Small Laccase Research Articles

Fabrication and application of a novel electrochemical biosensor based on a mesoporous carbon sphere@UiO-66-NH2/Lac complex enzyme for tetracycline detection.

The overuse of tetracycline results in a threat to human, poultry and livestock health. An enzymatic electrochemical biosensor is an ideal alternative method for accurate and rapid tetracycline detection, while the unstable and easily deactivated nature of the enzyme limits its development. To overcome these limitations, a highly sensitive enzymatic electrochemical biosensor for the determination of tetracycline is developed in this work based on a complex enzyme which was constructed using a mesoporous carbon sphere@UiO-66-NH2 (MCS@UiO-66-NH2) core-shell composite with embedded laccase (Lac). Compared to pure MCS and UiO-66-NH2, the MCS@UiO-66-NH2 core-shell composite has an advantageous mesoporous structure (pore diameter >8 nm), which is suitable for the immobilization of small laccase. The biosensor based on the complex enzyme exhibits a superior activity and enhanced stability as compared with that made using a pure enzyme because the mesoporous structure of the MCS@UiO-66-NH2 composite can effectively protect the laccase against inactivation and denaturation. Besides, its high specific surface area and good conductivity are beneficial to enzyme immobilization and electron transfer in the modified electrode. The biosensor based on this complex enzyme exhibits a relatively low detection limit of 8.94 × 10-7 mol L-1 and a detection range of 1.0 × 10-6-6.0 × 10-5 mol L-1 for tetracycline detection. Furthermore, the developed biosensor possesses good long-term stability, selectivity and reproducibility, indicating its potential application for tetracycline determination in actual food. This research work provides a prospective solution to resolve the stability and inactivation problems of enzymatic electrochemical biosensors in different application scenarios.

Characterization of whey protein isolate and pectin composite film catalyzed by small laccase from Streptomyces coelicolor

Abstract In this current study, whey protein isolate (WPI)/pectin biowaste composite film properties was investigated with addition of Streptomyces coelicolor-derived small laccase (SLAC). It was found that different SLAC concentrations has insignificant effect on the film color while best film transparency obtained with 4 U/100 mL SLAC. Optimum mechanical properties were obtained with 6 U/100 mL SLAC indicating best crosslinking effect on the WPI-pectin film. Lowest moisture content (MC), water vapor transmittance (WVP) and oxygen transmittance ( O 2 P) of the film obtained with 6 U/100 mL SLAC, while 4 U/100 mL SLAC resulted in lowest carbon dioxide transmittance (CO2P). Maximum increase in phase transition temperature and absorption peak shift according to DSC and FTIR results, respectively, indicated largest improvement in heat resistance and chemical bond strength of the film with 6 U/100 mL SLAC addition. The XRD results showed that the addition of SLAC was insignificant towards the film crystallinity. The work presents the development of new degradable composite film that can be derived from biowaste compounds for utilization in food processing application.

Chemical Exchange at the Trinuclear Copper Center of Small Laccase from Streptomyces coelicolor

The trinuclear copper center (TNC) of laccase reduces oxygen to water with very little overpotential. The arrangement of the coppers and ligands in the TNC is known to be from many crystal structures, yet information about possible dynamics of the ligands is absent. Here, we report dynamics at the TNC of small laccase from Streptomyces coelicolor using paramagnetic NMR and electron paramagnetic resonance spectroscopy. Fermi contact-shifted resonances tentatively assigned to histidine Hδ1 display a two-state chemical exchange with exchange rates in the order of 100 s−1. In the electron paramagnetic resonance spectra, at least two forms are observed with different gz-values. It is proposed that the exchange processes reflect the rotational motion of histidine imidazole rings that coordinate the coppers in the TNC.

Correlation between the T1 copper reduction potential and catalytic activity of a small laccase

Laccases are multicopper enzymes that catalyze oxidation of electron-rich substrates coupled to reduction of molecular oxygen to water. Since the Type 1 copper (T1 Cu) is the site where electrons are withdrawn from the substrate, it is assumed that the reduction potential of this copper correlates with enzyme activity. Herein, we studied the correlation of the T1 Cu reduction potential and the enzymatic activity of the small two-domain laccase Ssl1 from Streptomyces sviceus. For a systematic approach, we aimed to minimize any effects other than the reduction potential difference. To this end, we constructed a series of Ssl1 mutants with reduction potentials varying from <290 to 560 mV. Along with the hydrophobicity of the axial ligand of the T1 Cu also structural changes in the substrate binding site and additional hydrogen bonding increased the reduction potential. Enzyme activity experiments demonstrated that the T1 Cu reduction potential has a different effect on oxidation of different substrates. Whereas there was no obvious correlation between the T1 Cu reduction potential and kinetic parameters for the oxidation of syringaldazine (with a reduction potential of 390 mV), a good correlation was observed between the T1 Cu reduction potential and the conversion of substituted phenols with reduction potentials between 660 and 820 mV. This correlation was pronounced for the Ssl1 variants with reduction potentials above 470 mV, which demonstrated increased activities also during the oxidation of two dyes, alizarin red S and indigo carmine.

Comparative investigation of small laccase immobilized on carbon nanomaterials for direct bioelectrocatalysis of oxygen reduction

In this study, different carbon nanomaterials (carbon nanotubes, solid carbon spheres and graphene nanosheets) for immobilizing bacterial small laccase (SLAC) to enable direct bioelectrocatalysis of oxygen reduction were comparatively investigated. Highest bioelectrocatalytic current response was achieved with ethanol-assisted SLAC adsorption on single-walled carbon nanotubes. Direct electron transfer (DET) rates were determined and generally shown to be larger with carbon nanomaterials in smaller dimensions or higher surface curvature, i.e., 1.92, 1.05, 1.52, 0.62 and 0.63 s−1 for adsorbed SLAC on single-walled carbon nanotubes (d = ca. 1.5 nm), double-walled carbon nanotubes (d = 2–4 nm), thinner multi-walled carbon nanotubes (d = 8–15 nm), thicker multi-walled carbon nanotubes (d = 20–30 nm) and graphene nanosheets, respectively. No obvious catalytic current response was obtained with solid carbon spheres (d > 200 nm). Surface nanostructures on electrodes with decreased cylindrical diameter could enable a favorable protein orientation of immobilized SLAC for efficient electronic communication between the catalytic copper sites and carbon nanomaterials as revealed by surface vibrational spectroscopy. Moreover, such a difference in DET kinetics of SLAC could be an outcome of synergic effects of surface properties (curvature, oxygen-containing groups, etc.) and conductivity of carbon nanomaterials. It is expected to offer useful information to fabricating carbon-based oxygen-reducing biocathodes of wide working pH range and high tolerance to chloride.

Effects of small laccase from Streptomyces coelicolor on the solution and gel properties of whey protein isolate

Laccase from Streptomyces coelicolor (SLAC) can catalyze protein cross-linking without any phenolic acid assistance, and its effects on the solution and gel properties of whey protein isolate (WPI) were investigated in this study. WPI solution (5% w/v) was catalyzed by SLAC at 60 °C for 10 min at different enzymatic activities (0–20U). Results showed that, cross-linked by SLAC, the foaming capacity and emulsifying activity properties of WPI solution were improved. SLAC treated solutions had more negative charge in zeta potential, indicating more stable in protein solutions. SLAC-modified increased the apparent visicosities of WPI solutions. Rheological results showed that SLAC-treated WPI gels had higher dynamic storage modulus (G′) and took longer gelation time, compared to control (0U). SLAC treatment improved the strength of WPI gels and increased their water holding capacity (WHC). The results indicate that suitable SLAC catalysis could be used to modify whey protein isolate to improve its solution and gel properties.

Small laccase-catalyzed synthesis of a caffeic acid dimer with high antioxidant capacity

Abstract The increase in the demand for antioxidant compounds for application in industries such as food and pharmaceutical, has necessitated the need to investigate new production methods The application of laccase for antioxidant synthesis is gaining notable attention as a viable approach to address this need. In this study, a β-β caffeic acid dimer, phellinsin A, was synthesized using the small laccase, SLAC, a two-domain bacterial laccase native to Streptomyces coelicolor A3(2). Phellinsin A showed a 1.5-fold increase in 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and a 1.8-fold improvement in trolox equivalence antioxidant capacity (TEAC) when compared to caffeic acid. Phellinsin A also showed improved solubility properties in aqueous media and remarkable stability at acidic pH (pH 2.2 and pH 5.5). This study demonstrates the potential of the bacterial laccase, SLAC, as a catalyst for the biotransformation of natural phenolics into value-added compounds.

Whey protein isolate with improved film properties through cross-linking catalyzed by small laccase from Streptomyces coelicolor.

The effects of small laccase (SLAC) from Streptomyces coelicolor on the properties of whey protein isolate (WPI) films were studied. WPI was catalyze by SLAC without phenolic acid assistance. Particle size distribution results showed that some complexes with higher relative molecular weight formed in WPI samples treated with SLAC. The content of α-helixes decreased while those of β-sheets and random coils increased following SLAC treatment according to circular dichroism results. Fourier transform infrared spectral analysis suggested that some conformational changes occurred in WPI following SLAC treatment. Analysis of WPI films prepared by casting after SLAC treatment indicated that their film properties were all improved, including mechanical properties, solubility, water vapor, oxygen and carbon dioxide barrier properties, film color, light transmission, transparency and thermal properties. Compared with that of the control film, some obvious differences in the morphology of the WPI films were observed following SLAC treatment. This report demonstrates that laccase can directly catalyze protein cross-linking, which may be useful to improve the performance of protein films. In this study, SLAC was applied to WPI edible film during the film-making process. The results showed that SLAC can catalyze WPI cross-linking without phenolic acid assistance, and WPI film properties were improved after SLAC treatment. © 2018 Society of Chemical Industry.

Small laccase from streptomyces coelicolor-an ideal model protein/enzyme for undergraduate laboratory experience.

A one semester undergraduate biochemistry laboratory experience is described for an understanding of recombinant technology from gene cloning to protein characterization. An integrated experimental design includes three sequential modules: molecular cloning, protein expression and purification, and protein analysis and characterization. Students perform the tasks of cloning, expression, purification, analysis, and characterization of small laccase (SLAC) from Streptomyces coelicolor. SLAC is an extremely robust well-characterized protein/enzyme, which serves as an ideal model for undergraduate teaching laboratories. Also, this goal-oriented research-like approach helps students to unite the concepts of biochemistry and molecular biology and appreciate the utility of the methods more effectively. A student assessment before and after the course demonstrated an overall increase in learning and enthusiasm on the topic of modern protein chemistry. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):172-181, 2018.

Secretory expression of recombinant small laccase from Streptomyces coelicolor A3(2) in Pichia pastoris

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ± 10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding kcat values were 60.6 s−1, 25.36 s−1 and 27.84 s−1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.

Enhanced delignification of steam-pretreated poplar by a bacterial laccase

The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac’s substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes.

Designed protein aggregates entrapping carbon nanotubes for bioelectrochemical oxygen reduction.

The engineering of robust protein/nanomaterial interfaces is critical in the development of bioelectrocatalytic systems. We have used computational protein design to identify two amino acid mutations in the small laccase protein (SLAC) from Streptomyces coelicolor to introduce new inter-protein disulfide bonds. The new dimeric interface introduced by these disulfide bonds in combination with the natural trimeric structure drive the self-assembly of SLAC into functional aggregates. The mutations had a minimal effect on kinetic parameters, and the enzymatic assemblies exhibited an increased resistance to irreversible thermal denaturation. The SLAC assemblies were combined with single-walled carbon nanotubes (SWNTs), and explored for use in oxygen reduction electrodes. The incorporation of SWNTs into the SLAC aggregates enabled operation at an elevated temperature and reduced the reaction overpotential. A current density of 1.1 mA/cm(2) at 0 V versus Ag/AgCl was achieved in an air-breathing cathode system. Biotechnol. Bioeng. 2016;113: 2321-2327. © 2016 Wiley Periodicals, Inc.

Correlating the structures and activities of the resting oxidized and native intermediate states of a small laccase by paramagnetic NMR

Multicopper oxidases (MCO) are the fastest and most efficient known catalysts of the oxygen-reduction reaction. When all four copper ions are oxidized during catalysis, the native intermediate state (NI) decays in seconds to the resting oxidized state (RO), which returns to the catalytic cycle via reduction, but at a much slower rate than NI. We report the long-lived (months at 4°C) NI state of the small laccase (SLAC) MCO and the subsequent characterization of both its RO and NI states by paramagnetic 1H NMR. We find that the RO state of the trinuclear cluster (TNC) is best described as an isolated Type-3 dicopper site, antiferromagnetically coupled by a hydroxo group with −2J=500cm−1. The NI state is more complicated; we develop a theoretical treatment for the case in which all three copper ions in the TNC are coupled, and find that the results are consistent with three coupling constants of −2J=300, 240, and 160cm−1. These couplings result in a ground doublet state, a low-lying excited doublet state at 121cm−1, and a quartet excited state at 411cm−1, in good agreement with DFT models in which the Type-2 copper has a terminal hydroxo ligand.

New approaches to achieve high level enzyme production in Streptomyces lividans.

BackgroundActinomycetes are saprophytic soil bacteria, and a rich source of industrial enzymes. While some of these enzymes can be produced using well-characterized production platforms such as Escherichia coli or Bacillus subtilis,Streptomyces lividans may be the preferred host for proper folding and efficient secretion of active enzymes. A combination of promoters, signal peptides and hosts were tested in order to obtain the best protein expression in this actinomycete. The xylanase, Xys1, from S. halstedii, the α-amylase, Amy, from S. griseus and the small laccase, SLAC, from S. coelicolor were used as reporters.ResultsThe promoters xysAp from S. halstedii JM8 and pstSp from S. lividans were the most efficient among those tested. An improvement of 17 % was obtained in xylanase activity when the signal peptide of the α-amylase protein (Amy) of S. griseus IMRU3570 was used to direct its secretion. Enhanced expression of SsgA, a protein that plays a role in processes that require cell-wall remodelling, resulted in a improvement of 40 and 70 % of xylanase and amylase production, respectively. Deletion of genes SLI7232 and SLI4452 encoding putative repressors of xysAp provided improvement of production up to 70 % in the SLI7232 deletion strain. However, full derepression of this promoter activity was not obtained under the conditions assayed.ConclusionsStreptomyces lividans is a frequently used platform for industrial enzyme production and a rational strain-development approach delivered significant improvement of protein production by this host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0425-7) contains supplementary material, which is available to authorized users.

Functional interfaces for biomimetic energy harvesting: CNTs-DNA matrix for enzyme assembly

The development of 3D structures exploring the properties of nano-materials and biological molecules has been shown through the years as an effective path forward for the design of advanced bio-nano architectures for enzymatic fuel cells, photo-bio energy harvesting devices, nano-biosensors and bio-actuators and other bio-nano-interfacial architectures. In this study we demonstrate a scaffold design utilizing carbon nanotubes, deoxyribose nucleic acid (DNA) and a specific DNA binding transcription factor that allows for directed immobilization of a single enzyme.Functionalized carbon nanotubes were covalently bonded to a diazonium salt modified gold surface through carbodiimide chemistry creating a brush-type nanotube alignment. The aligned nanotubes created a highly ordered structure with high surface area that allowed for the attachment of a protein assembly through a designed DNA scaffold. The enzyme immobilization was controlled by a zinc finger (ZNF) protein domain that binds to a specific dsDNA sequence. ZNF 268 was genetically fused to the small laccase (SLAC) from Streptomyces coelicolor, an enzyme belonging to the family of multi-copper oxidases, and used to demonstrate the applicability of the developed approach. Analytical techniques such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and enzymatic activity analysis, allowed characterization at each stage of development of the bio-nano architecture. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6

An open reading frame of the Thermus thermophilus HJ6 hypothetical laccase, which composed of 729 bases, was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli SoluBL21™ cells. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 6M guanidine HCl. The solubilized protein was refolded by a simple on-column refolding procedure using Ni-chelation affinity chromatography and then the refolded protein was purified by gel filtration chromatography. It showed a single band with a molecular mass of 27kDa in SDS–PAGE. The results from UV–visible absorption and electron paramagnetic resonance (EPR) analysis suggested that the enzyme had the typical copper sites, type-1, 2, and 3 Cu(II) of laccase. The purified enzyme exhibited the laccase activity with the optimal catalytic temperature at 75°C. The optimum pH for the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and syringaldazine was 4.5 and 6.0, respectively. The recombinant protein showed high thermostability, and the half-life of heat inactivation was about 50min at 85°C. The enzyme oxidized various known laccase substrates, its lowest Km value being for syringaldazine, highest kcat value for guaiacol, and highest kcat/Km for 2,6-dimethoxy-phenol. The enzyme reaction was strongly inhibited by the metal chelators and the thiol compounds.

Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases found in many organisms and catalyses oxidation of primarily phenolic compounds by oxygen. A recently found bacterial laccase whose molecule is formed by two domains - the so called two-domain laccase (2DLac) or small laccase - has unusual resistance to inhibitors and an alkaline optimum of activity. The causes of these properties, as well as the biological function of two-domain laccases, are poorly understood. We performed an enzymatic and structural characterization of 2DLac from Streptomyces viridochromogenes (SvSL). It was cloned and overproduced in Escherichia coli. Phenolic compounds were oxidized in the presence of the enzyme under alkaline but not acidic conditions. Conversely, nonphenolic compounds were oxidized at acidic but not alkaline pH. SvSL catalysed oxidation of nonphenolic compounds more efficiently than that of phenols. Moreover, this two-domain laccase displayed a cytochrome c oxidase activity and exhibited no ferroxidase activity. The enzyme was resistant to specific inhibitors of copper-containing oxidases, such as NaN3 and NaF. We succeeded in generating X-ray quality crystals and solved their structure to a resolution of 2.4Å. SvSL is a homotrimer in its native state. Comparison of its structure with that of a three-domain laccase revealed differences in the second coordination sphere of the T2/T3 centre and solvent channels. The role of these differences in the resistance of the enzyme to inhibitors and the activity at alkaline pH is under discussion.

A click chemistry approach to site-specific immobilization of a small laccase enables efficient direct electron transfer in a biocathode.

Controlled orientation of a small laccase on a multi-walled carbon nanotube electrode was achieved via copper-free click chemistry mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer.

The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2)

Bacterial laccases show low activities but can be of biotechnological interest due to industrially suitable characteristics such as thermostability and tolerance to alkaline pH. In this study, three separate mutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small laccase (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparison with the native enzyme. The mutation, V290N showed approximately double the activity of SLAC when ABTS was used as substrate while the specific activity of SLAC-M298F was 4–5 times higher than that of SLAC when the assays were performed at ≥70°C. There was no significant difference in activity with 2,6-dimethoxyphenol (2,6-DMP); however, there was a significant shift in the optimal pH from pH 9.5 (SLAC) to 7.5 (SLAC-V290N). Optimal temperature for activity was not significantly altered but thermostability was reduced in all three mutants. The substrate range of the mutant variants remained largely unchanged, with the exception of SLAC-M298F which was unable to oxidise veratryl alcohol. Interestingly, the “typical” laccase inhibitor, sodium azide, had no significant inhibitory effect on the activity of SLAC-M298F, which also exhibited increased resistance to inhibition by sulfhydryl compounds. SLAC-V290N showed higher catalytic efficiency for 2,6-DMP (kcat/Km=2.226mM−1s−1) and ABTS (kcat/Km=1.874mM−1s−1) compared to SLAC (kcat/Km=1.615mM−1s−1 for 2,6-DMP and kcat/Km=1.611mM−1s−1 for ABTS). This study has shown that three ligands that are closely associated with the T1 copper in SLAC play a key role in maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourable characteristics such as thermostability, several favourable effects were observed. This study has also extended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a template for engineering with the aim of widening its potential range of industrial applications.

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.