Adventures in the Engineering of the Small Laccase (SLAC) from Streptomyces Coelicolor for Incorporation into Enzymatic Biofuel Cell Cathodes

The small laccase (SLAC) from Streptomyces coelicolor has many beneficial features for use in enzymatic biofuel cells including recombinant expression in E. coli and high activity at neutral pH [1].  We have previously used protein engineering to attempt to improve the enzyme for bioelectrocatalysis applications.  For example, we have previously engineered the SLAC enzyme to self-assemble into amorphous proteinaceous hydrogels and we included peptide-bound osmium complexes for high activity [2]. 

Three new protein engineering approaches are now being explored for the improved integration of small-laccase (SLAC) and carbon nanomaterials to improve cathode performance. The first approach is to genetically append the SLAC with a designed helical peptide, which binds to single-walled nanotube with high affinity. The peptide sequence was selected due to its ability to create a geometrically defined, virus-like coating on the surface of single walled nanotubes (SWNTs) [3].

The second approach relies on the interactions between zinc finger (Znf) protein domains and DNA. SLAC was genetically fused with Znf-268 and this fusion protein retained laccase activity while gaining the ability to bind DNA [4].  The DNA was attached to SNWTs through a specially designed 3D structured DNA scaffold.  This scaffold along with the highly specific Znf-DNA interaction provides a proof-of-concept for the nano-scale templating and arrangement of individual proteins during multi-enzyme immobilization with the goal of mimicking the natural organization of metabolic enzymes found in cells.

For the third approach, computationally designed biomolecular interactions were identified to engineer SLAC to self-assemble into crystalline-like assemblies.  This was accomplished by adding strategically placed disulfide bonds at protein/protein interfaces (Fig. 1).  The new protein assemblies spontaneously form in an oxidative environment after purification and they are robust. The protein design was performed using the Rosetta methodology, which can be used to predict protein structure from sequence, to phase crystal structures, to model and design protein interfaces, novel enzyme catalyst and new protein topologies [5].

All of the new SLAC mutant proteins have been expressed, purified, and kinetically characterized.  Different techniques have been explored to evaluate the assembly of the SLAC-SWNTs complexes and to characterize their electrochemical activities.  All three approaches have been successful in improving protein/nanomaterial interactions, leading to increased protein loading and improvements in overall activity in real electrochemical systems (Fig. 2).  The most recent electrochemical characterization of the new SLAC mutant proteins will be presented and the implications of these designs on biofuel cell performance will be discussed.

1.             Gallaway, J., et al., Oxygen-reducing enzyme cathodes produced from SLAC, a small laccase from Streptomyces coelicolor. Biosens Bioelectron, 2008. 23(8): p. 1229-35.

2.             Wheeldon, I.R., et al., Bioelectrocatalytic hydrogels from electron-conducting metallopolypeptides coassembled with bifunctional enzymatic building blocks. Proc Natl Acad Sci U S A, 2008. 105(40): p. 15275-80.

3.             Grigoryan, G., et al., Computational Design of Virus-Like Protein Assemblies on Carbon Nanotube Surfaces. Science, 2011. 332: p. 1071-1076.

4.             Géza R. Szilvay, S.B., Dimitri Ivnitski, Carol Li, Pablo DeLa Iglesia, Carolin Lau, Eva Chi, Margaret Werner-Washburne, Scott Banta, Plamen Atanassov, Engineering of a redox protein for DNA-directed assembly. Chem. Commun., 2011. 47: p. 7464-7466.

5.             Das, R. and D. Baker, Macromolecular modeling with rosetta. Annu Rev Biochem, 2008. 77: p. 363-382.