Characterization of the alkaline laccase Ssl1 from Streptomyces sviceus with unusual properties discovered by genome mining.
Characterization of the alkaline laccase Ssl1 from Streptomyces sviceus with unusual properties discovered by genome mining.
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DOI：
10.1371/journal.pone.0052360
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Urlacher VB
中科院分区：
文献类型：
--
作者：
Gunne M;Urlacher VB
真菌漆酶在沃茨和纺织品的漂白、纤维素脱木质素和有机合成等方面具有广泛的应用前景。然而，它们受限于酸性反应条件并且需要真核表达系统。这就提出了对没有这些限制的新型漆酶的需求。利用基因组挖掘技术构建的漆酶工程数据库LccED，从Sviceus链霉菌中筛选并克隆了漆酶Ssl 1基因，该基因能够克服真菌漆酶的局限性。ssl 1属于小漆酶家族，其仅包含少数特征酶。去除双精氨酸信号肽后，Ssl 1在大肠杆菌中得到了高效表达。杆菌Ssl 1是一种分子量为32.5 kDa的小分子漆酶，仅由两个铜氧还蛋白样结构域组成，在溶液中形成三聚体。Ssl 1氧化2，2 ′-连氮基-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）和酚类底物，如2，6-二甲氧基苯酚、愈创木酚和双羟甲基哌嗪。ABTS氧化的kcat值比其他底物高至少20倍。氧化反应的最佳pH值是底物依赖性的：对于酚类底物，在碱性条件下检测到最高活性（2，6-二甲氧基苯酚和愈创木酚的pH值为9.0，双羟甲基哌嗪的pH值为8.0），而与ABTS的最高反应速率在pH 4.0下观察到。尽管Ssl来源于嗜温生物，但其在高温（T1/2，60 °C = 88 min）和宽pH范围（pH 5.0至11.0）下表现出显著的稳定性。 值得注意的是，在pH 11下孵育5天后，酶保留80%的残余活性。去污剂和有机共溶剂不影响Ssl 1的稳定性。所描述的鲁棒性使得Ssl 1成为工业应用的潜在候选者，优选地在需要碱性反应条件的工艺中。
Fungal laccases are well investigated enzymes with high potential in diverse applications like bleaching of waste waters and textiles, cellulose delignification, and organic synthesis. However, they are limited to acidic reaction conditions and require eukaryotic expression systems. This raises a demand for novel laccases without these constraints. We have taken advantage of the laccase engineering database LccED derived from genome mining to identify and clone the laccase Ssl1 from Streptomyces sviceus which can circumvent the limitations of fungal laccases. Ssl1 belongs to the family of small laccases that contains only few characterized enzymes. After removal of the twin-arginine signal peptide Ssl1 was readily expressed in E. coli. Ssl1 is a small laccase with 32.5 kDa, consists of only two cupredoxin-like domains, and forms trimers in solution. Ssl1 oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and phenolic substrates like 2,6-dimethoxy phenol, guaiacol, and syringaldazine. The kcat value for ABTS oxidation was at least 20 times higher than for other substrates. The optimal pH for oxidation reactions is substrate dependent: for phenolic substrates the highest activities were detected at alkaline conditions (pH 9.0 for 2,6-dimethoxy phenol and guaiacol and pH 8.0 for syringaldazine), while the highest reaction rates with ABTS were observed at pH 4.0. Though originating from a mesophilic organism, Ssl demonstrates remarkable stability at elevated temperatures (T1/2,60°C = 88 min) and in a wide pH range (pH 5.0 to 11.0). Notably, the enzyme retained 80% residual activity after 5 days of incubation at pH 11. Detergents and organic co-solvents do not affect Ssl1 stability. The described robustness makes Ssl1 a potential candidate for industrial applications, preferably in processes that require alkaline reaction conditions.
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