than depicted.

DeFilippi, L. J., "Vapor Phase Biological Treatment Using Carbon Biomass Support" Applied Bioremediation 93, October 25-26, 1993, Fairfield, NJ.

DeFilippi, L. J., Koch, M. B., Voellinger, C. M., Winstead, D. R. and Lupton, F. L. "A Biological Air Treatment System Based Upon The Use of a Structured Biomass Support" IGT Symposium on Gas, Oil and Environmental Biotechnology, November 29 - December 1, 1993, Colorado Springs, CO.
Click here or here or here for abstract of this and many other papers relating to biofiltration.

Lupton, F. S., Sheridan, W. G. and DeFilippi L. J., "Combined Anaerobic In-Situ/Aerobic Ex-Situ Bioremediation of Chlorinated Ethenes Using an Immobilized Cell Bioreactor" (Presented as a poster session at the U.S. EPA sponsored Fifth Forum on Innovative Hazardous Waste Treatment Technologies: Domestic and International) May 3 to May 5, 1994, The Congress Hotel Chicago, Illinois.

BOOK CHAPTERS AND BOOK

DeFilippi, L. J., "Bioremediation of Hexavalent Chromium in Water, Soil and Slag Using Sulfate Reducing Bacteria" in D. L. Wise and D. J. Trantolo, eds., Remediation of Hazardous Waste Contaminated Soils, Marcel Dekker, New York, 1994.

DeFilippi, L. J. and Lupton, F. L., "Introduction to Microbial Degradation of Aqueous Waste and Its Application using a Fixed -film Reactor" in G. A. Lewandowski and L. J. DeFilippi, eds., Biological Treatment of Hazardous Wastes, Wiley Interscience (John Wiley and Sons), 1998.

Lewandowski, G. A. and DeFilippi, L. J., eds., Biological Treatment of Hazardous Wastes, Wiley Interscience (John Wiley and Sons), 1998.

DeFilippi, L. J., "Sulfate Reducing Bacteria and Other Biological Agents for Bioremediation of Hexavalent Chromium and Other Heavy Metals" in Wise, D. L, Trantolo, D. J. and Cichon, E. J. Inyang, H. I, and U. Stottmeister, Bioremediation of Contaminated Soils, Environmental Science and Pollution Series, Marcel Dekker, New York, 2000.

metaltox.jpg (51558 bytes)

Figure from Bioremediation of Contaminated Soils. Schematized and simple plot of organism health vs. relative concentration of metal, both in arbitrary units. The scales and units are relative and may have logarithmic character. The curve in the rear of the Figure represents a non-essential, but potentially toxic metal.

PATENTS

DeFilippi, L. J., "Process for Preparing Immobilized Enzymes", U.S. Patent 4,229,536 (1980).
This patent describes a process for preparing immobilized glucose isomerase using a high surface area alumina, polyethyleneimine and glutaraldehyde. The patent abstract reads: The activity and stability of immobilized enzymes may be improved by treating an immobilized enzyme comprising an enzyme covalently bound to polymeric material which is absorbed on an inorganic porous support material with a bifunctional monomeric material and a substrate followed by additional treatment with an enzyme and thereafter again treating the complex with additional bifunctional monomer and substrate.

DeFilippi, L. J., "Magnetic Support Matrix for Enzyme Immobilization", U.S. Patent 4,343,901 (1982).
This patent describes the process for preparing immobilized enzymes on an alumina base which is impregnated with magnetic materials (ferromagnetic particles dispersed throughout its interior ). The patent abstract reads: Surface-modified electrodes which may be used in electrochemical cells for production of electrical energy comprise an enzyme immobilized on a support. The support consists of at least a monolayer coating of a carbonaceous pyropolymer possessing recurring units containing at least carbon and hydrogen atoms composited on a high surface area refractory inorganic oxide such that the carbonaceous pyropolymer monolayer coating replicates the surface area and macropore volume of the inorganic oxide. The coated support is then treated by impregnation with a water-soluble polyamine followed by contact with a solution of a molar excess of a bifunctional monomeric material to form a copolymer which provides pendant bonding sites. The copolymer is entrapped and adsorbed in the pores of the support material to provide a permanent attachment thereto. The treated support is then contacted with an excess of an enzyme to effect the conjugate attachment of the enzyme to the treated support. The immobilized enzyme will act as a working electrode in the presence of a predetermined substrate such as glucose to provide electrical energy.

DeFilippi, L. J., "Antimicrobial Fabrics", U.S. Patent 4,496,363 (1985).
This patent describes the use of a electron beam to activate a polymeric surface for use in peptide and enzyme immobilization.

Malloy, T. P. and DeFilippi, L. J., "Surface Modified Electrodes", U.S. Patent 4,581,336 (1986).

Calcaterra, L. T., DeFilippi, L. J., Childs M. E. and Latos, E. J., "Antimicrobial Fabrics Using Graft Copolymers", U.S. Patent 4,810,567 (1989).

Lupton, F. S., DeFilippi, L. J., and Goodman, J. R., "Bioremediation of Chromium (VI) Contaminated Aqueous Systems by Sulfate Reducing Bacteria", U.S. Patent 5,062,956 (1991).

Lupton, F. S., DeFilippi, L. J., and Goodman, J. R., "Bioremediation of Chromium (VI) Contaminated Solid Residues", U.S. Patent 5,155,042 (1992).

Sanyal, S., Love, T. P., and DeFilippi, L. J., "Process and Apparatus for Removal of Organic Pollutants from Waste Water", U.S. Patent 5,217,616 (1993).

DeFilippi, L. J., Lupton, F. S. and Mashayekhi, M., "Process for Biological Remediation of Vaporous Pollutants", U.S. Patent 5,413,714 (1994).

DeFilippi, L. J., Lupton, F. S. and Mashayekhi, M., "Apparatus for Biological Remediation of Vaporous Pollutants", U.S. Patent 5,503,738 (1996). The patent abstract reads: This invention relates to a process for remediating vaporous pollutants which comprises passing a vaporous stream containing one or more of pollutants through a bioreactor, the bioreactor comprising a plurality of biologically active bodies, the biologically active body comprising a macroporous substrate and one or more of microorganisms capable of remediating one or more of said pollutants, wherein the substrate is fabricated from a decomposition-resistant material. The present invention further provides an apparatus for this process.

DeFilippi, L. J., "Support Containing Particulate Adsorbent and Microorganisms for Removal of Pollutants", U.S. Patent 5,580,770 (1996). The patent abstract reads: A biologically active support for removing pollutants from a fluid stream such as waste water is prepared. The support is formed of a polymeric foam substrate coated with a composition containing a particulate adsorbent which adsorbs, then releases pollutants, and a polymeric binder that binds the adsorbent to the surface of the substrate. The binder contains a suspension aid, and one or more pollutant-degrading microorganisms are adhered to the surface of the coated support. The binder preferably has a T.sub.g of lower than or equal to about 250 degree C and may be a latex. Examples of suspension aids are surfactants and polyanionic polypeptides such as ammonium caseinate. The adsorbent is preferably a carbon material such as coal, charcoal, carbon black and activated carbon. Other adsorbents are silica gel, active clays, zeolites, hydrophobic and ion exchange resins, and molecular sieves. To remove pollutants, the biologically active support is placed in a reactor and a fluid stream containing a pollutant such as phenol is passed through the reactor where the pollutant is degraded by the microorganism and adsorbed to the adsorbent. The adsorbent acts as a buffer by adsorbing excess pollutant from solution when the pollutant concentration increases and when the pollutant concentration decreases releases pollutant into solution where the microorganism degrades the pollutant.

DeFilippi, L. J. and Lupton, F. S. "Biologically Active Support Containing Bound Adsorbent Particles and Microorganisms for Waste Stream Purification", U.S. Patent 6,395,522 (2002).

Joseph A. Laszlo, David L. Compton, Louis J. DeFilippi, Steven Grall "Methods of making compositions comprising a UV-absorbing chromophore" USPTO Applicaton #: 20070077636 - Class: 435134000 (USPTO). The present disclosure relates generally to compositions comprising UV-absorbing chromophores such as phytochemicals and methods of production of same. More specifically, the present disclosure relates to fat soluble compositions comprising feruloylated vegetable oils and methods for the acylation of polyols.

Joseph A. Laszlo, David L. Compton, Louis J. DeFilippi, Steven Grall "Compositions comprising a UV-absorbing chromophore" USPTO Applicaton #: 20070077214 - Class: 424059000. Disclosed herein is a chemical composition comprising a linker agent and a compound comprising at least one UV-absorbing chromophore

Louis J. DeFilippi, Steven G. Grall, James F. Kinney, Joseph A. Laszio, David L. Compton "Formulations with feruloyl glycerides and methods of preparation" USPTO Applicaton #: 20080050321 - Class: 424059000

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