Research Article | Published:

Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents

Nature Biotechnologyvolume 14pages458467 (1996) | Download Citation



Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50–60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (<2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a ‘rational’ design effort.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    Chen, K. and Arnold, F. 1993. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc. Natl. Acad. Sci. USA 90: 5618–5622.

  2. 2

    You, L. and Arnold, F.H. 1995. Directed evolution of Subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9: 77–83.

  3. 3

    Brannon, D.R., Mabe, J.A., and Fukuda, D.S. 1976. De-esterification of cephalosporin para-nitrobenzyl esters by microbial enzymes. J. Antibiotics 29: 121–124.

  4. 4

    U.S. Patent 3,725,359 [1975].

  5. 5

    Zock, J., Cantwell, C., Swartling, J., Hodges, R., Pohl, T., Sutton, K., Rosteck Jr., P., McGilvray, D., and Queener, S. 1994. The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase—cloning, sequence and high-level expression in Escherichia coli . Gene 151: 37–43.

  6. 6

    Cooper, R.D.G. 1992. The carbacephems: a new beta-lactam antibiotic class. Am. J. Med. 92 Supplement 6A: S2–S6.

  7. 7

    Arnold, F.H. 1996 Directed evolution: creating biocatalysts for the future. Chem. Eng. Science. In press.

  8. 8

    Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91: 10747–10751.

  9. 9

    Stemmer, W.P.C. 1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391.

  10. 10

    Crameri, A., Whitehorn, E., Tate, E., and Stemmer, W.P.C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology 14: 315–319.

  11. 11

    Leung, D.W., Chen, E., and Goeddel, D.V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1: 11–15.

  12. 12

    Eckert, K.A. and Kunkel, T.A. 1991. DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Applic. 1: 17–24.

  13. 13

    Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Applic. 2: 28–33.

  14. 14

    Chen, K. and Arnold, F. 1991. Enzyme engineering for nonaqueous solvents: random mutagenesis to enhance activity of subtilisin E in polar organic media. Bio/Technology 9: 1073–1077.

  15. 15

    Moore 1996. PhD thesis, Cal. Inst. of Tech., Pasadena, CA.

  16. 16

    Pohlenz, H.D., Boidol, W., Schuttke, I., and Streber, W.R. 1992. Purification and properties of an arthrobacter-oxydans p52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide-sequence of the corresponding gene. J. Bacteriol. 174: 6600–6607.

  17. 17

    Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Siliman, I. 1991. Atomic structure of acetylcholinesterase from Torpedo califor-nica: a prototypic acetylcholine-binding protein. Science 253: 872–879.

  18. 18

    Schrag, J.D. and Cygler, M. 1993. 1.8 angstroms refined structure of the lipase from Geotrichum candidum . J. Mol. Biol. 230: 575–591.

  19. 19

    Sali, A. and Blundell, T.L. 1993. Comparative modelling by satisfaction of spatial restraints. J. Mol. Biol. 234: 779–815.

  20. 20

    Sali, A. and Overington, J.P. 1994. Derivation of rules for comparative modeling from a database of protein structure alignments. Prot Sci. 3: 1582–1596.

  21. 21

    U.S.Patent Application No.07,739,2801.

  22. 22

    Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. pp. 1.82–1.84 in Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

  23. 23

    Chen, Y., Usui, S., Queener, S.W., and Yu, C. 1995. Purification and properties of p-nitrobenzyl esterase from Bacillus subtilis . J. Ind. Micro. 15: 10–18.

  24. 24

    Jbilo, O., L'Hermite, Y., Talesa, V., Toutant, J.P., and Chatonnet, A. 1994. Acetylcholinesterase and butyrylcholinesterase expression in adult-rabbit tissues and during development. Eur. J. Biochem. 225: 115–124.

  25. 25

    Jbilo, O. and Chatonnet, A. 1990. Complete sequence of rabbit butyrylcholinesterase. Nucleic Acids Res. 18: 3990.

  26. 26

    Ozols, J. 1989. solation, properties, and the complete amino-acid sequence of a 2nd form of 60-kda glycoprotein esterase—orientation of the 60-kda proteins in the microsomal membrane. J. Biol. Chem. 264: 12533–12545.

  27. 27

    Bomblies, L., Biegelmann, E., Doering, V., Gerisch, G., Krafft-Czepa, H., Noegel, A.A., Schleicher, M., and Humbel, B.M. 1990. Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases. J. Cell Biol. 110: 669–679.

  28. 28

    Hwang, C.-S. and Kolattukudy, P.E. 1993. Molecular cloning and sequencing of thioesterase B cDNA and stimulation of expression of the thioesterase B gene associated with hormonal induction of peroxisomal proliferation. J. Biol. Chem. 268: 14278–14284.

  29. 29

    Lorti, M., Grandori, R., Fusetti, F., Longhi, S., Brocca, S., Tramontano, A., and Alberghina, L. 1993. Cloning and analysis of Candida cylindracealipase sequences. Gene 124: 45–55.

  30. 30

    Kaiser, R., Erman, M., Duax, W.L., Ghosh, D., and Joernvall, H. 1994. Monomeric and dimeric forms of cholesterol esterase from Candida cylindracea . Primary structure, identity in peptide patterns, and additional microheterogeneity. FEBS Lett. 337: 123–127.

Download references

Author information

Author notes

  1. Frances H. Arnold: Corresponding author (e-mail:


  1. Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA, 91125

    • Jeffrey C. Moore


  1. Search for Jeffrey C. Moore in:

  2. Search for Frances H. Arnold in:

About this article

Publication history



Issue Date


Further reading