竹黄菌漆酶对工业染料的脱色降解及其基因分析  

Shiraia laccase:decolorization of industrial dyes and its gene analysis

作　　者：郭艳华 王玥 李信萍 周建芹 王剑文[1] 郑丽屏 GUO Yanhua;WANG Yue;LI Xinping;ZHOU Jianqin;WANG Jianwen;ZHENG Liping(College of Pharmaceutical Sciences,Soochow University,Suzhou 215123,China;Gold Mantis School of Architecture,Soochow University,Suzhou 215123,China)

机构地区：[1]苏州大学药学院,江苏苏州215123 [2]苏州大学金螳螂建筑学院,江苏苏州215123

出　　处：《生物加工过程》2023年第6期642-651,共10页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金(82073955、81773696);江苏高校优势学科建设工程(RAPD)。

摘　　要：竹黄菌Shiraia sp.的子座或子实体是我国传统中药——竹黄,具有化痰止咳、活血祛风、利湿的功效,其主要活性成分为苝醌类化合物——竹红菌素,竹红菌素具有抗肿瘤、抗病毒的光敏活性。从短穗竹Brachystachyum densiflorum茎秆中分离筛选到1株内生竹黄菌Shiraia sp. S8,其无性菌丝培养可产胞外漆酶。在自然pH、28℃、150 r/min振荡摇瓶培养8 d后,胞外漆酶的酶活达1 361 U/L。胞外漆酶粗酶液的最适反应温度为60℃,最适反应pH为4.0且在pH为6.0~7.0时具有较好的稳定性,108 h后残余酶活为50%。竹黄菌S8菌株胞外漆酶在33.33μmol/L 2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)或266.66μmol/L乙酰丁香酮介导作用下,可使活性蓝19、孔雀石绿和中性红快速脱色,60 min内脱色率分别可达85.06%、88.55%和81.13%。在竹黄菌转录组数据分析的基础上,克隆得到编码漆酶的cDNA序列(lcc1)。该cDNA编码的蛋白具备完整的真菌漆酶保守结构域,前18个N末端氨基酸残基为典型信号肽序列,lcc1全长1 785 bp,开放阅读框(ORF)全长1 002 bp,编码594个氨基酸,预测其分子量为6.53×10^(4),pI为6.05。本研究为真菌漆酶的生产提供了新的菌株资源,并为漆酶在染料废水处理上的应用提供了参考。The Shiraia stromas or fruiting bodies are used as traditional Chinese medicine(TCM)to invigorate blood circulation and relieve rheumatic pains.Due to the existence of hypocrellins as the major bioactive components,they can also serve as potential photosensitizers with anti-cancer and anti-viral activities.Shiraia sp.S8,which was isolated from the short tissues of Brachystachyum densiflorum and its mycelium culture,could produce extracellular laccase in the medium.The crude laccase in cultural broth could reach its peak activity of 1361 U/L on the 8 th day.The crude laccase showed good stability at pH 6.07.0 as>50%laccase activity was maintained after 108 h under this pH.The optimal pH and temperature for the enzymatic activity of laccase turned out to be pH 4.0 and 60℃,respectively.The decolorization of industrial dyes,including reactive blue 19,malachite green and neutral red,was carried out with the crude laccase under the mediation by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate)(ABTS)at 33.33μmol/L or acetosyringone(AS)at 266.66μmol/L.As a result,the decolorization percentages in 60 min reached 85.06%,88.55%and 81.13%,respectively.Based on the transcriptome data of the fungus,the full-length cDNA(1785 bp)was cloned for laccase and named as lcc1 with 1002 bp in open reading frame(ORF).The protein encoded by lcc1 would be composed of 594 amino acid residues,including a complete conserved domain(first 18 amino acid residues in the N-terminal)of fungal laccase.In addition,the isoelectric point of this enzyme would be around 6.05 and the calculated molecular weight was about 6.53×10^(4).This study demonstrated the decolorization of industrial dyes by crude laccase and provided gene analysis for such a novel strain of fungal enzyme.

关 键 词：竹黄菌 漆酶 染料脱色 基因分析 Brachystachyum densiflorum 

分 类 号：Q93[生物学—微生物学]