Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis
Abstract
:1. Introduction
2. Results
2.1. Purification of Laccases
Chromatographic Method | Chromatographic Fraction by Steps | Yield (mg) | Total Activity (U) a | Specific Activity (U/mg) b | Recovery of Activity (%) | Purification Fold c |
---|---|---|---|---|---|---|
None | Crude extract | 23040 | 2853 | 0.12 | 100 | 1 |
DEAE-cellulose anion exchange | D2 (Lac 1 and Lac2) | 3200 | 1323 | 0.41 | 46.38 | 3.3 |
D3 (Lac3) | 175 | 689 | 3.94 | 24.14 | 32 | |
CM-cellulose cation exchange | D2C1 (Lac1) | 62 | 33 | 0.53 | 1.16 | 4.2 |
D2C2 (Lac2) | 32 | 297 | 9.31 | 10.42 | 75 | |
D3C (Lac3) | 29 | 64 | 2.24 | 2.24 | 18 | |
Q-Sepharose anion exchange | D2C1Q (Lac1) | 31 | 18.6 | 0.59 | 0.65 | 4.8 |
D2C2Q (Lac2) | 4.2 | 49.8 | 11.85 | 1.74 | 96 | |
D3CQ (Lac3) | 9.6 | 27 | 2.81 | 0.95 | 23 | |
Superdex 75 size exclusion | D2C1QS (Lac1) | 1.58 | 10.8 | 6.86 | 0.38 | 55 |
D2C2QS (Lac2) | 1.13 | 25.6 | 22.76 | 0.90 | 183 | |
D3CQS (Lac3) | 0.13 | 22.4 | 171 | 0.79 | 1379 |
2.2. Effects of pH and Temperature on Laccase Activity and Stability
2.3. Substrate Specificity of Isolated Laccases
Substrate | Wavelength (nm) | Relative Laccase Activity (%) | ||
---|---|---|---|---|
Lac1 | Lac2 | Lac3 | ||
ABTS | 420 | 100 ± 1.0 | 100 ± 0.0 | 100 ± 1.5 |
Guaiacol | 460 | 16 ± 0.08 | 24 ± 0.08 | 100 ± 0.04 |
Dimethylphthalate | 470 | 22.83 ± 2.17 | 100 ± 3.0 | 92.39 ± 3.26 |
Syringaldazine | 525 | 100 ± 4.23 | 89.44 ± 4.93 | 84.51 ± 1.41 |
l-DOPA | 470 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
Ferulic acid | 318 | 72.73 ± 3.64 | 3.64 ± 0.0 | 100 ± 1.82 |
Caffeic acid | 318 | 34.55 ± 1.82 | 12.5 ± 0.0 | 100 ± 1.82 |
Vanillic acid | 261 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
p-Coumaric acid | 318 | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.0 ± 0.0 |
Hydroquinone | 289 | 25 ± 1.56 | 6.25 ± 0.0 | 100 ± 3.125 |
Catechol | 276 | 100 ± 0.14 | 92.86 ± 0.56 | 42.86 ± 0.28 |
p-Phenylenediamine | 250 | 18.2 ± 0.0 | 54.55 ± 0.27 | 100 ± 0.09 |
2.4. Kinetic Constants for Laccases
Laccase Isoforms | Km (mM) | Kcat (min−1) | Kcat/Km (min−1·mM−1) |
---|---|---|---|
Lac1 | 0.16 | 0.958 | 5.99 |
Lac2 | 0.55 | 1.65 | 3.00 |
Lac3 | 0.104 | 0.264 | 2.54 |
2.5. Peptides Identified by Gel-Fractionation LC-LTQ-Orbitrap-MS
Laccaes Isoforms | |||
---|---|---|---|
Lac1 | Lac2 | Lac3 | |
Peptides | RNDVVSPDGFERR | KVIQPDGFSRS | KGDNFQLNVVNQLSDTTMLKD |
RRAITVNGIFPGTPVILQKN | SAVLAGGSYPGPLIKG | RYAGGPTSPLAIINVESNKRY | |
KNDKVQINTINELTDPGMRR | RLYDVDDESTVLTVGDWYHAPSLSLTGVPHPDSTLFNGLGRS | RANPNLGSTGFDGGINSAILRY | |
RSTSIHWHGLFQHKT | SLNGPASPLYVMNVVK | ||
RYKGAPAVEPTTVATTGGHKL | RYSLVLNANQAVGNYWIRA | ||
RKPQDFLPSEQVIILPANKL | ANPNSGDPGFANQMNSAILRY | ||
RTSNSDVVNLVNPPRR | REYNLRPLIKK | ||
RDVLPINGGNTTFRF | RDAHDLAPAGSIYDIKL | ||
RTLCPAYDGLAPEFQ | LGDVVEITMPALVFAGPHPLHLQWHTFAVVRS | ||
SAGSSTYNYENPVRRD | |||
DVVSIGDDPTDNVTIRF |
3. Discussion
4. Experimental Section
4.1. Chemical Reagents and Microorganism
4.2. Enzyme Activity Assay
4.3. Purification of Laccase isoforms
4.4. Biochemical Characterization of the Purified Laccase Isoforms
4.4.1. Effects of Temperature and pH on Laccase Activity and Stability
4.4.2. Assay for Substrate Specificity
4.4.3. Kinetic Constants of Laccase Isoforms
4.5. Molecular Weight Determination and Peptide Identification with Gel-Fractionation LC-LTQ-Orbitrap-MS
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Yuan, X.; Tian, G.; Zhao, Y.; Zhao, L.; Wang, H.; Ng, T.B. Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis. Molecules 2016, 21, 203. https://doi.org/10.3390/molecules21020203
Yuan X, Tian G, Zhao Y, Zhao L, Wang H, Ng TB. Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis. Molecules. 2016; 21(2):203. https://doi.org/10.3390/molecules21020203
Chicago/Turabian StyleYuan, Xianghe, Guoting Tian, Yongchang Zhao, Liyan Zhao, Hexiang Wang, and Tzi Bun Ng. 2016. "Biochemical Characteristics of Three Laccase Isoforms from the Basidiomycete Pleurotus nebrodensis" Molecules 21, no. 2: 203. https://doi.org/10.3390/molecules21020203