A highly stable laccase obtained by swapping the second cupredoxin domain.
A highly stable laccase obtained by swapping the second cupredoxin domain.
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DOI：
10.1038/s41598-018-34008-3
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发表时间：
2018-10-23
影响因子：
4.6
通讯作者：
Camarero S
中科院分区：
文献类型：
--
作者：
Pardo I;Rodríguez-Escribano D;Aza P;de Salas F;Martínez AT;Camarero S
高氧化还原潜力漆酶的稳健性通过将其第二个铜还蛋白结构域与另一种真菌漆酶的铜还蛋白结构域交换来增强，这在不影响酶功能的情况下在蛋白质序列中引入了一系列中性突变。新漆酶对温度、pH值(2-9)和有机溶剂表现出良好的稳定性，同时保持了氧化高氧化还原电位底物的能力。通过对信号肽进行工程处理，提高了酿酒酵母的酶分泌水平，为工程酶的进一步鉴定奠定了基础。纯化的结构域交换漆酶在乙醇或甲醇存在下表现出更高的活性，在50-70 °C有较好的半衰期，在酸性pH下的稳定性得到改善，对DMP的催化效率相似，但对ABTS的催化效率较低(由于最适pH的变化)。一个新的N-糖基化位点和一个可能的新的表面盐桥被评估为通过定点突变提高稳定性的可能决定因素。虽然这两种酶似乎都不是提高热稳定性的严格原因，但新的盐桥被发现显著有助于交换酶在广泛的pH范围内的高稳定性。最后，通过对工业木质素硫酸盐木质素在高温、中性pH和短时间培养条件下的酶处理，展示了新型漆酶的应用潜力。
The robustness of a high-redox potential laccase has been enhanced by swapping its second cupredoxin domain with that from another fungal laccase, which introduced a pool of neutral mutations in the protein sequence without affecting enzyme functionality. The new laccase showed outstanding stability to temperature, pH (2–9) and to organic solvents, while maintaining the ability to oxidize high-redox potential substrates. By engineering the signal peptide, enzyme secretion levels in Saccharomyces cerevisiae were increased, which allowed to purify the engineered enzyme for further characterization. The purified domain-swap laccase presented higher activity in the presence of ethanol or methanol, superior half-lives at 50–70 °C, improved stability at acidic pH, and similar catalytic efficiency for DMP albeit a lower one for ABTS (due to a shift in optimum pH). A new N-glycosylation site and a putative new surface salt-bridge were evaluated as possible determinants for the improved stability by site-directed mutagenesis. Although neither seemed to be strictly responsible for the improved thermostability, the new salt bridge was found to notably contribute to the high stability of the swapped enzyme in a broad pH range. Finally, the application potential of the new laccase was demonstrated with the enzymatic treatment of kraft lignin, an industrially relevant lignin stream, at high temperature, neutral pH and short incubation times.
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DOI：
10.1007/978-1-60761-652-8_1
发表时间：
2010-01-01
期刊：
IN VITRO MUTAGENESIS PROTOCOLS, THIRD EDITION
影响因子：
--
作者：
Alcalde, Miguel
通讯作者：
Alcalde, Miguel
影响因子：
--
作者：
Mate, Diana;Garcia-Burgos, Carlos;Alcalde, Miguel
通讯作者：
Alcalde, Miguel
DOI：
10.1073/pnas.0901522106
发表时间：
2009-06-16
影响因子：
11.1
作者：
Bloom, Jesse D.;Arnold, Frances H.
通讯作者：
Arnold, Frances H.
影响因子：
3.8
作者：
Pardo, Isabel;Isabel Vicente, Ana;Camarero, Susana
通讯作者：
Camarero, Susana
影响因子：
46.9
作者：
Ostermeier, M;Shim, JH;Benkovic, SJ
通讯作者：
Benkovic, SJ