2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine, SGZ), 2,6-dimethoxyphenol (2,6-DMP), 3', 5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone (ACS)) and indigocarmine (IC) were purchased from Sigma-Aldrich. All other chemicals were standard reagent grade (Sigma Aldrich).

B. pumilus DSM 27 was obtained from the German collection of microorganisms (DSMZ). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(r_K ^-, m_K ⁺), relA1, supE44, λ^-, Δ(lac-proAB), (F', traD36, proAB, lacI ^qZΔM15)] and the pQE-60 expression vector were purchased from Promega (Madison, USA) and Qiagen (Valencia, USA), respectively. The origin of replication in pQE-60 is ColE1 (pBR322) and transcription of the inserted gene is controlled by the bacteriophage T5 promoter (recognized by the E. coli housekeeping RNA polymerase) and two lac operator sequences (conferring inducibility by IPTG). For efficient repression the host strain JM109 which over-expresses the repressor LacI was used.

B. pumilus was grown overnight at 30°C and 150 rpm in SRB medium (20 g/L yeast extract

25 g/L tryptone, 3 g/L K₂HPO₄). E. coli was routinely grown in LB medium at 37°C and 150 rpm. For plasmid selection 0.1 mg/mL ampicillin was added to LB agar plates and liquid medium.

The putative laccase gene (spore coat protein A, cotA) of B. pumilus was identified by Protein Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the CotA sequence of B. subtilis as search template (gene bank accession no. NP_388511). The cotA gene of B. pumilus (accession no. ZP_03054403) was PCR amplified from genomic DNA with forward primer 5'- CGT

CC ATG G

AAGCTT

E. coli JM109 was transformed with pBpL6 for expression and purification of B. pumilus laccase.

To obtain a fully copper loaded enzyme, the method reported by Durao et al. 2008 was used for CotA production 26 . Briefly, the recombinant E. coli strain was cultivated in TB medium supplemented with 0.1 mg/mL ampicillin at 30°C and 120 rpm. Starting from an isolated colony, an overnight pre-culture was diluted 1:50 into a 700 mL volume in a 2 L Erlenmeyer flask. At an optical density of 1-2 (ΔOD₆₀₀), laccase expression was induced by adding 0.1 mM IPTG. At the same time, 0.25 mM CuCl₂ was added and the temperature was decreased to 25°C. The cells were further incubated for 4 h at 120 rpm and then without shaking for additional 16 hours. Durao et al. (2008) showed that static incubation improved the cellular copper content, thus yielding a fully copper loaded protein population. Cells were harvested by centrifugation at 4°C for 30 min at 4,495 × g, washed in 20 mM Tris buffer pH 7.6, centrifuged again and subsequently stored at -20°C.

Frozen cells were thawed on ice and resuspended in 20 mM Tris buffer pH 7.6 containing 1 mg/mL lysozyme and protease inhibitor mix (Roche Complete Protease Inhibitior Mix, EDTA-free) and re-frozen at -80°C. Cells were thawed, Benzonase^® Nuclease (Roche) was added and the suspension incubated for 1 h at 37°C at 120 rpm. The suspension was subjected to twelve 10 s rounds of sonication with a Branson sonicator equipped with a microtip at a setting of 80%. Cellular debris was removed by centrifugation at 4°C for 40 min, 47,000 × g. The crude cell lysate was incubated at 70°C for 20 min and soluble protein was collected by centrifugation at 4°C for 40 min and 47,000 × g. Purification was performed on an Äkta purifier FPLC system (GE-Healthcare). The sample was loaded onto a 27 mL Q-Sepharose FF anion exchange chromatography column (GE-Healthcare), previously equilibrated with 20 mM Tris buffer pH 7.6. Proteins were eluted with a NaCl gradient from 0 to 1 M. Fractions displaying laccase activity, as detected using an ABTS oxidation assay, were pooled and concentrated by ultrafiltration using a 30 kDa cut-off. The sample was loaded onto a Superdex 75 column (GE-Healthcare), previously equilibrated with 20 mM Tris buffer containing 0.1 M NaCl pH 7.6. Fractions with laccase activity were pooled, concentrated by ultrafiltration and stored in 20 mM Tris buffer pH 7.6 at -80°C. The purity of the sample was analyzed by SDS-PAGE using a 10% polyacrylamide gel. The copper content was determined by ICP-OES (Inductive coupled plasma optical emission spectroscopy) on a Perkin Elmer Optima 3000 instrument. The protein solution was treated with nitric acid in a microwave digestion system prior to optical analysis at Cu lines 327.393 nm and 324.752 nm.

Total protein concentration was determined by the method of Bradford 27 with bovine serum albumin (BSA) as standard and by UV-Vis absorbance measurements with a Nanodrop ND-1000 spectrophotometer using an extinction coefficient of 77,303 M^-1cm^-1 (calculated from the amino acid sequence).

Spectrophotometric laccase activity assays were routinely carried out in a 96-well plate at 25°C with 0.5 mM ABTS in McIlvaine buffer at pH 4 (mixture of 0.1 M citric acid and 0.2 M K₂HPO₄) using a Varian Cary 50 Bio or a Bio Tek Synergy Mx spectrophotometer and initiated by adding enzyme solution. The assay volume was either 200 or 300 μL.

Oxidation of ABTS was monitored at 420 nm (ε = 36,000 M^-1cm^-1), of SGZ at 525 nm (ε = 65,000 M^-1cm^-1) and of 2,6-DMP at 468 nm (ε = 37,500 M^-1cm^-1). Laccase activity as a function of pH was performed in McIlvaine buffer in the pH range 2-8 for ABTS (0.5 mM), SGZ (0.05 mM) and 2,6-DMP (0.3 mM). The temperature optimum was recorded between 20°C and 75°C by following ABTS or 2,6-DMP oxidation in an assay volume of 3 mL using a magnetically stirred, temperature controlled cuvette device. The effect of DMSO on laccase activity was determined by adding 0.4-50% (v/v) of solvent to the reaction mixture and following the oxidation of ABTS, SGZ or 2,6-DMP. The stability of laccase was tested by incubating the enzyme in McIlvaine buffer at pH 5 or 7, potassium phosphate buffer pH 7 and in ddH₂O at 4, 25, 45 and 65°C and subsequently performing an ABTS assay.

Kinetic parameters of purified laccase were determined in McIlvaine buffer at 25°C against ABTS (10 mM-0.6 μM, pH 4), 2,6-DMP (10 mM-0.6 μM, pH 7) and SGZ (1.25 mM-0.04 μM plus 25% (v/v) DMSO, pH 6.5). Enzymatic assays were performed in triplicate. Oxidation of ACS was monitored at 340 nm by the decrease of substrate absorbance (ε = 1,000 M^-1cm^-1) in 0.1 M potassium phosphate buffer (2.5 mM-0.04 mM, pH 6.5). The initial rates, recorded within 3 to 10 min, were approximated by non-linear regression algorithms according to the Michaelis-Menten equation using SIGMA-PLOT Enzyme Kinetics software. One unit was defined as the amount of enzyme that oxidized 1 μmol of substrate per minute.

Purified laccase was subjected to a substrate screening. The screening was based on the direct oxidation of the substrate and on the coupled decolorization of IC. Therefore, potential laccase substrates were dissolved in water and 5% (v/v) DMSO at a concentration of 10 mM and diluted to a final concentration of 1 mM. Reactions were performed in 96-well plates in 0.1 M potassium phosphate buffer, pH 6.5 or 7.8 at 37°C with shaking. The reaction was initiated by adding 0.23 U/mL purified laccase. One unit was defined as the amount of enzyme that oxidized 1 μmol of ABTS per minute at room temperature. A UV-Vis scan between 230-700 nm was recorded prior to laccase addition and after 48 hours reaction time.

To evaluate the ability of the purified laccase to decolorize IC, 1 mM of the dye was dissolved in water and incubated with 0.1 mM of the potential mediator substrate. The reaction was initiated by the addition of laccase which was substituted with water in the control reaction. The reaction was followed over a period of 48 h at intervals of 30 min and was performed at 37°C. The change in absorbance (for λ = 350, 420, 550, 570, 610 and 650 nm) was recorded to follow IC decolorization. Although IC showed a maximum absorbance at 610 nm, decolorization was calculated based on the shoulder at 650 nm. In this way it was possible to follow starting concentrations of 1 mM as the Lambert-Beer law was still valid under these conditions. Furthermore, no by-product was detected at 650 nm as opposed to 610 nm. The decolorization was further assessed by varying the IC to ACS ratio in the presence and absence of laccase.

Decolorization (%) was calculated based on the difference in absorbance at 650 nm in reactions with and without laccase and defined as:

Decolorization ( % ) = 100 − a b s o r b a n c e t 48 _ l a c c a s e × 100 a b s o r b a n c e t 48 _ c o n t r o l

Decolorization reactions were carried out in the presence and in the absence of laccase. The absorbance measured after 48 h in reactions lacking laccase was then defined as 100% (absorbance_{t48_control}).

A Protein Blast search for uncharacterized bacterial laccases was performed using the CotA amino acid sequence of Bacillus subtilis as template. Out of numerous homologues, a hypothetical protein annotated as "spore coat protein A" found in the fully sequenced genome of Bacillus pumilus ATCC 7061 was selected for cloning and recombinant expression in E. coli due to its relatively close relation to other already characterized Bacillus laccases. The B. pumilus DSM 27 sequence encoding a CotA-like protein was cloned into the expression vector pQE-60 as described in Methods. The cloned sequence exhibited 99.8% amino acid identity to the CotA sequence of B. pumilus ATCC7061, with K79E being the only difference. CotA of B. pumilus DSM 27 exhibited 68% amino acid identity and 79% similarity to CotA of B. subtilis, which is the best studied bacterial laccase 22 28 . Amino acid identity and similarity to the second previously characterized CotA laccase from B. licheniformis ATCC 7061 (accession no. YP_077905, 25 ) was 62% and 76%, respectively. CotA of B. pumilus consists of 510 amino acids (hypothetical molecular weight: 58.6 kDa), which is close to the length of 513 amino acids for both the B. subtilis and B. licheniformis laccases. The ligands which bind T1 copper (M502, H419, C492, H497), T2 copper (H105, H422) and T3 copper (H107, H153, H155, H424, H491, H493) in B. subtilis CotA 22 were all conserved in B. pumilus CotA. The cysteine residues C229 and C322 which have been proposed to form a disulfide bond in B. subtilis CotA 29 were also conserved in B. pumilus CotA.

For production of B. pumilus laccase with E. coli JM109 (pBpL6) special growth and induction procedures were applied which had been described previously 26 . Similarly to B. subtilis CotA, the use of reduced cultivation temperature before and after induction, the addition of high concentrations of Cu²⁺ at induction and static incubation in the second phase of CotA expression as stated in materials and methods yielded maximal amounts of fully copper-loaded, active B. pumilus enzyme.

The supernatant from cells expressing laccase was incubated at 70°C for 20 min, as described for other Bacillus laccases expressed in E. coli 25 30 . This treatment results in denaturation of most E. coli proteins and is used to enrich heat resistant proteins such as spore-coat associated laccases. Above 90% of the initial laccase activity was recovered in the supernatant after centrifugation of heat-treated lysate. Subsequent column purifications included anion exchange and size exclusion chromatography, yielding a single protein band of approximately 30 kDa, as shown by SDS-PAGE analysis in Figure 1. The apparent molecular weight of the expressed laccase was much lower than the expected one of approximately 58 kDa, when proteins were separated by SDS-PAGE under standard conditions (5 min, 95°C, in buffer containing 6% SDS and 10% β-mercapthoethanol). However, when the protein was boiled for 20 min, the band migrated at the expected molecular weight, suggesting that full denaturation of the protein required extra heat.

Purified protein samples showed a strong blue color, and copper content determination revealed a molar copper to protein ratio of 3.9, affirming a fully copper-loaded enzyme. The pH optima of CotA activity were determined to be around pH 4 for ABTS, pH 7 for 2,6-DMP and pH 6.5 for SGZ. These values were very similar to those found for other Bacillus CotA laccases 25 28 . The temperature optimum for ABTS oxidation was determined to be between 55-75°C. For 2,6-DMP oxidation a steady increase of activity up to 70°C was monitored, again demonstrating the high temperature tolerance of CotA laccase. For detailed temperature-activity and pH-activity profiles see Additional file 2.

Kinetic parameters of recombinant B. pumilus laccase for ABTS, 2,6-DMP and SGZ oxidation were determined and are shown in Table 1. Michaelis-Menten plots for all substrates are given in Additional file 3. The values obtained for ABTS and 2,6-DMP were more similar to those obtained for B. subtilis than for B. licheniformis laccase 25 26 . It was not possible to determine the kinetic parameters for SGZ due to its low solubility. The observed kinetics did not fit a Michaelis-Menten equation. Instead, only a linear correlation was observed. This was true either when low SGZ (6-78 μM) and DMSO concentrations (< 0.8% v/v) or high SGZ (0.08-1.25 mM) and DMSO (25% v/v, leading to maximal rates of SGZ oxidation, see Fig. 2) concentrations were used. Hence, no K _M can be given for SGZ oxidation. Nevertheless, a rough estimate of k _cat was deduced from the highest observed reaction rates measured with either < 0.8% (v/v) or 25% (v/v) DMSO (data marked with * in Table 1).

SGZ required the presence of DMSO to enable its solubility. The effect of increasing concentrations of DMSO upon laccase activity was therefore investigated. To this end, ABTS, 2,6-DMP and SGZ oxidations were measured in the presence of increasing DMSO concentrations (Figure 2). It was found that SGZ oxidation rates were enhanced with increasing concentrations of DMSO with an optimum at around 25% (v/v). Concentrations above 50% (up to 90%) did not result in further enhanced activity, but rather deactivated the enzyme rapidly (data not shown). For ABTS and 2,6-DMP any addition of DMSO led to a gradually decreased oxidation rate; however, the effect was much more severe for ABTS.

The kinetic stability upon storage of the enzyme was also investigated. Protein samples were incubated in McIlvaine buffer at pH 5 and 7, potassium phosphate buffer at pH 7 and ddH₂O at 4, 25, 45 and 65°C. ABTS oxidation was measured periodically. The half-life of laccase is summarized in Table 2. Within 1 hour, all laccase preparations in McIlvaine buffer pH 5 showed over 75% loss of activity. Enzyme samples stored in either McIlvaine or phosphate buffer at pH 7 showed over 50% loss in activity after 1 h. Unexpectedly, this significant loss was not detected for the samples stored in deionized water.

The catalytic activity of purified CotA was screened against a panel of 37 structurally different potential substrates of both synthetic and natural origin. The activity was measured at a substrate concentration of 1 mM. A UV-Vis scanning between 230-700 nm was recorded prior to laccase addition and after 48 h reaction time. By this method only substrates leading to a changed UV spectrum upon oxidation were detectable. From the 37 compounds tested, a change of absorbance was detected for 18 compounds (Table 3). Control reactions lacking laccase were performed in parallel, to exclude the possibility of non-enzymatic oxidation.

In addition, all substrates were tested for their ability to function as mediator in the decolorization of the dye IC (Table 3). The relative decrease of absorbance was determined spectrophotometrically as a measure of the degree of mediated oxidation of IC. For the mediator screen, 1 mM IC was combined with 0.1 mM of the substrate. The results are summarized in Table 3.

Amongst the 37 compounds tested, 18 showed a change of absorbance as a result of laccase promoted oxidation. In turn, three of these compounds, ABTS (37), ACS (18) and syringaldehyde (20) functioned as mediators and completely decolorized IC within 3 hours. Within 48 hours, two compounds promoted decolorization of 25% (5) and 40% (14), respectively. Laccase alone lead to a decolorization of 20% within 48 hours at pH 6.8. All other compounds did not lead to a decolorization of more than 20% and therefore cannot be considered as mediators for IC oxidation.

ACS was selected for further studies and its potential as a mediator for the decolorization of IC was evaluated. In the screening, experiments were conducted with 0.1 mM ACS and 1 mM IC (1:10 ratio). This ratio was decreased to 1:100 (Figure 3A and 3B) and 1:1000 (Figure 3C and 3D) with IC starting concentrations of 0.25 mM and 1 mM, respectively. Furthermore, the pH effect was evaluated by performing the reactions at pH 6.5 and 7.8. Direct decolorization of IC by laccase was monitored in reactions lacking ACS.

Both mediated and non-mediated reactions were more efficient at higher pH. Although IC was partially (at pH 6.5) and completely (at pH 7.8) decolorized by laccase alone within 48 h, the mediated reactions proceeded faster, most notably at IC:ACS ratio of 1:100. It was found that mediated reactions at IC:ACS ratio of 1:100 completely decolorized IC at both pH values. However, at IC:ACS ratio of 1:1000, only reactions at pH 7.8 were completed. Independently of the pH, the rates of ACS mediated reactions performed at an IC:ACS ratio of 1:1000 and non-mediated reactions were almost the same. This indicated that ACS concentrations significantly below the K _m, which was determined to be 30 mM, had no influence on the reaction kinetics.

Decolorization reactions were also conducted with 10 mM IC and 1:100 and 1:1000 ratios of IC:ACS at pH 6.5 and 7.8. As a control, direct oxidation was monitored in reactions lacking ACS. Although the initial absorbance could not be detected spectrophotometrically (OD_{650 nm} > 4), complete decolorization within 72 hours was observed for the reaction performed at an IC:ACS ratio of 1:100 and pH 7.8. No measurable bleaching was observed for the non-mediated reaction or any of the other control reactions conducted. In reactions which lead to complete decolorization of IC at λ = 650 nm, a red soluble reaction product with an absorbance maximum at λ = 550 nm was formed. The formation of a red product during laccase-catalyzed IC transformation has previously been reported and presumably is due to the condensation of two IC molecules 31 32 .