Yao Yanhua, Zhou Guimei, Lin Yonghui, Xu Xinqi, Yang Jie
Fujian Key Laboratory of Marine Enzyme Engineering, College of Biological Sciences and Technology, Fuzhou University, Fuzhou, 350116, Fujian, China.
GRG Metrology & Test Fuzhou Co., Ltd, Fuzhou, 350003, Fujian, China.
AMB Express. 2020 Oct 2;10(1):178. doi: 10.1186/s13568-020-01118-z.
Laccases are a class of multi-copper oxidases with important industrial values. A thermotolerant laccase produced by a basidiomycete fungal strain Cerrena unicolor CGMCC 5.1011 was studied. With glycerin and peptone as the carbon and nitrogen sources, respectively, a maximal laccase activity of 121.7 U/mL was attained after cultivation in the shaking flask for 15 days. Transcriptomics analysis revealed an expressed laccase gene family of 12 members in C. unicolor strain CGMCC 5.1011, and the gene and cDNA sequences were cloned. A glycosylated laccase was purified from the fermentation broth of Cerrena unicolor CGMCC 5.1011 and corresponded to Lac2 based on MALDI-TOF MS/MS identification. Lac2 was stable at pH 5.0 and above, and was resistant to organic solvents. Lac2 displayed remarkable thermostability, with half-life time of 1.67 h at 70 ºC. Consistently, Lac2 was able to completely decolorize malachite green (MG) at high temperatures, whereas Lac7 from Cerrena sp. HYB07 resulted in accumulation of colored MG transformation intermediates. Molecular dynamics simulation of Lac2 was conducted, and possible mechanisms underlying Lac2 thermostability were discussed. The robustness of C. unicolor CGMCC 5.1011 laccase would not only be useful for industrial applications, but also provide a template for future work to develop thermostable laccases.
漆酶是一类具有重要工业价值的多铜氧化酶。对担子菌真菌菌株单色蜡蘑CGMCC 5.1011产生的一种耐热漆酶进行了研究。分别以甘油和蛋白胨作为碳源和氮源，在摇瓶中培养15天后，漆酶的最大活性达到121.7 U/mL。转录组学分析揭示了单色蜡蘑菌株CGMCC 5.1011中一个由12个成员组成的表达漆酶基因家族，并克隆了该基因和cDNA序列。从单色蜡蘑CGMCC 5.1011的发酵液中纯化出一种糖基化漆酶，基于基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF MS/MS）鉴定，其对应于Lac2。Lac2在pH 5.0及以上时稳定，并且对有机溶剂具有抗性。Lac2表现出显著的热稳定性，在70℃下的半衰期为1.67小时。同样，Lac2能够在高温下完全使孔雀石绿（MG）脱色，而来自蜡蘑属HYB07菌株的Lac7则导致有色MG转化中间体的积累。对Lac2进行了分子动力学模拟，并讨论了Lac2热稳定性的潜在机制。单色蜡蘑CGMCC 5.1011漆酶的稳健性不仅对工业应用有用，而且为未来开发耐热漆酶的工作提供了模板。
