Directed laccase evolution for improved ionic liquid resistance
Directed laccase evolution for improved ionic liquid resistance
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DOI：
10.1039/c3gc36899h
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发表时间：
2013-01-01
期刊：
影响因子：
9.8
通讯作者：
Schwaneberg, Ulrich
中科院分区：
文献类型：
--
作者：
Liu, Haifeng;Zhu, Leilei;Schwaneberg, Ulrich
在自然界中，木质素的生物降解是一个具有挑战性的过程，因为木质素是高度交联的且水溶性差。漆酶（EC 1.10.3.2，苯酐：氧氧化还原酶）在木质素的酶促降解中起关键作用，并且离子液体（IL）已成功地用于溶解木质素。使用漆酶降解木质素的一个限制是它们在IL存在下的低活性/抗性。为了提高漆酶在白细胞介素中的抗性，建立了一种基于ABTS（2，2 '-连氮基-双（3-乙基苯并噻唑啉-6-磺酸））筛选的定向进化方法。1-乙基-3-甲基咪唑乙基硫酸盐（[EMIM] [EtSO 4]）能有效溶解木质素，其阴离子对漆酶无抑制作用。在[EMIM] [EtSO 4]存在下，ABTS自由基阳离子的稳定性不受影响。因此，[EMIM] [EtSO 4]是一种适合于漆酶定向进化的共溶剂。从T.变色栓菌（Trametes versicolor）在酿酒酵母中表达，最后选择lcc 2作为起始点，因为其在[EMIM] [EtSO 4]存在下具有上级抗性和活性。在两轮定向进化后，lcc 2变体M3（Phe 265 Ser/Ala 318 Val）在15%（v/v）[EMIM] [EtSO 4]存在下显示出比lcc 2野生型（WT）高约4.5倍的活性，并且在缓冲液中显示出比lcc 2 WT高3.5倍的活性。[EMIM] [EtSO 4]对M3的IC 50值从392 mM（lcc 2 WT）增加至497 mM。
In nature, the biodegradation of lignin is a challenging process since lignin is highly cross-linked and poorly water-soluble. Laccases (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) play a key role in the enzymatic degradation of lignin and ionic liquids (ILs) have been used successfully to dissolve lignin. One limitation in lignin degradation using laccases is their low activity/resistance in the presence of ILs. In order to improve the resistance of laccase in IL, a directed evolution protocol based on the ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid))-screening assay in 96-well microtiter plate format was developed. 1-Ethyl-3-methylimidazolium ethylsulfate ([EMIM] [EtSO4]) can dissolve lignin efficiently and its anion does not inhibit laccase. The stability of the ABTS radical cation was not affected in the presence of [EMIM] [EtSO4]. Therefore, ([EMIM] [EtSO4]) is a suitable cosolvent for directed laccase evolution. Four laccases (lcc1_2005, lcc1_1997, lcc2 and CVLG1) from T. versicolor (Trametes versicolor) were expressed in Saccharomyces cerevisiae and finally lcc2 was selected as the starting point due to its superior resistance and activity in presence of [EMIM] [EtSO4]. After two rounds of directed evolution, the lcc2 variant M3 (Phe265Ser/Ala318Val) displayed about 4.5-fold higher activity than the lcc2 wild type (WT) in the presence of 15% (v/v) [EMIM] [EtSO4] and a 3.5-fold higher activity than lcc2 WT in buffer. The IC50 value of [EMIM] [EtSO4] towards M3 increases from 392 mM (lcc2 WT) to 497 mM.