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Edgardo T. Farinas, Ph.D.

                               

New Jersey Institute of Technology,  Department of Chemistry and Environmental Science, University Heights, Newark, NJ 07102

Field of Interest:

Evolutionary and rational approaches for enzyme design.

 

Research Experience:

New Jersey Institute of Technology

 

Currently, I am an assistant professor in the Department of Chemistry and Environmental Science.  Our research interests are in engineering enzymes with improved properties using directed evolution and rational approaches.  Also, we are developing novel protein display technologies. 

 

University of Texas at Austin

 

I was involved in postdoctoral research concerning the directed evolution of protease substrate specificity with Profs. George Georgiou and Brent Iverson from June 2003 to June 2004.  The goal of the project was to screen protease libraries for substrate specificity using fluorescent activated cell sorting (FACS). The protease OmpT, which is expressed on the outer membrane of Escherichia coli, was used as a model system. OmpT has a preference to cleave peptides at Arg-Arg  in the P1 and P1’ positions, respectively.  The target substrate was an unnatural peptide containing a D-amino acid at the cleavage site, Ala-(D)Arg.  Accomplishments include creating gene libraries that target the 22 the amino acids found in the substrate-binding pocket.  The libraries were generated using a PCR method that can perform saturation mutagenesis at all the sites simultaneously.  A FACS screen was developed and optimized to screen for the desired substrate while deselecting for the native substrate.

 

California Institute of Technology

 

I was involved in postdoctoral research concerning the directed evolution of cytochrome P450 for alkane oxidation with Prof. Frances Arnold from April 1999 to April 2003.  The goal of the project was to evolve P450 BM-3 for alkane oxidation.  Unlike most monooxygenases, which are membrane bound, difficult to express, and require ancillary proteins for activity, P450 BM-3 is a soluble monooxygenase and contains the monooxygenase and reductase domain in a single polypeptide chain.  The natural substrates are fatty acids with carbon chain lengths varying from C12 to C18.  Accomplishments include design of a high-throughput kinetic assay screen for alkane hydroxylase activity. Mutant libraries of P450 BM-3 were created using error prone PCR and recombination. The evolved enzyme has been reengineered to accept alkanes with chain lengths as small as C3. The evolved P450 is the fastest known alkane hydroxylase that has been reported to date.

 

Yale University

 

I was involved in postdoctoral research in metalloprotein design with Prof. Lynne Regan from February 1997 to April 1999. The goal of this project was to rationally design four cysteine (Cys4) residues into small, simple, and well characterized protein scaffolds that will bind iron in a tetrahedral array ([Fe(Cys-S)­­4]-1,-2). The [Fe(Cys-S)­­4]-1,-2 site was modeled after that found in the electron transfer protein rubredoxin. Accomplishments include the expression, purification, and spectroscopic characterization of designed proteins capable of binding transition metals tetrahedrally.

 

 

University of California, Santa Cruz

 

Graduate research in inorganic/bioinorganic chemistry with the Prof. Pradip K. Mascharak from September 1991 to February 1997.  The research objective was to design synthetic analogues of Co(III)-Bleomycin, which bind to DNA in a sequence selective manner and cleave the helix upon UV illumination.  Another goal was to ascertain the origin of the DNA sequence specificity. The final aim was to synthesize other cobalt(III) complexes that are capable of oxidizing organics including alkanes upon UV activation.  Accomplishments include: design, syntheses, and characterization of several multistep organic ligands; design, syntheses, and characterization of cobalt(III) complexes of such ligands;  the determination of the DNA sequence specificity of the model complexes using radiolabled DNA restriction fragments;  the DNA binding characteristics of the synthetic analogues to designed oligomers probed by standard 1D and 2D NMR techniques;  syntheses and characterization of model complexes of Co(II)-Bleomycin using EPR techniques;  syntheses of several mononuclear cobalt(III) alkylperoxide complexes which are capable of oxidizing alkanes upon UV illumination.

 

Education:

            Postdoctoral Research        University of Texas at Austin (6/2003 - 6/2004)

            Postdoctoral Research        California Institute of Technology (4/99 - 6/2003)

Postdoctoral Research        Yale University (2/97 - 4/99)

            Ph.D. Chemistry                   University of California, Santa Cruz (2/97)      

            B.S. Chemistry                      Loyola University of Chicago (12/90)

 

Honors and Awards:

NSF Career Award (2008-2013)

NSF (2005-2006)

NSF postdoctoral fellowship (1999-2001)

            NIH postdoctoral fellowship (1998, declined)

            Ford Fellowship (1998)

            Finn Wold Travel Award (1998)

 

Publications

Refereed journal articles

14. Nirupama Gupta, Frederick S. Lee, Edgardo T. Farinas, Laboratory evolution for substrate specificity, Journal of Molecular Catalysis B: Enzymatic, 2010, 62, 230-234.

13. Nirupama Gupta and Edgardo T. Farinas, Narrowing Laccase Substrate Specificity Using Active Site Saturation Mutagenesis, Combinatorial Chemistry and High Throughput Screening, 2009, 12, 269-274

 

12. Miguel Alcalde*, Edgardo T. Farinas*, Frances H. Arnold, Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450BM-3 variant 139-3,  Journal of Biomolecular Screening, 2004,  9, 141-146.

*These author contributed equally

 

11. Alkene epoxidation catalyzed by cytochrome P450 BM-3  139-3.  Edgardo T. Farinas, Miguel Alcalde, and Frances Arnold, Tetrahedron, 2003, 60, 525-528.

 

10. Laboratory Evolution of a soluble, self-sufficient, highly active alkane hydroxylase.  Anton Glieder*, Edgardo T. Farinas*, Frances H. Arnold.  Nature Biotechnology,  2002, 20, 1135-1139.

*These author contributed equally

 

9.  Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation. Edgardo T. Farinas, Ulrich Schwaneberg, Anton Glieder, Frances H. Arnold. Advanced Synthesis and Catalysis, 2001, 343, 601-606.

 

8.  Cost-effective Whole-cell assay for Laboratory Evolution of Hydroxylases in Escherichia coli.  Ulrich Schwaneberg, Christopher Otey, Patrick C. Cirino, Edgardo Farinas, and Frances H. Arnold. Journal of Biomolecular Screening, 2001, 6, 111-118.

 

7.  The de novo design of a Rubredoxin-like Fe site.  Edgardo T. Farinas and Lynne Regan. Protein Science,  1998, 7, 1939-1946.

 

6.  Photoinduced Oxidation of Hydrocarbons with Cobalt(III)-alkylperoxy Complexes. Edgardo T. Farinas, Cattien Nguyen, and Pradip K. Mascharak. Inorganica Chimica Acta,1997, 263, 17-21.

 

5.  Photoinduced DNA Cleavage Reactions by Designed Analogues of Co(III)-Bleomycin, The Metalated Core is the Primary Determinant of Sequence Specificity.  Edgardo T. Farinas, Jennifer D. Tan, and Pradip K. Mascharak.  Inorganic Chemistry, 1996, 35, 2637.

 

4.  Structural Features that Control Oxygen Activation at the Non-Heme Iron Site in Fe(II)-Bleomycin:  An Analogue Study.  Richard J. Guajardo, Ferman Chavez, Edgardo T. Farinas, and Pradip K. Mascharak.  Journal of the American Chemical Society, 1995, 117, 3883.

 

3.  Electron Paramagnetic Resonance Studies on the Formation and Decomposition of the Oxygenated Product of [CoII(PMA)]+, a Synthetic Analogue of Co(II)-Bleomycin. Edgardo T. Farinas, Narayan Baidya and Pradip K. Mascharak.  Inorganic Chemistry 1994, 33, 5970.

 

2.  NMR Evidence of Sequence Specific DNA Binding by a Co(III)-Bleomycin Analogue with Tethered Acridine.  Jennifer D. Tan, Edgardo T. Farinas, Sheila S. David, and Pradip K. Mascharak. Inorganic Chemistry , 1994, 33, 4295.

 

1.  A Designed Synthetic Analogue of Co(III)-Bleomycin with Enhanced DNA-Binding and Photocleaving Activity. Edgardo T. Farinas, Jennifer D. Tan, Narayan Baidya, and Pradip K. Mascharak. Journal of the American Chemical Society  1993, 115, 2996.

 

Patents

 

1. Cytochrome P450 Monooxygenases,  Edgardo T. Farinas, Frances H. Arnold, Ulrich Schwaneberg, Anton Glieder U.S. Patent 7,226,768, June 5, 2007

 

                Invited articles/book chapters

 

6. Fluorescence activated cell sorting for enzymatic activity, Edgardo T. Farinas, Combinatorial Chemistry and High Throughput Screening, vol 9, no 4, 2006, 321-328

 

5. Combinatorial Chemistry and High Throughput Screening: Edgardo T. Farinas,  Editorial, Combinatorial Chemistry and High Throughput Screening, vol 9, no 4, 2006, 235-236 (editor)

 

4. Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes.  Direct Evolution Methods  Book II: Methods in Molecular Biology Series. Eds.  F. H. Arnold and G. Georgiou. Humana Press, Totowa, New Jersey.  2003, 149-155.

 

3. Directed Enzyme Evolution. Edgardo T. Farinas, Thomas Bulter, Frances H. Arnold. Current Opinions in Biotechnology, 2001, 12, 545-551.

 

2.  Syntheses, Structures and Reactivities of Designed Analogues of Cobalt(III)-Bleomycins:  Insight into the Mechanism of Sequence - specific DNA Cleavage upon Illumination.  Edgardo T. Farinas and Pradip K. Mascharak.  Proceedings of Indian Academy of Science (Chem. Soc.), 1995, 107, 459.

 

1.  Structure and Reactivity of Fe(II)-SAr Complexes:  Relevance to the Active Site Of Isopenicillin N Synthase, Edgardo T. Farinas and Pradip K. Mascharak. ChemTracts.  1995, 7, 33.

 

Presentations

27.  Directed Evolution of Laccase:  Controlling Substrate Specificity. Nirupama Gupta and Edgardo T. Farinas, Bioactive Systems Symposium, Polytech University, Brooklyn, New York 06/12/2008

 

26. Protein Engineering: Evolution in a Test Tube. American Institute of Chemical Engineers, NJIT Student Chapter, April 28, 2008.

 

25.  Noninvasive bio-sensors for pharmaceutical, bio-defense and protein engineering, Camelia Prodan and Edgardo T. Farinas, NJIT ADVANCE Interdisciplinary Research Seminar, New Opportunities in Bio Applications Research, NJIT, Eberhardt 112, 03/10/2008.

 

24. Evolution in a Test Tube: Controlling Substrate Specificity, Nirupama Gupta and Edgardo T. Farinas, Poster presentation, Baekeland Symposium, NJIT 11/15/2007.

 

23. Evolution in a Test Tube: Controlling Substrate Specificity, Nirupama Gupta and Edgardo T. Farinas, Poster presentation, NJIT Graduate Student Research Day, 11/14/2007, Awarded Outstanding Graduate Student Research Award

 

22. Laboratory  Evolution Of Laccase: Controlling Substrate Specificity, Nirupama Gupta, Da Jeong Shim, and Edgardo T. Farinas, The Third Annual LRIG Student Poster Contest technology exposition at the Hilton East Brunswick, May 17, 2007 , 1st prize

 

21. Directed Evolution of Laccase: Controlling Substrate Specificity, Unum Rahmat and Edgardo T. FarinasVerbal Presentation at The Fifty-Ninth Annual Research Conference of the Intercollegiate Council of the American Chemical Society Student Affiliate Chapters, North Jersey Section; April 27, 2007 Drew University

 

20. Proteins Designed to Order: Evolution in a Test Tube. Nirupama Gupta and Edgardo T. Farinas.Graduate Student Research Day, NJIT, November 6, 2006

 

19.  Purification Process of Laccase and Colorimetric Assays for Screening. Davendra Beni, Edgardo T. Farinas, Nirupama Gupta, Daniel Ramirez. ACS SEED program, Seton Hall University, September 25, 2006.

 

18. Biodegradation of Polycyclic Aromatic Hydrocarbons by Bacterial Laccases Using Laccase Mediator Systems.  Nirupama Gupta and Edgardo T. Farinas. The Laboratory Robotics Interest Group of the American Chemical Society Meeting at Montclair State University on March 23rd, 2006.

 

17. Development of a High-Throughput Screen for Increased Redox Potential in Laccase,  Murtuza Jaffari, Edgardo T. Farinas, Nirupama Gupta.    The Laboratory Robotics Interest Group of the American Chemical Society Meeting at Montclair State University on March 23, 2006.

 

16. Directed evolution of enzymes, Minority Biomedical Research Support program: Directed evolution of enzymes; Edgardo T. Farinas, Rutgers-Newark September 26, 2005 (invited).

 

15. Laboratory Evolution of a Soluble, Self-Sufficient, Highly Active Alkane Hydroxylase”. Oral Presentation, Edgardo T. Farinas, NJIT Mathematical Biology Seminar, Newark NJ, February 1, 2005 (invited).

 

14.  “Laboratory Evolution of a Soluble, Self-Sufficient, Highly Active Alkane Hydroxylase”. Edgardo T. Farinas. Oral Presentation. NJIT Mathematical Biology Seminar, Newark NJ, February 1, 2005.

 

13.  “Directed evolution of monooxygenases”. Edgardo T. Farinas. Oral presentation. Fall 2004 NJIT Chemical Engineering Seminar Series. Newark, NJ December 9, 2004.

 

12.  “Laboratory Evolution of P450 BM-3 for Alkane Hydroxylation”.  Edgardo T. Farinas, Anton Glieder, Frances H. Arnold.  Poster presentation. Sixth International Symposium on Cytochrome P450 Biodiversity. Los Angeles, CA August 20-25, 2002.

 

11.  “Laboratory Evolution of Cytochrome P450 BM-3 into an Efficient Alkane Hydroxylase”.  Edgardo T. Farinas, Anton Glieder, Frances H. Arnold. California Catalysis Society, Pasadena, CA October 24-25, 2001.

 

10. “Directed Evolution of Cytochrome P450 BM3: Design of an enzymatic system to make a diol from an alkane”.  Edgardo Farinas and Frances Arnold.  An oral presentation. The 2000 Conference of Ford Fellows (Academic  Exchange Session), Irvine, California, October 13-14, 2000.

 

9.  “The de novo design of a Rubredoxin-like Fe site”. A poster presentation. The Protein Society 12th Symposium.  San Diego, CA July 25 - 29, 1998.

 

8.  “Design of an Electron Transfer Metalloprotein” Edgardo Farinas and Lynne Regan. An oral presentation.  Chemical Biology Seminar Series. Yale University, New Haven CT, May 1, 1998.

 

7.  “Design of de novo [Fe(Cys)­4]-1,-2 Sites in Proteins” Edgardo Farinas and Lynne Regan. Poster presentation. 1997 23rd Annual Symposium of the Department of Molecular Biophysics and Biochemistry. The Swope Center Marine Biological Laboratories, Woods Hole, October 17 -19, 1997

 

6.  “Photoinduced Alkane Functionalization with Mononuclear Cobalt(III) Alkylperoxy Complexes.”  Edgardo T. Farinas, Cattien Nguyen, and Pradip K. Mascharak. Poster presentation. 1996 Pacific Conference.  San Francisco, California.  October 29 - November 1, 1996

 

5.  “In Photoinduced DNA Cleavage Reactions by Designed Analogues of Co(III)-Bleomycin, The Metallated Core is the Primary Determinant of Sequence Specificity.”  Edgardo T. Farinas and Pradip K. Mascharak.  An oral presentation by Pradip K. Mascharak for Mechanisms of Metal Mediated Biopolymer Cleavage a Symposium at the 212th American Chemical Society National Meeting.  Orlando, Florida.  August 25 - 27, 1996.

 

4.  “In Photoinduced DNA Cleavage Reactions by Designed Analogues of Co(III)-Bleomycin, The Metallated Core is the Primary Determinant of Sequence Specificity.”  Edgardo T. Farinas, Jennifer D. Tan, and Pradip K. Mascharak. Poster presentation. Seventh International Conference on Bioinorganic Chemistry.  Lubeck, Germany.  September 3 - 8, 1995

 

3. “Sequence-specific DNA Binding and Cleavage by a Co(III)-Bleomycin Analogue With Tethered Acridine.”  Edgardo T. Farinas, Jennifer D. Tan, Sheila S. David, and Pradip K. Mascharak. Poster presentation. 208th American Chemical Society National Meeting, Washington DC.  August 21 - 25, 1994.

 

2.  “Electron Paramagnetic Resonance Studies on a Synthetic Analogue of Cobalt-Bleomycin.”  Edgardo T. Farinas, Narayan Baidya and Pradip K. Mascharak. Poster presentation. Sixth International Conference on Bioinorganic Chemistry.  University of California, San Diego.  August 23-27, 1993.

 

1.  “Enhanced DNA Photocleavage by a New Synthetic Analogue of Co(III)-Bleomycin.” Edgardo T. Farinas, Jennifer D. Tan, Narayan Baidya, and Pradip K. Mascharak. Poster presentation. 205th. American Chemical Society National Meeting. Denver, Colorado.   March 28- April 2, 1993.

 

Collaborations:

Frank Jordan (Rutgers-Newark), Camelia Prodan (NJIT), Ligia O. Martins (Universidade Nova de Lisboa)           Stephen Marino (Max Plank Inst.), Patrick Eichenberger (NYU)