Directed Evolution of Enzymes for Industrial Biocatalysis

Abstract

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are
increasingly being sought as environmentally f riendly and cost-effective alternatives to
conventional catalysts used in industries ranging from bioremediation to applications in
medicine and pharmaceutics. Despite the benefits, they are not without their limitations.
Many naturally occurring enzymes are not suitable for use outside of their native cellu lar
environments. However, protein engineering can be used to generate enzymes tailored for
specific industrial applications. Directed evolution is particularly useful and can be employed
even when lack of structural information impedes the use of rational design. The aim of this
review is to provide an overview of current industrial applications of enzyme technology and
to show how directed evolution can be used to modify and to enhance enzyme properties.
This includes a brief discussion on library generat ion and a more detailed focus on library
screening methods, which are critical to any directed evolution experiment .

Introduction

Directed evolution has emerged in just a few years as one of the most effective
approaches to adapting biocatalysts to the performance requirements of industrial and
medical applications. Directed evolution mimics the processes of Darw inian evolution in a
test tube, combining random mutagenesis and/or recombination with screening or selection
for enzyme variants that have the desired properties1. A recent conference* that convened
practitioners from industry and academia surveyed current efforts and highlighted the rapid
progress the field has enjoyed. In addition to impressive demonstrations of enzyme
engineering, there were a variety of challenging problems and approaches to laboratory
evolution In fact, although enzymes are very active and selective biocatalysts, for industrial
application, a very common reason to engineer them is to increase their stability under
reaction conditions, because those ones needed for a biocatalytic procedure may differ
dramatically from those present in a cell, therefore demanding high temperatures, extremes
of pH, high substrate and/or product concentrations, oxidants, and organic co-solvents.
Protein engineering is concerned with the design and construction of novel enzymes w ith
tailored functional properties, including stability, catalytic activity, reaction product
inhibition and substrate specificity. Two broad approaches have been used for enzyme
engineering, namely, rational design and directed evolution.Representative reports of
improved catalytic activity, enzyme stability, and specificity are discussed. Finally, an insight
is given on recent technological developments to make enzyme s more suitable for
biocatalysis. These new technologies could favor the switch of numerous processes from
chemistry to biocatalysis soon.vity improvements of up to 920-fold were achieved while the
(S)-enantioselectivity was maintained or increased to 99% ee.

Isomerases, ligases, and transporters For isomerases, error-prone PCR was performed on a
metallic l-arabinose isomerase from Escherichia coli and a non- metallic tagatose-6-
phosphate isomerase f rom Staphylococcus aureus (SATI), respectively (Kim et al. , 2010). A
SATI variant with 190% increased isomerization activity toward the unnatural substrate
galactose was obtained. The authors also compared the results between the metallic and
non- metallic enzymes, and found that the latter was a better template f or directed evolution.

Cellular transport systems, such as sugar transporters and efflux pumps, play an important
role in many biological processes. However, they are rarely targeted for directed evolution.
It is partly because they are membrane proteins a nd have no use in single step
biotransformation, but may greatly increase the efficiency of a cell factory. Recent examples
of efflux pump engineering may pave the way for directed evolution of this class of proteins.
Cellular export systems, such as efflux pumps, provide a direct mechanism for reducing
biofuel toxicity (Dunlop et al., 2011).

Discussion

Enzymes have the potential to catalyse a wide variety of chemical reactions. They are
increasingly being sought as environmentally friendly and cost ‐effective alternatives to
conventional catalysts used in industries ranging from bioremediation to applications in
medicine and pharmaceutics. Despite the benefits, they are not without their limitations.
Many naturally occurring enzymes are not suitable f or use outside of their native cellular
environments. However, protein engineering can be used to generate enzymes tailored for
specific industrial applications. Directed evolution is particularly useful and can be employed
even when lack of structural information impedes the use of rational design. The aim of this
review is to provide an overview of current industrial applications of enzyme technology and
to show how directed evolution can be used to modify and to enhance enzyme properties.
This includes a brief discussion on library generation and a more detailed focus on library
screening methods, which are critical to any directed evolution experiment.Enzyme
engineering is widely used to transform enzymes into better catalysts for improved
specificity, selectivity, stability, and new chemistry. Methods available to produce the
genetic diversity necessary to discover variants with improved characteristics range from
focussed point mutations to a completely randomized approach .. The high catalytic
efficiency and excellent stability of xylanases are prerequisites for their successful
application in biotechnology. However, wild-type enzymes often do not have both favorable
enzymatic properties and high catalytic efficiency, which limit their application in so me
industries. In the studies attempting to improve the properties of these enzymes, most of
the substitution of modules or regions were limited to the N terminus of the proteins and
there are no reports showing the replacement of internal peptides in prot eins, especially for
the xylanases f rom the GH10 familyThe present review seeks to highlight the major fields of
enzyme application and to provide an updated overview on previous protein engineering
studies wherein natural enzymes were modif ied to meet the operational conditions required
for industrial application. The powerful and revolutionary techniques so far developed for
protein engineering provide excellent opportunities for the design of industrial enzymes w ith
specific properties and production of high-value products at lower production costs.

Conclusion

Biocatalysts have been increasingly used in practical synthesis of chemicals. As one of

the key enabling technologies in biocatalyst development, directed evolution has been
demonstrated to be hig hly effective in altering or improving almost all kinds of protein
properties for all six different classes of enzymes. Continuing development of new library
creation methods and high throughput screening methods will make directed evolution even
more powerful, which will lead to broader applications of biocatalysis in pharmaceutical,
agriculture, food, chemical, and energy industries.

References

Clarke A. R., Atkinson T. and Holbrook J. J. (1989) From analysis to synthesis – new
ligand-binding sites on the lactate dehydrogenase framework. 2. Trends Biochem. Sci. 14:
145–148

Clarke A. R., Atkinson T. and Holbrook J. J. (1989) From analysis to synthesis – new
ligand-binding sites on the lactate dehydrogenase framework. 2. Trends Biochem. Sci. 14:
101105

van den Heuvel R. H. H., Fraaije M. W., Ferrer M., Mattevi A. and van Berkel W. J. H.
(2000) Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl. Acad. Sci. USA
97: 9455–9460
Scrutton, N. S., A. Berry and R. N. Perham (1990) Redesign of the coenzyme specificity
of a dehydrogenase by protein engineering. Nature 343: 38–43

Seebeck, F. P. and Hilvert D. (2003) Conversion of a PLP -dependent racemase into an

aldolase by a single active site mutation. J. Am. Chem. Soc. 125: 10158–10159

Osborne, M. J., Schnell J., Benkovic S. J., Dyson H. J. and Wright P. E. (2001) Backbone
dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic
mechanism. Biochemistry 40: 9846–9859

Rajagopalan P. T. R., Lutz S. and Benkovic S. J. (2002) Coupling interactions of distal

residues enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer
rates. Biochemistry 41: 12618–12628

Agarwal P. K., Billeter S. R., Rajagopalan P. T. R., Benkovic S. J. and Hammes -Schiffer
S. (2002) Network of coupled promoting motions in enzyme catalysis. Proc. Natl. Acad. Sci.
USA 99: 2794–2799

Wang L., Brock A., Herberich B. and Schultz P. G. (2001) Expanding the genetic code of
Escherichia coli. Science 292: 498 –500 10 Liebeton K., Zonta A., Schimossek K., Nardini
M., Lang D., Dijkstra B. W. et al. (2000) Directed evolution of an enantioselective lipase.
Chem. Biol. 7: 709–718