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Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis
Journal of Bioscience and Bioengineering VOL. xxx No. xxx, xxx, xxxx www.elsevier.com/locate/jbiosc
Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis Kai-Zhong Xu, Hao-Ran Wang, Ya-Jing Wang, Jing Xia, Hui Ma, Yu-Jie Cai, Xiang-Ru Liao, and Zheng-Bing Guan* The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China Received 28 June 2019; accepted 29 September 2019 Available online xxx
CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7e11. Mutant S208G/F227A showed optimal temperature at 80 C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Site-directed mutagenesis; CotA-laccase; Bacillus pumilus; Catalytic efficiency; Dye decolorization]
Acidic dyes are widely used in wool dyeing (1). The pH of dyeing wastewater ranges from 2.5 to 3.5 when wool fabric is dyed with strong acid dyes under conventional process conditions (2). What’s more, acidic dyeing wastewater have great environmental pollution (3). The traditional process of treating acidic dyeing wastewater is physical adsorption treatment (4e6). Bio-enzymatic decolorization is a new method of dye decolorization (7). At present, there are many reports about the enzymatic decolorization of acid dye wastewater. Horseradish peroxidase enzyme is used to treat Acid blue 225 and Acid violet 109 (8). Peroxidase from Coprinus cinereus NBRC 30628 has good decolorization effect on Acid orange 7 (9). The decolorization of Acid blue 80 by laccase DENILITE II shows remarkable effect (10). Among these enzymes, CotA-laccase has attracted much attention because of its wide range of substrates and good treatment effect (11). Laccases (EC 1.10.3.2) are multi-copper oxidases which can catalyze many phenolic compounds and oxidize aromatic compounds. They belong to the blue multi-copper oxidase family and they are regarded as green and environmentally friendly products because they use oxygen molecules as electron acceptors during oxidation reaction and convert them into H2O without any byproducts (12). Accordingly, laccases are important biocatalysts for environmental pollution treatment (13). CotA-laccases from Bacillus perform excellent thermal stability and alkaline endurance in particular (14). However, the applications of CotA-laccase are limited by their low catalytic efficiency relative to fungal laccases (15). The catalytic
efficiency of CotA-laccase from Bacillus subtilis PAP1158 with ABTS as substrate is 611.89 L$mmol1$s1 (16). On the other hand, the catalytic efficiency of fungal laccase from Pycnoporus sanguineus RP15 is 3140.1 L$mmol1$s1 (17). Many studies have reported the modification of CotA-laccases by genetic engineering. Gupta and Farinas (18) used saturation mutagenesis to improve the substrate specificity of CotA-laccase from B. subtilis. A recent study showed that Arg416 of CotA-laccase from B. subtilis plays an irreplaceable role in substrate oxidation (19). The D501G mutation of CotA-laccase from Bacillus amyloliquefaciens presents excellent stability and catalytic efficiency (20). Chen et al. (21) constructed a mutant L386W/G417L by site-directed mutagenesis to overcome the low catalytic efficiency of CotA-laccase of Bacillus pumilus. Modifying natural CotAlaccases via site-directed mutagenesis has become a research hotspot. At present, only a few reports indicate that the role of S210G is related to the catalytic efficiency of CotA-laccase (22). There is no report on the effect of mutations at 208 and 227 of CotA-laccase. Our laboratory had isolated a novel CotA-laccase gene from B. pumilus W3. This CotA-laccase can tolerate alkaline environment and the optimum reaction temperature is 80 C when ABTS is used as substrate (23). In order to improve its catalytic efficiency, the site-directed mutagenesis was invited to modify the B. pumilus W3 CotA-laccase. This study compared the homologous sequence between B. subtilis and B. pumilus W3 CotA-laccase and used sitedirected mutagenesis to change the amino acid residues. In this study, the effects of S208G and F227A mutations on the catalytic efficiency of CotA-laccase were studied.
* Corresponding author. Tel./fax: þ86 510 8532 7725. E-mail address: [email protected] (Z.-B. Guan).
1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.09.020
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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Chemical reagents 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) was purchased from SigmaeAldrich (St. Louis, MO, USA). Ampicillin (Amp), isopropyl b-D-thiogalactoside (IPTG) were purchased from Takara (Dalian, China). TransStarRFastPfu PCR SuperMix and gel extraction kit were purchased from TransGen (Peking, China). Methyl red was purchased from Klamar (Shanghai, China). Acid blue 129 and malachite green were purchased from Chemsky (Shanghai, China). Methyl blue was purchased from HWRK Chem (Peking, China). All chemical reagents were analytically pure. Bacterial strains, plasmid, and growth conditions The CotA-laccase gene (GenBank: AGO57931.1) of B. pumilus W3 was cloned into pColdII plasmids. The strain B. pumilus W3 was stored at the China Center for Type Culture Collection (CCTCC No. M2015018). The cloning strain Escherichia coli DH5a was purchased from Takara. The expression strain E. coli BL21(DE3) was purchased from Miaolingbio (Wuhan, China). All primers were synthesized by BGI (Shenzhen, China). LuriaeBertani (LB) medium was adopted to culture strains. All strains were grown at 37 C and induced expression at 15 C. Bioinformatics analysis The CotA-laccase from B. pumilus W3 shared 67.38% identity with the CotA-laccase from B. subtilis (PDB code: 1GSK) (Fig. 1). The sequence alignment of amino acid residues was compared by DNAMAN software from Lynnon Biosoft (San Ramon, CA, USA). The 3D structure of CotA-laccases was built on the Swiss Model (https://www.swissmodel.expasy.org). Molecular docking was performed by Discovery Studio and AutoDock software (24,25). Site-directed mutagenesis The wide-type laccase gene was used as template and the site-directed mutagenesis was based on the QuikChange method (26). All site directed primers were designed and synthesized (Table 1). The PCR products were incubated at 37 C with Dpn I enzyme. The Dpn I is a kind of restriction nuclease which can cut the methylated DNA chain specificity. E. coli DH5a was used as host to subclone PCR products. Mutant CotA-laccase genes were confirmed by DNA sequencing. The correct plasmids were transformed into E. coli BL21(DE3) to express recombinant protein. Culture conditions and purification The recombinant strains were incubated in 5 ml LB medium at 37 C. Amp (100 mg/ml) was adding into the medium. Transfer 1 ml of bacterial solution to 50 ml of fresh LB medium after 10 h of shaking culture. Continue to culture until the optical density at 600 nm reaches 0.3e0.5. The medium was induced with IPTG (0.1 mM) after incubating for 30 min at 15 C (27). Meanwhile, CuSO4 (0.25 mM) was added into the medium. Keep culturing at 15 C for 24 h. The recombinant strains were harvested by centrifugation (10 min, 5000 rpm, 4 C) and resuspended in 20 mM phosphate buffer (pH 7.0). The cells were smashed by ultrasonic on the ice. Then removal of cell debris in the broken liquid by centrifugation (15 min, 8000 rpm, 4 C). The suspension was heated in water bath at 70 C for 10 min (28). In order to purification, a His6 tag was fused to the N-terminus of the CotAlaccase. His6 tag is a small tag that barely had bad effect on target protein (29). Consequently, this recombinant laccase could be purified by Niþ-affinity chromatography. The Ni-NTA column was purchased from GE Healthcare (Uppsala, Sweden). Ni-NTA column was equilibrated with 20 mM phosphate buffer (pH 7.0) containing imidazole (20 mM) and NaCl (500 mM). Serial gradient elution buffer solutions [phosphate buffer (20 mM) containing imidazole (0e500 mM) and NaCl (500 mM)] were used to wash the column. The proteins which corresponded to penetration peak and eluting peak were measured activity by using ABTS as substrate (30). Then the mixture containing CotA-laccases was desalted by Amicon Ultra-15 (Merck, Shanghai, China) (31). The concentration of CotA-laccase protein was tested by Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) (32). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the purity of purified CotA-laccase protein (33). Enzyme activity assay The activity of CotA-laccase was measured at 50 C. ABTS (0.3 mM) was used as substrate. Citrate/phosphate (pH 3.5) was used as pH buffer solution. The ultraviolet absorbance of ABTS was 420 nm (ε ¼ 36,000 M1cm1) (34). The assays were measured at 50 C and pH 3.5 using different concentrations (0.02e0.5 mM) of ABTS as substrates when calculated the kinetic parameters. One unit was defined as 1 mmol of substrate was oxidized by how many amounts of enzyme every minute. Kinetic parameters Km and kcat were calculated by MichaeliseMenten equation and LineweavereBurk plot (35). All assays were performed at least in triplicate.
Optimum pH and pH stability The optimum pH was measured at 50 C. The tests used ABTS (final concentration 0.5 mM) as substrate and performed at series pH values (pH 2.0e6.0) of citrate/phosphate buffer. The purified CotA-laccase was incubated at 4 C and different pH values (pH 3.0e12.0) for 10 h before measuring the residual activity. The residual activity test used ABTS as substrate. All assays were performed at least in triplicate. Optimum temperature and thermal stability The optimum temperature of CotA-laccase activity was measured at different temperatures (20e90 C). The tests used ABTS as substrate with citrate/phosphate buffer (pH 3.5). The residual activity was determined at 50 C and pH 3.5 using ABTS as substrate. The CotAlaccases were incubated at different temperatures (60 C, 70 C, 80 C) and their residual activity was measured at intervals (30 min, 60 min, 90 min, 180 min and 300 min). The residual activity was measured at 90 C after different times (5 min, 10 min, 15 min, 20 min, 30 min and 60 min). All assays were performed at least in triplicate. Dye decolorization Methyl red, Acid blue 129, malachite green and methyl blue were used to assess the dye decolorization ability of wild-type CotA-laccase and mutant CotA-laccases. The chemical structures of dyes used in this experiment are shown in Fig. S1 . Enzyme (5 U/ml) was added into a 3 ml reaction system containing citrate/phosphate buffer (100 mM, pH 3.5) and 0.025 mg/ml of dyes. ABTS (0.5 mM) was used as redox mediator. The system was incubated at 50 C for 5 h. Contrast with the system with heat-inactivated enzymes and without ABTS. The decolorization rate was calculated spectrophotometrically as the relative decrease in absorbance at each maximal absorbance wavelength of dyes (28). The test maximal absorbance wavelengths for methyl red, Acid blue 129, malachite green and methyl blue were 410 nm (36), 629 nm (37), 619 nm and 664 nm, respectively (38,39). Dye decolorization was calculated by Eq. 1: Relative decolorization ð%Þ ¼ 100
ðA0 A1 Þ A0
(1)
where A1 is the absorbance of the reaction system after incubation for 5 h and A0 is the absorbance of the control group after incubation for 5 h.
RESULTS Design, cloning, and expression The 3D structure of CotAlaccase was built by homologous on Swiss Model and molecular docking with ABTS to study the interactions between laccase and ABTS. The result of molecular docking showed that ABTS had hydrogen bonds with amino acid residues of wild-type CotAlaccase like Asn211, Asn212, Phe227, Phe228, Cys229. In addition, ABTS could form conjugation forces with amino acid residues Pro210, Pro226 and Phe227 (Fig. 2B). The homologous between B. pumilus W3 CotA-laccase and B. subtilis CotA-laccase (PDB code: 1GSK) showed that the amino acid at position 227 was different between the two CotA-laccases. The position 227 of B. subtilis CotA-laccase was Ala while the corresponding position of B. pumilus W3 CotA-laccase was Phe (Fig. 1). Phe227 had more steric effects with ABTS than Ala227. Ala227 had contribution to the stable binding of benzothiazole with ABTS (Fig. 2A). Besides, Ser208 was closed to the active pocket. Ser208 located in a region rich in Pro fragments. Increasing the flexibility of the Pro area may conducive to the combination of enzymes and substrates (40). The amino Ser208 from wild-type CotA-laccase did not show any interactions with ABTS (Fig. 2B). Amino Gly208 participates in the active hydrophilic part of the benzothiazole group, which binds with ABTS molecules (Fig. 3B). Therefore, we mutated Phe227 into Ala227 and turn Ser208 into Gly208 by sitedirected mutagenesis. In this study the mutants S208G, F227A,
FIG. 1. Alignment with the CotA-laccase sequences of B. pumilus W3 and B. subtilis (PDB code: 1GSK).
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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TABLE 1. Primer sequences. Gene name S208G S208G F227A F227A S208G/F227A S208G/F227A a
Description Ser208 replaced by Gly Ser208 replaced by Gly Phe227 replaced by Ala Phe227 replaced by Ala Ser208 replaced by Gly and Phe227 replaced by Ala Ser208 replaced by Gly and Phe227 replaced by Ala
Nucleotide sequences (50 -30 )a
Primer S208G-F S208G-R F227A-F F227A-R SF-F SF-R
0
5 -CATTCCAGGAGGATGGCGCACTATTTTATCCAG-30 50 -GTTATTTGGTCTACCTGGATAAAATAGTGC GCCA-30 50 -TAACACACCAGAAGATAGTGACCTTCCAGATCCCTCTA TCGTGCCAGC-30 50 -GTTTCCCCGCAAAAAGCTGGCACGATAGAGGGATCTGGAA GGTCA-30 50 -TAACACACCAGAAGATAGTGACCTTCCAGATCCCTCTA TCGTGCCAGC-30 50 -GTTTCCCCGCAAAAAGCTGGCACGATAGAGGGATCTGGAA GGTCA-30
Bold and underlined letters were mutant sites and cloning sites.
and S208G/F227A were successfully constructed by site-directed mutagenesis. Purification of CotA-laccases All enzymes were purified by nickel ion affinity chromatography in order to compare the property of mutants and wild-type CotA-laccase. The purified enzyme molecular weight was w65 kDa (Fig. 4). Kinetic parameters of CotA-laccases The kcat and Km values of wild-type CotA-laccase and mutants to ABTS are listed in Table 2. The catalytic efficiency (kcat/Km) of S208G increased by 1.3 times in comparison with that of the wild-type laccase. The catalytic efficiency of F227A increased by 2.11-fold in comparison with that of the wild-type laccase. Moreover, the kcat/Km value of the double mutant strain S208G/F227A increased by 5.13-fold. The Km values of the mutants to ABTS were 0.172, 0.123 and 0.073 mM, and that of the wild-type was 0.241 mM. Effect of pH on enzyme activity and stability The optimal pH values of wild-type CotA-laccase and mutants were determined to be about 3.5 using ABTS as substrate (Fig. 5A). The wild-type CotA-laccase still had more than 90% residual activity after incubation for 10 h at pH 6e10. The residual activity of mutant CotA-laccase was more than 80% after incubated for 10 h at pH 7e11 (Fig. 5B). Effect of temperature on enzyme activity and stability The wild-type CotA-laccase and mutant S208G/F227A had the same optimal temperature at 80 C with ABTS as substrate. The mutant S208G and F227A held the same optimal temperature at 70 C (Fig. 6A). The half-lives of wild-type laccases were 2.31, 1.52, and 0.83 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The residual activity of wild-type laccase was more
than 70% after incubated 5 min at 90 C. The half-lives of S208G were 1.57, 1.34, and 0.56 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The half-lives of F227A were 1.12, 0.47, and 0.38 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The half-lives of S208G/F227A were 1.35, 0.51 and 0.44 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. Dye decolorization The purified enzymes were compared for the decolorization ability using four dyes (methyl red, Acid blue 129, malachite green and methyl blue). This study was carried out with mediator ABTS. Methyl red, Acid blue 129, malachite green and methyl blue were decolorized by CotAlaccases (Fig. 7A). The decolorization rates of methyl red by double mutant S208G/F227A reached 69.8%. Compared with the wild-type CotA-laccase, the decolorization rate of methyl red increased by 26%. The decolorization ability of the mutant (F227A and S208G/F227A) to methyl red was obviously improved compared with the wild-type. CotA-laccase can decolorize Acid blue 129 and malachite green efficiently without mediator ABTS (Fig. 7B). Mutant S208G/F227A still had nearly 80% relative activity after incubation in methyl red dye decolorization system for 5 h, while wild-type had 72% relative activity (Fig. 7C). DISCUSSION The selection of mutation sites is a key point in site-directed mutagenesis (41). In this study, molecular docking and homology analysis were used to determine the mutation sites. As shown in Fig. 3A, Ser208 was closed to the active pocket. Ser208 located in a region rich in Pro fragments. It had been mentioned that increasing the flexibility of the Pro area may
FIG. 2. The 3D model (A) and 2D pattern (B) of the key amino acid residues of wild-type CotA-ABTS. (A) The red structure indicates ABTS and the blue structure shows amino acid residues around ABTS. (B) The interactions between wild-type CotA-laccase and ABTS.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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FIG. 3. The 3D model (A) and 2D pattern (B) of the key amino acid residues of mutant CotA-ABTS. (A) The red structure indicates ABTS and the blue structure shows amino acid residues around ABTS. (B) The interactions between mutant S208G/F227A and ABTS.
conducive to the combination of enzymes and substrates (22). What is more, ABTS had a hydrophilic tendency (42) while Gly208 participates in the active hydrophilic part of the benzothiazole group, which bound with ABTS molecules (Fig. 3B). ABTS could form van der Waals force with Gly208, but it had no direct interaction with Ser208. The Km value of CotA-laccase to ABTS decreased from 0.241 to 0.172 after the amino acid at 208 position was mutated from Ser to Gly. This indicated that the enzyme affinity of S208G to ABTS increased. Besides, amino acid at 227 was also in an important position. Both Phe227 and Ala227 could form Pi-Alkyl interaction with ABTS. Ala227 could also form hydrogen bonds with ABTS. The side chain benzene ring of Phe227 was close to the ABTS binding channel, which may affect the binding and release of ABTS to laccase. Ala227 had less steric effects with ABTS than Phe227 (Fig. 3B). The Km value of CotA-laccase to ABTS decreased from 0.241 to 0.123 after the amino acid at 227 position was mutated from Phe to Ala. This indicated that Ala227 may have contribution to the stable binding of benzothiazole with ABTS.
Current studies on laccase are mostly about the first and third copper ion centers (15,43,44), while less studies about the second copper ion centers (40,45,46). ABTS binding pocket is located in the second copper ion center (47). In this study, mutant sites (Ser208 and phe227) are near ABTS binding pocket. In this study, the mutants S208G, F227A, and S208G/F227A were constructed by site-directed mutagenesis to improve their catalytic efficiency. The molecular weight of the mutants and the wild-type laccase were all w65 kDa, which were consistent with laccase from B. subtilis WD23 (48) and Cyanobacteria (49). The kcat/Km value of the double mutant strain S208G/F227A increased compared with the laccase reported by DONG (50). The optimum pH of the mutants and the wild-type was 3.5 using ABTS as substrate. Showing that mutants had better catalytic activity under acidic conditions compared with laccase from Agaricus bisporus CU13 exhibited optimum pH at 5.0 using ABTS as substrate (51). The thermal stability of the mutant laccase was lower than that of the wild-type. The decrease of intermolecular van der Waals force and the change of conjugation force after mutation may be the reasons for the lower thermal stability (52e54). And the increase of molecular flexibility may also have bad impact on thermal stability (55). But the mutants still had higher than 40% residual activity after incubating at 90 C for 5 min. These results indicate that mutants have thermal stability similar to laccases from B. subtilis cjp3 (56) and from Pandoraea sp. ISTKB (57). Despite the decline in stability, mutants can still meet the needs of industrial applications. In this study, the decolorization effect of wild-type laccase on methyl red was lower than that of mutant S208G/F227A (Fig. 7A). This might be due to the increased specificity of the mutant S208G/F227A to methyl red. The molecular docking results of CotA-laccase and methyl red showed that
TABLE 2. Kinetic parameters for CotA-laccase using ABTS as substrate. Enzyme
FIG. 4. SDS-PAGE of cell fragmentation supernatant and purified CotA-laccase. Lane M is protein marker; lanes 1, 2 and 3 show the cell transformed with empty pColdII plasmids, cell transformed with pColdII-CotA and the purified CotA-laccase, respectively. The molecular weight of CotA-laccase was w65 kDa.
WT S208G F227A S208G/F227A
kcat (s1)
Km (mM)a
kcat/Km (s1 mM1)
40.26 38.46 43.44 56.52
0.241 0.172 0.123 0.073
167.05 223.60 353.17 856.97
a Laccase activity was calculated at 50 C with 100 mM citrate/phosphate buffer (pH 3.5). ABTS was chosen as substrate. Km and kcat were determined by MichaeliseMenten equation and LineweavereBurk plot.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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FIG. 5. Effect of pH on the activity (A) and stability (B) of the purified CotA-laccase enzymes (wild-type, S208G, F227A, and S208G/F227A) at 50 C using ABTS as substrate. (A) The test on enzyme activity was measured at 50 C. The tests used ABTS (0.5 mM) as substrate and performed at a series of different pH values (pH 2.0e7.0) with citrate/phosphate buffer. (B) The purified CotA-laccase was measured the residual activity incubated after incubating at 4 C and different pH values (pH 3.0e12.0) for 10 h before. The residual activity was calculated at 50 C and pH 3.5 using ABTS as substrate.
FIG. 6. Effect of temperature on the activity (A) and stability (B) of the purified CotA-laccases (wild-type, S208G, F227A, and S208G/F227A) using ABTS as substrate at pH 3.5. (A) CotA-laccase activity was measured at different temperatures (20e90 C). The tests used ABTS as substrate with citrate/phosphate buffer (pH 3.5). (B) The purified enzyme was incubated at four different temperatures (60 C, 70 C, 80 C, and 90 C) before measuring the residual activity. The residual activity was determined at 50 C and pH 3.5 using ABTS as substrate.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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J. BIOSCI. BIOENG., In conclusion, the catalytic efficiency of CotA-laccase was improved by site-directed mutagenesis. Results of this study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis. The expression of CotAlaccase during high-density fermentation will continue to be studied in the future. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.09.020.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31472003), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.
References
FIG. 7. (A) Decolorization of synthetic dyes (0.025 mg/ml) by the wild-type and mutants (S208G, F227A, and S208G/F227A) were determined at pH 3.5 and 50 C for 5 h with ABTS as mediator. (B) Decolorization of synthetic dyes (0.025 mg/ml) by the wildtype and mutants (S208G, F227A, and S208G/F227A) were determined at pH 3.5 and 50 C for 5 h without mediator. (C) Tolerance of enzyme in different dyes. The purified enzyme was incubated at reaction system containing citrate/phosphate buffer (100 mM, pH 3.5) and 0.025 mg/ml of dyes for 5 h at 50 C. The residual enzyme activity was measured with ABTS as substrate.
the active position and hydrogen bonds of CotA-laccase rotated after the mutation (Figs. 2 and 3). The methyl red molecule could enter the catalytic pathway more easily when the amino acid at 227 mutated from Phe to Ala. Besides, mutant S208G/F227A could form more hydrogen bonds with methyl red, which made their binding more stable (Fig. S3).
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Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis Kai-Zhong Xu, Hao-Ran Wang, Ya-Jing Wang, Jing Xia, Hui Ma, Yu-Jie Cai, Xiang-Ru Liao, and Zheng-Bing Guan* The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China Received 28 June 2019; accepted 29 September 2019 Available online xxx
CotA-laccases are potential enzymes that are widely used in decolorization of dyes and degradation of toxic substances. In this study, a novel CotA-laccase gene from Bacillus pumilus W3 was applied for rational design. After a series of site-directed genetic mutations, the mutant S208G/F227A showed a 5.1-fold higher catalytic efficiency (kcat/Km) than the wild-type CotA-laccase did. The optimal pH of S208G/F227A was 3.5 with ABTS as substrate. The residual activity of mutant S208G/F227A was more than 80% after incubated for 10 h at pH 7e11. Mutant S208G/F227A showed optimal temperature at 80 C with ABTS as substrate. The thermal stability of mutant laccase S208G/F227A was lower than that of wild-type CotA-laccase. This study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis. Ó 2019, The Society for Biotechnology, Japan. All rights reserved. [Key words: Site-directed mutagenesis; CotA-laccase; Bacillus pumilus; Catalytic efficiency; Dye decolorization]
Acidic dyes are widely used in wool dyeing (1). The pH of dyeing wastewater ranges from 2.5 to 3.5 when wool fabric is dyed with strong acid dyes under conventional process conditions (2). What’s more, acidic dyeing wastewater have great environmental pollution (3). The traditional process of treating acidic dyeing wastewater is physical adsorption treatment (4e6). Bio-enzymatic decolorization is a new method of dye decolorization (7). At present, there are many reports about the enzymatic decolorization of acid dye wastewater. Horseradish peroxidase enzyme is used to treat Acid blue 225 and Acid violet 109 (8). Peroxidase from Coprinus cinereus NBRC 30628 has good decolorization effect on Acid orange 7 (9). The decolorization of Acid blue 80 by laccase DENILITE II shows remarkable effect (10). Among these enzymes, CotA-laccase has attracted much attention because of its wide range of substrates and good treatment effect (11). Laccases (EC 1.10.3.2) are multi-copper oxidases which can catalyze many phenolic compounds and oxidize aromatic compounds. They belong to the blue multi-copper oxidase family and they are regarded as green and environmentally friendly products because they use oxygen molecules as electron acceptors during oxidation reaction and convert them into H2O without any byproducts (12). Accordingly, laccases are important biocatalysts for environmental pollution treatment (13). CotA-laccases from Bacillus perform excellent thermal stability and alkaline endurance in particular (14). However, the applications of CotA-laccase are limited by their low catalytic efficiency relative to fungal laccases (15). The catalytic
efficiency of CotA-laccase from Bacillus subtilis PAP1158 with ABTS as substrate is 611.89 L$mmol1$s1 (16). On the other hand, the catalytic efficiency of fungal laccase from Pycnoporus sanguineus RP15 is 3140.1 L$mmol1$s1 (17). Many studies have reported the modification of CotA-laccases by genetic engineering. Gupta and Farinas (18) used saturation mutagenesis to improve the substrate specificity of CotA-laccase from B. subtilis. A recent study showed that Arg416 of CotA-laccase from B. subtilis plays an irreplaceable role in substrate oxidation (19). The D501G mutation of CotA-laccase from Bacillus amyloliquefaciens presents excellent stability and catalytic efficiency (20). Chen et al. (21) constructed a mutant L386W/G417L by site-directed mutagenesis to overcome the low catalytic efficiency of CotA-laccase of Bacillus pumilus. Modifying natural CotAlaccases via site-directed mutagenesis has become a research hotspot. At present, only a few reports indicate that the role of S210G is related to the catalytic efficiency of CotA-laccase (22). There is no report on the effect of mutations at 208 and 227 of CotA-laccase. Our laboratory had isolated a novel CotA-laccase gene from B. pumilus W3. This CotA-laccase can tolerate alkaline environment and the optimum reaction temperature is 80 C when ABTS is used as substrate (23). In order to improve its catalytic efficiency, the site-directed mutagenesis was invited to modify the B. pumilus W3 CotA-laccase. This study compared the homologous sequence between B. subtilis and B. pumilus W3 CotA-laccase and used sitedirected mutagenesis to change the amino acid residues. In this study, the effects of S208G and F227A mutations on the catalytic efficiency of CotA-laccase were studied.
* Corresponding author. Tel./fax: þ86 510 8532 7725. E-mail address: [email protected] (Z.-B. Guan).
1389-1723/$ e see front matter Ó 2019, The Society for Biotechnology, Japan. All rights reserved. https://doi.org/10.1016/j.jbiosc.2019.09.020
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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XU ET AL.
J. BIOSCI. BIOENG., MATERIALS AND METHODS
Chemical reagents 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; ABTS) was purchased from SigmaeAldrich (St. Louis, MO, USA). Ampicillin (Amp), isopropyl b-D-thiogalactoside (IPTG) were purchased from Takara (Dalian, China). TransStarRFastPfu PCR SuperMix and gel extraction kit were purchased from TransGen (Peking, China). Methyl red was purchased from Klamar (Shanghai, China). Acid blue 129 and malachite green were purchased from Chemsky (Shanghai, China). Methyl blue was purchased from HWRK Chem (Peking, China). All chemical reagents were analytically pure. Bacterial strains, plasmid, and growth conditions The CotA-laccase gene (GenBank: AGO57931.1) of B. pumilus W3 was cloned into pColdII plasmids. The strain B. pumilus W3 was stored at the China Center for Type Culture Collection (CCTCC No. M2015018). The cloning strain Escherichia coli DH5a was purchased from Takara. The expression strain E. coli BL21(DE3) was purchased from Miaolingbio (Wuhan, China). All primers were synthesized by BGI (Shenzhen, China). LuriaeBertani (LB) medium was adopted to culture strains. All strains were grown at 37 C and induced expression at 15 C. Bioinformatics analysis The CotA-laccase from B. pumilus W3 shared 67.38% identity with the CotA-laccase from B. subtilis (PDB code: 1GSK) (Fig. 1). The sequence alignment of amino acid residues was compared by DNAMAN software from Lynnon Biosoft (San Ramon, CA, USA). The 3D structure of CotA-laccases was built on the Swiss Model (https://www.swissmodel.expasy.org). Molecular docking was performed by Discovery Studio and AutoDock software (24,25). Site-directed mutagenesis The wide-type laccase gene was used as template and the site-directed mutagenesis was based on the QuikChange method (26). All site directed primers were designed and synthesized (Table 1). The PCR products were incubated at 37 C with Dpn I enzyme. The Dpn I is a kind of restriction nuclease which can cut the methylated DNA chain specificity. E. coli DH5a was used as host to subclone PCR products. Mutant CotA-laccase genes were confirmed by DNA sequencing. The correct plasmids were transformed into E. coli BL21(DE3) to express recombinant protein. Culture conditions and purification The recombinant strains were incubated in 5 ml LB medium at 37 C. Amp (100 mg/ml) was adding into the medium. Transfer 1 ml of bacterial solution to 50 ml of fresh LB medium after 10 h of shaking culture. Continue to culture until the optical density at 600 nm reaches 0.3e0.5. The medium was induced with IPTG (0.1 mM) after incubating for 30 min at 15 C (27). Meanwhile, CuSO4 (0.25 mM) was added into the medium. Keep culturing at 15 C for 24 h. The recombinant strains were harvested by centrifugation (10 min, 5000 rpm, 4 C) and resuspended in 20 mM phosphate buffer (pH 7.0). The cells were smashed by ultrasonic on the ice. Then removal of cell debris in the broken liquid by centrifugation (15 min, 8000 rpm, 4 C). The suspension was heated in water bath at 70 C for 10 min (28). In order to purification, a His6 tag was fused to the N-terminus of the CotAlaccase. His6 tag is a small tag that barely had bad effect on target protein (29). Consequently, this recombinant laccase could be purified by Niþ-affinity chromatography. The Ni-NTA column was purchased from GE Healthcare (Uppsala, Sweden). Ni-NTA column was equilibrated with 20 mM phosphate buffer (pH 7.0) containing imidazole (20 mM) and NaCl (500 mM). Serial gradient elution buffer solutions [phosphate buffer (20 mM) containing imidazole (0e500 mM) and NaCl (500 mM)] were used to wash the column. The proteins which corresponded to penetration peak and eluting peak were measured activity by using ABTS as substrate (30). Then the mixture containing CotA-laccases was desalted by Amicon Ultra-15 (Merck, Shanghai, China) (31). The concentration of CotA-laccase protein was tested by Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) (32). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the purity of purified CotA-laccase protein (33). Enzyme activity assay The activity of CotA-laccase was measured at 50 C. ABTS (0.3 mM) was used as substrate. Citrate/phosphate (pH 3.5) was used as pH buffer solution. The ultraviolet absorbance of ABTS was 420 nm (ε ¼ 36,000 M1cm1) (34). The assays were measured at 50 C and pH 3.5 using different concentrations (0.02e0.5 mM) of ABTS as substrates when calculated the kinetic parameters. One unit was defined as 1 mmol of substrate was oxidized by how many amounts of enzyme every minute. Kinetic parameters Km and kcat were calculated by MichaeliseMenten equation and LineweavereBurk plot (35). All assays were performed at least in triplicate.
Optimum pH and pH stability The optimum pH was measured at 50 C. The tests used ABTS (final concentration 0.5 mM) as substrate and performed at series pH values (pH 2.0e6.0) of citrate/phosphate buffer. The purified CotA-laccase was incubated at 4 C and different pH values (pH 3.0e12.0) for 10 h before measuring the residual activity. The residual activity test used ABTS as substrate. All assays were performed at least in triplicate. Optimum temperature and thermal stability The optimum temperature of CotA-laccase activity was measured at different temperatures (20e90 C). The tests used ABTS as substrate with citrate/phosphate buffer (pH 3.5). The residual activity was determined at 50 C and pH 3.5 using ABTS as substrate. The CotAlaccases were incubated at different temperatures (60 C, 70 C, 80 C) and their residual activity was measured at intervals (30 min, 60 min, 90 min, 180 min and 300 min). The residual activity was measured at 90 C after different times (5 min, 10 min, 15 min, 20 min, 30 min and 60 min). All assays were performed at least in triplicate. Dye decolorization Methyl red, Acid blue 129, malachite green and methyl blue were used to assess the dye decolorization ability of wild-type CotA-laccase and mutant CotA-laccases. The chemical structures of dyes used in this experiment are shown in Fig. S1 . Enzyme (5 U/ml) was added into a 3 ml reaction system containing citrate/phosphate buffer (100 mM, pH 3.5) and 0.025 mg/ml of dyes. ABTS (0.5 mM) was used as redox mediator. The system was incubated at 50 C for 5 h. Contrast with the system with heat-inactivated enzymes and without ABTS. The decolorization rate was calculated spectrophotometrically as the relative decrease in absorbance at each maximal absorbance wavelength of dyes (28). The test maximal absorbance wavelengths for methyl red, Acid blue 129, malachite green and methyl blue were 410 nm (36), 629 nm (37), 619 nm and 664 nm, respectively (38,39). Dye decolorization was calculated by Eq. 1: Relative decolorization ð%Þ ¼ 100
ðA0 A1 Þ A0
(1)
where A1 is the absorbance of the reaction system after incubation for 5 h and A0 is the absorbance of the control group after incubation for 5 h.
RESULTS Design, cloning, and expression The 3D structure of CotAlaccase was built by homologous on Swiss Model and molecular docking with ABTS to study the interactions between laccase and ABTS. The result of molecular docking showed that ABTS had hydrogen bonds with amino acid residues of wild-type CotAlaccase like Asn211, Asn212, Phe227, Phe228, Cys229. In addition, ABTS could form conjugation forces with amino acid residues Pro210, Pro226 and Phe227 (Fig. 2B). The homologous between B. pumilus W3 CotA-laccase and B. subtilis CotA-laccase (PDB code: 1GSK) showed that the amino acid at position 227 was different between the two CotA-laccases. The position 227 of B. subtilis CotA-laccase was Ala while the corresponding position of B. pumilus W3 CotA-laccase was Phe (Fig. 1). Phe227 had more steric effects with ABTS than Ala227. Ala227 had contribution to the stable binding of benzothiazole with ABTS (Fig. 2A). Besides, Ser208 was closed to the active pocket. Ser208 located in a region rich in Pro fragments. Increasing the flexibility of the Pro area may conducive to the combination of enzymes and substrates (40). The amino Ser208 from wild-type CotA-laccase did not show any interactions with ABTS (Fig. 2B). Amino Gly208 participates in the active hydrophilic part of the benzothiazole group, which binds with ABTS molecules (Fig. 3B). Therefore, we mutated Phe227 into Ala227 and turn Ser208 into Gly208 by sitedirected mutagenesis. In this study the mutants S208G, F227A,
FIG. 1. Alignment with the CotA-laccase sequences of B. pumilus W3 and B. subtilis (PDB code: 1GSK).
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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TABLE 1. Primer sequences. Gene name S208G S208G F227A F227A S208G/F227A S208G/F227A a
Description Ser208 replaced by Gly Ser208 replaced by Gly Phe227 replaced by Ala Phe227 replaced by Ala Ser208 replaced by Gly and Phe227 replaced by Ala Ser208 replaced by Gly and Phe227 replaced by Ala
Nucleotide sequences (50 -30 )a
Primer S208G-F S208G-R F227A-F F227A-R SF-F SF-R
0
5 -CATTCCAGGAGGATGGCGCACTATTTTATCCAG-30 50 -GTTATTTGGTCTACCTGGATAAAATAGTGC GCCA-30 50 -TAACACACCAGAAGATAGTGACCTTCCAGATCCCTCTA TCGTGCCAGC-30 50 -GTTTCCCCGCAAAAAGCTGGCACGATAGAGGGATCTGGAA GGTCA-30 50 -TAACACACCAGAAGATAGTGACCTTCCAGATCCCTCTA TCGTGCCAGC-30 50 -GTTTCCCCGCAAAAAGCTGGCACGATAGAGGGATCTGGAA GGTCA-30
Bold and underlined letters were mutant sites and cloning sites.
and S208G/F227A were successfully constructed by site-directed mutagenesis. Purification of CotA-laccases All enzymes were purified by nickel ion affinity chromatography in order to compare the property of mutants and wild-type CotA-laccase. The purified enzyme molecular weight was w65 kDa (Fig. 4). Kinetic parameters of CotA-laccases The kcat and Km values of wild-type CotA-laccase and mutants to ABTS are listed in Table 2. The catalytic efficiency (kcat/Km) of S208G increased by 1.3 times in comparison with that of the wild-type laccase. The catalytic efficiency of F227A increased by 2.11-fold in comparison with that of the wild-type laccase. Moreover, the kcat/Km value of the double mutant strain S208G/F227A increased by 5.13-fold. The Km values of the mutants to ABTS were 0.172, 0.123 and 0.073 mM, and that of the wild-type was 0.241 mM. Effect of pH on enzyme activity and stability The optimal pH values of wild-type CotA-laccase and mutants were determined to be about 3.5 using ABTS as substrate (Fig. 5A). The wild-type CotA-laccase still had more than 90% residual activity after incubation for 10 h at pH 6e10. The residual activity of mutant CotA-laccase was more than 80% after incubated for 10 h at pH 7e11 (Fig. 5B). Effect of temperature on enzyme activity and stability The wild-type CotA-laccase and mutant S208G/F227A had the same optimal temperature at 80 C with ABTS as substrate. The mutant S208G and F227A held the same optimal temperature at 70 C (Fig. 6A). The half-lives of wild-type laccases were 2.31, 1.52, and 0.83 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The residual activity of wild-type laccase was more
than 70% after incubated 5 min at 90 C. The half-lives of S208G were 1.57, 1.34, and 0.56 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The half-lives of F227A were 1.12, 0.47, and 0.38 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. The half-lives of S208G/F227A were 1.35, 0.51 and 0.44 h after incubating for 5 h at 60 C, 70 C and 80 C, respectively. Dye decolorization The purified enzymes were compared for the decolorization ability using four dyes (methyl red, Acid blue 129, malachite green and methyl blue). This study was carried out with mediator ABTS. Methyl red, Acid blue 129, malachite green and methyl blue were decolorized by CotAlaccases (Fig. 7A). The decolorization rates of methyl red by double mutant S208G/F227A reached 69.8%. Compared with the wild-type CotA-laccase, the decolorization rate of methyl red increased by 26%. The decolorization ability of the mutant (F227A and S208G/F227A) to methyl red was obviously improved compared with the wild-type. CotA-laccase can decolorize Acid blue 129 and malachite green efficiently without mediator ABTS (Fig. 7B). Mutant S208G/F227A still had nearly 80% relative activity after incubation in methyl red dye decolorization system for 5 h, while wild-type had 72% relative activity (Fig. 7C). DISCUSSION The selection of mutation sites is a key point in site-directed mutagenesis (41). In this study, molecular docking and homology analysis were used to determine the mutation sites. As shown in Fig. 3A, Ser208 was closed to the active pocket. Ser208 located in a region rich in Pro fragments. It had been mentioned that increasing the flexibility of the Pro area may
FIG. 2. The 3D model (A) and 2D pattern (B) of the key amino acid residues of wild-type CotA-ABTS. (A) The red structure indicates ABTS and the blue structure shows amino acid residues around ABTS. (B) The interactions between wild-type CotA-laccase and ABTS.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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FIG. 3. The 3D model (A) and 2D pattern (B) of the key amino acid residues of mutant CotA-ABTS. (A) The red structure indicates ABTS and the blue structure shows amino acid residues around ABTS. (B) The interactions between mutant S208G/F227A and ABTS.
conducive to the combination of enzymes and substrates (22). What is more, ABTS had a hydrophilic tendency (42) while Gly208 participates in the active hydrophilic part of the benzothiazole group, which bound with ABTS molecules (Fig. 3B). ABTS could form van der Waals force with Gly208, but it had no direct interaction with Ser208. The Km value of CotA-laccase to ABTS decreased from 0.241 to 0.172 after the amino acid at 208 position was mutated from Ser to Gly. This indicated that the enzyme affinity of S208G to ABTS increased. Besides, amino acid at 227 was also in an important position. Both Phe227 and Ala227 could form Pi-Alkyl interaction with ABTS. Ala227 could also form hydrogen bonds with ABTS. The side chain benzene ring of Phe227 was close to the ABTS binding channel, which may affect the binding and release of ABTS to laccase. Ala227 had less steric effects with ABTS than Phe227 (Fig. 3B). The Km value of CotA-laccase to ABTS decreased from 0.241 to 0.123 after the amino acid at 227 position was mutated from Phe to Ala. This indicated that Ala227 may have contribution to the stable binding of benzothiazole with ABTS.
Current studies on laccase are mostly about the first and third copper ion centers (15,43,44), while less studies about the second copper ion centers (40,45,46). ABTS binding pocket is located in the second copper ion center (47). In this study, mutant sites (Ser208 and phe227) are near ABTS binding pocket. In this study, the mutants S208G, F227A, and S208G/F227A were constructed by site-directed mutagenesis to improve their catalytic efficiency. The molecular weight of the mutants and the wild-type laccase were all w65 kDa, which were consistent with laccase from B. subtilis WD23 (48) and Cyanobacteria (49). The kcat/Km value of the double mutant strain S208G/F227A increased compared with the laccase reported by DONG (50). The optimum pH of the mutants and the wild-type was 3.5 using ABTS as substrate. Showing that mutants had better catalytic activity under acidic conditions compared with laccase from Agaricus bisporus CU13 exhibited optimum pH at 5.0 using ABTS as substrate (51). The thermal stability of the mutant laccase was lower than that of the wild-type. The decrease of intermolecular van der Waals force and the change of conjugation force after mutation may be the reasons for the lower thermal stability (52e54). And the increase of molecular flexibility may also have bad impact on thermal stability (55). But the mutants still had higher than 40% residual activity after incubating at 90 C for 5 min. These results indicate that mutants have thermal stability similar to laccases from B. subtilis cjp3 (56) and from Pandoraea sp. ISTKB (57). Despite the decline in stability, mutants can still meet the needs of industrial applications. In this study, the decolorization effect of wild-type laccase on methyl red was lower than that of mutant S208G/F227A (Fig. 7A). This might be due to the increased specificity of the mutant S208G/F227A to methyl red. The molecular docking results of CotA-laccase and methyl red showed that
TABLE 2. Kinetic parameters for CotA-laccase using ABTS as substrate. Enzyme
FIG. 4. SDS-PAGE of cell fragmentation supernatant and purified CotA-laccase. Lane M is protein marker; lanes 1, 2 and 3 show the cell transformed with empty pColdII plasmids, cell transformed with pColdII-CotA and the purified CotA-laccase, respectively. The molecular weight of CotA-laccase was w65 kDa.
WT S208G F227A S208G/F227A
kcat (s1)
Km (mM)a
kcat/Km (s1 mM1)
40.26 38.46 43.44 56.52
0.241 0.172 0.123 0.073
167.05 223.60 353.17 856.97
a Laccase activity was calculated at 50 C with 100 mM citrate/phosphate buffer (pH 3.5). ABTS was chosen as substrate. Km and kcat were determined by MichaeliseMenten equation and LineweavereBurk plot.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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FIG. 5. Effect of pH on the activity (A) and stability (B) of the purified CotA-laccase enzymes (wild-type, S208G, F227A, and S208G/F227A) at 50 C using ABTS as substrate. (A) The test on enzyme activity was measured at 50 C. The tests used ABTS (0.5 mM) as substrate and performed at a series of different pH values (pH 2.0e7.0) with citrate/phosphate buffer. (B) The purified CotA-laccase was measured the residual activity incubated after incubating at 4 C and different pH values (pH 3.0e12.0) for 10 h before. The residual activity was calculated at 50 C and pH 3.5 using ABTS as substrate.
FIG. 6. Effect of temperature on the activity (A) and stability (B) of the purified CotA-laccases (wild-type, S208G, F227A, and S208G/F227A) using ABTS as substrate at pH 3.5. (A) CotA-laccase activity was measured at different temperatures (20e90 C). The tests used ABTS as substrate with citrate/phosphate buffer (pH 3.5). (B) The purified enzyme was incubated at four different temperatures (60 C, 70 C, 80 C, and 90 C) before measuring the residual activity. The residual activity was determined at 50 C and pH 3.5 using ABTS as substrate.
Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020
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J. BIOSCI. BIOENG., In conclusion, the catalytic efficiency of CotA-laccase was improved by site-directed mutagenesis. Results of this study showed that Gly208 and Ala227 play key roles in catalytic efficiency and it is possible to improve catalytic efficiency of CotA-laccase through site-directed mutagenesis. The expression of CotAlaccase during high-density fermentation will continue to be studied in the future. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbiosc.2019.09.020.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (31472003), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.
References
FIG. 7. (A) Decolorization of synthetic dyes (0.025 mg/ml) by the wild-type and mutants (S208G, F227A, and S208G/F227A) were determined at pH 3.5 and 50 C for 5 h with ABTS as mediator. (B) Decolorization of synthetic dyes (0.025 mg/ml) by the wildtype and mutants (S208G, F227A, and S208G/F227A) were determined at pH 3.5 and 50 C for 5 h without mediator. (C) Tolerance of enzyme in different dyes. The purified enzyme was incubated at reaction system containing citrate/phosphate buffer (100 mM, pH 3.5) and 0.025 mg/ml of dyes for 5 h at 50 C. The residual enzyme activity was measured with ABTS as substrate.
the active position and hydrogen bonds of CotA-laccase rotated after the mutation (Figs. 2 and 3). The methyl red molecule could enter the catalytic pathway more easily when the amino acid at 227 mutated from Phe to Ala. Besides, mutant S208G/F227A could form more hydrogen bonds with methyl red, which made their binding more stable (Fig. S3).
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Please cite this article as: Xu, K.-Z et al., Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2019.09.020