Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green

Laccases can catalyze monoelectronic oxidation and have shown to have an increasing value in industrial application. In this study, as identified by Native-PAGE and ESI-MS/MS, ascomycetous fungus Setosphaeria turcica produced three laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and after being renaturated from the inclusion body, the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 °C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. It is suggested that Stlac2 has potential industrial applications. Importance Setosphaeria turcica, an ascomycetous fungus causes northern corn leaf blight, product three laccase isozymes identified by Native-PAGE and ESI-MS/MS. The major expression laccase gene StLAC2 was expression in both eukaryotic and prokaryotic expression systems, which found incorrect glycosylation at Asn97 may result in the depression of laccase activity. The heterologous laccase Stlac2 decolorize organic dye malachite green, which had a potential industrial application.


Introduction
were investigated and identified by Native-PAGE and ESI-MS/MS. The major expression laccase gene tested by SDS-PAGE. BCA Protein Assay kits (Sangon, China) were used to analyze the protein concentration. 146

Deglycosylation assay and identification of glycosylation sites 147
For N-glycan removal, eukaryotic recombinant Stlac2 was denatured with Glycoprotein denaturing 148 buffer at 100 ºC for 10 min to be deglycosylated using PNGase F (NEB, USA) according to instructions. 149 The deglycosylated recombinant laccase was separated by SDS-PAGE. Stained band of recombinant 150 laccase expressed in P. pastoris was used for identification of the sites of glycosylation and were 151 conducted in accordance with the batch procedures outlined by Beijing Protein Innovation Co., Ltd.

Assay of laccase activity and biochemical characterization of recombinant Stlac2 161
ABTS was used as a substrate for assaying recombinant Stlac2 activity at 420 nm (ε420=36 mM -1 /cm -1 ). 162 One unit (U) of laccase activity was defined as the oxidation of 1 μmol ABTS in one minute. 163 The enzyme activity was assayed in the pH range of 3.0-6.5 using 0.1 M of citrate buffer. The effect of 164 temperature was determined by incubating the reaction mixture at different temperatures varying from 165 relative activity with respect to maximum activity, which was considered as 100%. The effects of various metal ions were determined by incubating the recombinant Stlac2 at a final concentration of 5 and 10 168 mM for 5 min at 60 ºC. The enzyme activity was expressed as percent relative activity with respect to no 169 additional ions. 170

MG decolorization by recombinant Stlac2 171
To evaluate the application, triarylmethane dye MG was used to analyze the degradation by Stlac2. The 172 reaction mixture contained 100 mM Tris buffer with 20, 50, 100, and 200 mg/L MG were conducted and 173 50 μg purified recombinant Stlac2 in 0.5 mL reaction volume, and incubated at room temperature. The 174 decolorization of MG was followed by measuring absorbance at 618 nm, where the degradation 175 efficiency was calculated as a percentage (Yang et al. 2017). All experiments were performed in triplicate; controls were not treated with recombinant Stlac2. 179

Isolation and identification of laccase isozymes 181
Intracellular and extracellular crude protein were collected from S. turcica in liquid CM culture 7d using 182 for isolation of laccase isozymes by Native-PAGE staining with ABTS and 2,6-DMP. It is more sensitive 183 than staining with ABTS than with 2,6-DMP (Fig. S1). There are two bands in the intracellular crude 184 protein and one in concentrated extracellular crude protein staining with ABTS (Fig.1a). The two bands 185 were tested by ESI-MS/MS to identify laccase isozymes, and the peptide fingerprints were compared 186 with the protein database of S. turcica downloaded from JGI (Fig.1b). The peptide sequence of proteins matched with Stlac2 (Gi: 482813379) and Stlac6 (Gi: 482808389) of band I; Stlac1 (Gi: 482805212), and Stlac6 (Gi: 482808389) of bandⅡ, all of which are classified to be laccase sensu stricto in S. turcica. 189 The molecular weight of the laccase isozymes did not match the specific bands of protein markers shown. 190 This is attributable to the fact that protein separation in Native-PAGE depended not only on molecular 191 size, but also the charge the protein carried. 192

Recombinant laccase Stlac2 expression in PichiapastorisKM71H 193
To characterize laccase Stlac2, the coding sequence was cloned and inserted into the plasmid pPICZaA 194 including α-factor signal and 6 histidine tag (Fig. S2b). The constructed plasmid pPICZα-StLAC2 was 195 transformed into P. pastorisKM71H, and the transformants grew well on the YPD medium containing 196 zeocin(100 μg/mL) was verified by PCR ( Fig. S1d) using primers of α-factor and AOX, indicating that 197 pPICZα-StLAC2 was integrated into the AOX sites of P. pastoris. confirmed by Western blot (Fig. 2b). The molecular weight of recombinant laccaseStlac2 was about 100 203 kDa compared with the protein marker, but Stlac2 (554 amino acids) had the predicted molecular weight 204 of 61.64 kDa. In order to exclude the possibility of protein dimer, the reducing agents of 20% β-mercapto 205 ethanol and 1.0 mol/L DTT was added to depolymerize. The results showed that the single apparent band 206 did not change (data not shown), meaning that recombinant laccase Stlac2 was monomer with a much 207 larger molecular weight than predicted. the plasmid pET32a without signal peptide (Fig. S2c). The constructed plasmid pET32-StLAC2 was 211 transformed into E. coli Rossetta (DE3), and the transformants growing on the LB medium containing 212 ampicillin (50 μg/mL) was verified by PCR (Fig. S1e) using T7 primers. 213 Recombinant laccase Stlac2 with an apparent molecular weight about 80 kDa with histidines tag but not 214 signal peptide was detected in the whole-cell extract of cultural cells after IPTG induction compared with 215 non-induction (Fig. 3a). The recombinant laccase was purified by Ni-IDA (Fig. 3b), and the specific band 216 of Stlac2 was present in the precipitate, indicating that the expressed protein was in the inclusion bodies 217 of about 70% of purity. The target protein was purified by denaturation and renaturation from inclusion 218 bodies with the purity of 87% and the concentration of 1.03 mg/mL. The activity of purified recombinant 219 Stlac2 using ABTS as substrate at pH4.5 citrate buffer and room temperature was 28.23 U/mg. 220

Glycosylation of Stlac2 recombinant protein in P. pastoris 221
When the activity of Stlac2 recombinant protein in P. pastoris was detected using ABTS, the color of 222 reaction mixture did not change, compared with the same concentration of protein of Stlac2 recombinant 223 protein in E. coli and crude protein from S. turcica, which changed the color of ABTS to dark green and 224 increased over time (Fig. 4a). The glycosylation of Stlac2 recombinant protein in P. pastoris was detected 225 (Fig. 4b). After the deglycosylation using PNGase F, there were two bands with molecular weight of 226 approximately 80 kDa and 90 kDa, respectively, compared with 100 kDa before deglycosylation, 227 suggesting that recombinant Stlac2 in P. pastoris was glycosylated at two sites of Asn-X-Ser/Thr. 228 7 potential N-glycosylation (Asn-X-Ser/Thr) sites were detected by NetNGlyc 1.0 server, including Asn4, 229 Asn97, Asn206, Asn373, Asn382, Asn383, and Asn475. From these, Asn382 was indicated as non-230 glycosylated with the 'potential' score is less than the default threshold of 0.5. In order to identify the glycosylation sites affecting laccase activity, the stained band of recombinant laccase Stlac2 expressed 232 in P. pastoris was identified by peptide mass fingerprinting (ESI-MS/MS analysis) and positive peptide 233 was located in the sequence of Stlac2, as shown in Fig.5. The peptides containing the predicted 234 glycosylation sites Asn206, Asn373, and Asn475 was detected with no glycosylation, but the peptides 235 containing Asn4, Asn97, Asn382, and Asn383 were not covered. Stlac2 and the laccases of which N-236 glycosylation sites were known were aligned to analyze potential glycosylation sites of fungal laccase.  Three-dimensional structural simulation of Stlac2 showed that, when the carbohydrate chain of N-acetyl-240 D-glucosamine attaches to Asn97, it may block the egress for the water molecules, resulting from the 241 reduction of the dioxygen molecule, potentially affecting the activity of laccase. 242

The activity of the recombinant laccase under different temperatures, pH values, and ions 243
The oxidation of ABTS by the purified laccase in 0.1 M citrate buffer was evaluated at different levels 244 of pH, temperature, and metal ions. The purified recombinant laccase showed the highest activity at pH 245 4.5 (Fig. 6a) and 60 ºC (Fig. 6b). The effects of different concentrations of metal ions on purified laccase 246 activity were tested by evaluating oxidation of ABTS at the optimum pH and temperature for 5 min in 247 the presence of different ions (Fig. 6c). The results showed that the activity of Stlac2 was relatively stable 248 in the presence of Cu 2+ , which is a structural component of the catalytic center. The relative activity of 249 Stlac2 increased to 315% when the concentration of Fe 3+ increased up to 10 mM. 5 mM Mn 2+ increased 250 the laccase activity, while the activity decreased when the concentration of Na + , Mg 2+ , Ca 2+ , and Mn 2+ 251 was 10 mM.
symptoms. The decolorization activity of recombinant Stlac2 on malachite green was investigated to 255 demonstrate its industrial applicability. The decolorization of recombinant Stlac2 were analyzed after 256 being treated at 20, 50, 100, and 200 mg/L malachite green for 15 min, 30 min, 45 min, 1 h, 1.5 h, and 3 257 h (Fig.7). Stlac2 displays an excellent decolorization activity without any redox mediators under 20 mg/L 258 malachite green, as Stlac2 decolorized 67.08% MG in 15 min and more than 70% of 50 mg/L malachite 259 green after 3 h of incubation. With the increase of MG concentration, the decolorization rates showed the 260 trend of gradual reduction. The decolorization efficiency was not significantly different when the 261 concentration of MG was more than 100 mg/L. 262

Discussion 263
In this work, using Native-PAGE and ESI-MS/MS identification, there are 3 laccase isozymes produced 264 by S. turcica, while 9 laccase-like multicopper oxidases were found in the genome using a Hidden highly expressed with different degrees when detected by q-PCR. They have the similarly predicted 278 molecular weight of 60.83-68.10 kDa and pI, signifying that it may be difficult to separate. Native-PAGE 279 was used to separate isozymes here to find out the active laccase in S. turcica. The results showed that in 280 S. turcica, the bands are the same when stained with ABTS and DMP. One band was identified as Stlac2 281 and Stlac6. Stlac2 has a lower predicted molecular weight of 61.64 kDa and low predicted pI of 5.00, 282 and it was mostly secreted intracellularly. The other band was identified as Stlac1 and Slac6, which were 283 detected both intracellularly and extracellularly. Stlac1 and Stlac6 shared the highest identity of 48.70% 284 in 9 laccase-like multicopper oxidases of S. turcica; had a similar predicted molecular weight of 65.73 285 kDa and 65.85 kDa, respectively. Since predicted pI of Stlac6 is 5.07, lower than 5.65 of Stlac1, and 286 predicted molecular weight larger than Stlac2, the band of Stlac6 should be detected between Stlac1 and 287 were not detected. Different cultural conditions and additions of aromatic compounds may also lead to 290 differential production of laccase isozymes (Kumar et al. 2017). Thus, further understanding of laccase 291 isozymes in S. turcica for basic and applied purposes will be investigated in future studies. 292 Stlac2 was heterologously expressed in eukaryotic expression system P. pastoris KM71H, and the results 293 showed that recombinant Stlac2 is incapable of oxidizing the substrates ABTS and DMP (data of DMP 294 was not showed). Most fungal laccases are reported as glycoprotein with a carbohydrate content of 10%-295 increased the thermostability of fungi enzymes, but depending on the glycosylation position it might lead 299 to increased or reduced catalytic activity(Ergün and Çalık 2015). In our case, recombinant Stlac2 can be 300 expressed in P. pastoris, detected extracellularly, but it is inactive. The higher molecular weight of 301 recombinant Stlac2 than predicted is attributable to the glycosylation status. There are a predicted 7 302 potential N-glycosylation, and the glycosylation of 3 potential glycosylation sites were excluded by ESI-303 MS/MS analysis of recombinant Stlac2. The glycosylation site of Asn382 is conserved compared with 304 the glycosylation sites of the known laccase proteins, revealing that its glycosylation has no obvious 305 influence on enzyme activity. When a three-dimensional structural simulation of Stlac2 was used for the 306 analysis, it was shown that the glycosylation of Asn97 the undetected glycosylation site may result in the 307 depression of laccase activity by blocking the release route of water molecules in the channel of dioxygen 308 reduction (Bento et al. 2005). When the prokaryotic expression system E. coli was used, recombinant 309 protein without glycosylation achieved the activation of ABTS and even the degradation of MG, which 310 seems like glycosylation of Stlac2 inessential for function. laccase, Stlac2 has similar activation with temperature and pH. Stlac2 has more tolerance to metal ions 322 like Na + , Mg 2+ , and Ca 2+ with the concentration of 5 mM, inhibiting the activity of Stlac4. However, 323 when the concentration is increased to 10 mM, the activity of Stlac2 was decreased, similar to Stlac4. 324 The effect of Cu 2+ on Stlac2 was not as obvious as Stlac4. When 10 mMCu 2+ was added, the relative 325 activity of Stlac2 was 108.0%, while that of Stlac4 was 217.4%. The activity of Stlac2 is increased by 326 adding up to 10 mM of Fe 3+ mM, which is consistent with Stlac4, but contrary to other laccases where

Ethical approval 346
This article does not contain any studies with human participants or animals performed by any of the 347 authors. 348

Competing interests 349
The authors declare that they have no competing interests. The results of laccase isozymes confirmed by ESI-MS/MS. 470 a The optimal pH was determined at pH from 3.0 to 6.5 at room temperature. Enzyme activity is 503 plotted as percentage (% relative activity) relative to the maximum value. 504 b The optimal temperature was measured with temperatures from 30 to 80 ºC in 0.1 M citrate-505 phosphate buffer of pH 4.5. Enzyme activity is plotted as percentage (% relative activity) 506 relative to the maximum value. 507 c Concentration (1, 5, and 10 mM) of metal ions (Na + , Mg 2+ , Ca 2+ , Mn 2+ , Fe 3+ , and Cu 2+ ) on the activity is plotted as percentage (% relative activity) relative to the value of samples without 510 metal ions. 511 Error bars correspond to standard error of mean. 512

Fig. 7 Decolorization of MG by recombinant Stlac2. 513
Error bars correspond to standard error of mean for triplicates. 514 The construction of pPICZα-StLAC2 expression vector . 518 c The construction of pET32-StLAC2 expression vector. 519 d The confirmation of transformants with α-factor primer and 3'AOX primer. 520 e The confirmation of transformants with T7 primers.   T  A  T  L   I  I  I  I  I   179  1GW0_A  220  3PPS_A  209  3SQR_A  165  5LM8_A 175 StLAC2