生物工程学报  2017, Vol. 33 Issue (4): 565-577
http://dx.doi.org/10.13345/j.cjb.160352
中国科学院微生物研究所、中国微生物学会主办
0

文章信息

宁娜, 谭慧军, 孙新新, 倪金凤
Ning Na, Tan Huijun, Sun Xinxin, Ni Jinfeng
真核生物来源漆酶的异源表达研究进展
Advance of heterologous expression study of eukaryote-origin laccases
生物工程学报, 2017, 33(4): 565-577
Chinese Journal of Biotechnology, 2017, 33(4): 565-577
10.13345/j.cjb.160352

文章历史

Received: September 21, 2016
Accepted: January 9, 2017
真核生物来源漆酶的异源表达研究进展
宁娜, 谭慧军, 孙新新, 倪金凤     
山东大学 微生物技术国家重点实验室,山东 济南 250100
收稿日期:2016-09-21; 接收日期:2017-01-09; 网络出版时间:2017-01-16
基金项目:国家重点基础研究发展计划 (973计划) (No. 2011CB707402),国家自然科学基金 (Nos. 31272370, 30870085) 资助
摘要: 漆酶属于多铜氧化酶家族中的一种,广泛存在于昆虫、植物、真菌和细菌中。由于其作用的底物范围较广,因此在纺织、制浆、食品以及木质素的降解等方面有广阔的应用前景。但是自然界中的漆酶存在表达量和酶活低、高温易失活等问题,限制了它的应用。对漆酶进行大量高效的异源表达,是解决这一问题的有效途径。近年来,越来越多不同来源的漆酶基因被克隆,并在不同宿主中异源表达。但这些大多局限于实验室研究,还未达到工业化生产的水平。笔者对真核生物来源漆酶的异源表达研究进展进行综述,重点介绍了真核生物来源的漆酶在不同表达系统中的异源表达情况以及在酵母细胞中表达漆酶时提高表达量和酶活性能的方法,以期为研究者们提供参考。
关键词真核漆酶     异源表达     酶活力     稳定性    
Advance of heterologous expression study of eukaryote-origin laccases
Na Ning, Huijun Tan, Xinxin Sun, Jinfeng Ni     
State Key Laboratory of Microbial Technology, Shandong University, Ji'nan 250100, Shandong, China
Abstract: Laccases are enzymes belonging to the group of multi-copper oxidases. These enzymes are widely distributed in insects, plants, fungi and bacteria. In general, laccases can oxidize an exceptionally high number of substrates, so they have broad applications in textile, pulp, food and the degradation of lignin. However, low yield, low activity and thermo-instability of laccase in nature limit the application of laccase. High efficient heterologous expression of the protein is an effective way for solving this problem. Here, we summarize the research advances of heterologous expression of eukaryote-origin laccases. We focus on the overexpression of eukaryote-origin laccases using different expression system and the method for improving the production yield and enzyme activity in yeast cells. Information provided in this review would be helpful for researchers in the field.
Key words: eukaryote-origin laccases     heterologous expression     enzyme activity     stability    

漆酶 (Laccase,苯二酚:双氧氧化还原酶;EC 1.10.3.2) 与抗坏血酸氧化酶和各种铁氧化酶同属于多铜氧化酶家族[1]。漆酶最早被发现于植物中[2],随着分子生物学和生物信息学技术的发展,人们陆续在细菌[3]、真菌以及昆虫[4]中发现了漆酶。不同来源的漆酶具有不同的体内功能。细菌漆酶主要与细菌色素的形成和金属离子抵抗有关[5-6]。也有报道称芽孢杆菌中的漆酶,在抗紫外线孢子的发育中起作用[7]。目前研究最多的是真菌漆酶,主要集中于担子菌和子囊菌中的漆酶,这类菌株多为白腐真菌[8]。白腐真菌是目前所知道的唯一能够利用自身氧化酶系统将木质素降解为二氧化碳的微生物[9],漆酶在此过程中起了重要作用。另外,真菌漆酶还是一些致病真菌的毒性成分,在真菌的分化和色素形成中起重要作用[10]。植物漆酶虽然发现较早,但研究却相对较少。植物中的漆酶主要与木质素的合成以及损伤部位的修复有关[2]。也有少量的报道证明植物中的漆酶在植物种间竞争[11]以及植物抵抗微生物的侵染[12]中起作用。昆虫漆酶根据生理作用的不同可以分为漆酶1和漆酶2。目前昆虫漆酶的研究多集中于漆酶2在昆虫外骨骼的硬化、表皮色素沉积以及角质层鞣化方面的作用,而对可能在木质素降解或食物脱毒中起作用的漆酶1的报道较少[13]

漆酶底物范围广泛,主要包括酚类化合物、芳香族化合物、脂肪胺和无机阳离子等[14]。漆酶可以利用铜离子特有的氧化还原能力,对还原性底物进行单电子氧化,同时传递4个电子,将作为第二底物的氧气还原成水[15]。基于漆酶底物范围的广泛性以及副产物只有水的环境友善性,使得漆酶在木质素降解、造纸工业、染料脱色、食品和饮料业等方面都具有潜在的应用价值[16]。然而,自然界中分离到的野生菌株的漆酶有产量和活性较低、纯化困难、高温易失活、不易操作等缺点,很大程度上阻碍了对漆酶生理生化性质的基础研究及其工业化应用[10, 17-18]。近年来,越来越多不同来源的漆酶基因被克隆,并在不同宿主中异源表达。但这些大都局限于实验室研究,还未达到工业化生产的水平。提高漆酶的表达量、重组蛋白的耐热性和pH稳定性是目前漆酶工业应用需要面对的问题。对于细菌漆酶的研究进展已经有学者对其进行了总结[19-20],因此文中将结合笔者的研究工作,对近年来真核来源的漆酶 (植物、真菌和昆虫来源的漆酶) 的异源表达情况进行概述,以期为相关研究者们提供参考。

1 真核生物来源的漆酶在不同表达系统中的表达

目前为止,异源表达真核生物来源的漆酶 (以下简称真核漆酶) 所用表达宿主主要有细菌、酵母、丝状真菌和昆虫杆状细胞,其中使用最为广泛的宿主是毕赤酵母Pichia pastoris。下面根据所使用表达系统的不同,对真核漆酶的异源表达情况进行介绍。

1.1 真核漆酶在细菌中的表达

由于细菌具有好操作、培养周期短、经济实惠等特点,经常被用来表达漆酶。其中大肠杆菌表达系统是应用最广泛的原核表达系统。表 1显示了真核漆酶在细菌中的表达情况。杨建强等[21]将野生革耳Panus rudis来源的漆酶基因在大肠杆菌中表达,得到可溶性漆酶蛋白,在可溶漆酶蛋白中添加CuSO4并在室温下孵育复性,获得有活性的漆酶,这是首次报道的真菌来源的漆酶基因在大肠杆菌中实现表达。另外来自黑蛋巢菌Cyathus bulleri [22]和白腐担子菌类木硬孔菌Rigidoporus lignosus [23]的真菌漆酶也在大肠杆菌中成功异源表达。

表 1 漆酶在细菌中的表达 Table 1 The expression of laccase genes in bacteria
Laccase originExpression hostsSubstratesActiveReferences
Panus rudis Escherichia coliABTSYes[21]
Cyathus bulleri Escherichia coliABTSYes[22]
Rigidoporus lignosus Escherichia coliSyringaldazineYes[23]
Termitomyce albuminosus Escherichia coliABTSNo[24]
Pleurotus eryngii Lactobacillus buchneriNRNR[25]
ABTS: 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported.

然而,潘程远等[24]在大肠杆菌中表达来源于鸡枞菌Termitomyce albuminosus的漆酶cDNA (talcc) 时,得到重组漆酶蛋白,但未检测到漆酶活性。大肠杆菌虽然操作简便,但一般不具备对重组蛋白进行二级结构修饰的能力,而漆酶是一种高糖基化修饰的酶,因此在大肠杆菌中表达真核来源的漆酶时往往会得到没有酶活性的重组蛋白。

1.2 真核漆酶在酵母细胞中的表达

酵母是最低等的真核生物。具有操作简单、生长速率快、可进行高密度发酵;培养基廉价,适用于工业生产;不含有病原体、热原体和病毒包涵体;可对重组蛋白进行蛋白酶解、形成二硫键和糖基化等翻译后修饰的优点。但是对在酵母中异源表达的漆酶进行糖基化分析,发现酵母对漆酶的糖基化有时会影响甚至破坏漆酶的酶活性质和酶活力[26-27]

尽管有一些酵母菌会产生自己的漆酶[28-29],但是很多不同的酵母,例如酿酒酵母Saccharomyces cerevisiae、毕赤酵母Pichia pastoris、乳酸克鲁维酵母Kluyveromyces lactis、解脂耶氏酵母Yarrowia lipolytica、隐球酵母Cryptococcus sp.作为宿主,都成功表达了其他真菌 (担子菌门和子囊菌门) 来源的漆酶。表 2总结了2007年以来截止到目前在酵母中成功表达的部分真核漆酶。非常有趣的是,漆酶的表达水平因酵母宿主和漆酶亚型的不同而不同。例如,在乳酸克鲁维酵母K. lactis和酿酒酵母S. cerevisiae中表达来自于糙皮侧耳Pleurotus ostreatus中的漆酶POX3时,酿酒酵母中表达的漆酶酶活性比在乳酸克鲁维酵母中的高[30]。Gu等[31]在毕赤酵母P. pastoris中表达来自于毛头鬼伞Coprinus comatus的漆酶Lac3和Lac4时,Lac4的酶活可达到1 465 U/L,而Lac3的酶活为689 U/L。表达漆酶时在培养基中加入不同浓度的硫酸铜 (0.1-1.0 mmol/L),可以促进漆酶的表达并提高酶活力。Pezzella等[30]在酿酒酵母中表达来自于糙皮侧耳P. ostreatus中的漆酶,不加硫酸铜时酶活为30 U/L,加入0.6 mmol/L硫酸铜时酶活提高至75 U/L。另外在培养过程中,达到最大酶活的时间差异较大,从48-504 h不等,这种差异主要与漆酶亚型、表达载体以及培养规模等相关。酵母菌产生的重组漆酶在应用方面的研究主要集中于染料脱色方面 (表 2),也有关于漆酶对酚类化合物的降解作用[32]以及对植物生长的促进作用[33]的相关研究。

表 2 漆酶在酵母细胞中的表达 Table 2 Expression of laccase genes in yeast cells
OriginLaccasesExpression hostsExpression plasmidsCuSO4 concentration (mmol/L)Culture time (h)Maximum activity (U/L)Applications of the recombinant laccasesReferences
Colletotrichum lagenariumCILACII Pichia pastoris GS115pPIC9KNRNRNRDecolorization of dyes[34]
Moniliophthora perniciosa FA553LacMP Pichia pastoris X33pPICZaA-6AA-LacMP0.496232NR[35]
Volvariella volvaceaVvlcc3 Pichia pastoris GS115pPIC9K-Vvlcc30.3504296.83NR[36]
Basidiomycete cerrena sp.Lac1 Pichia pastoris X33pPICZα-Lac11.02166 300Decolorization of dyes[37]
Phytophthora capsiciPCLAC2 Pichia pastoris GS115pPIC9K/ Pclac2NR16884 000NR[38]
Coprinopsis cinereaCcLcc2 Pichia pastoris GS115pYM7898NR72NRDyes decolorization[39]
Okayama-7#130
Coprinus comatusLac3 Pichia pastoris KM71HpPICZαB-10AALac30.5336689Dyes decolorization[31]
Lac4pPICZαB-10AALac41 465
Phomopsis liquidambariLACB3 Schizosacchar-omyces pombepESP-3-lacB3NRNRNRGrowth promotion of plants[33]
Volvaria volvaceavv-lac1 Pichia pastoris GS115pPIC9K-vv-lac1NRNR333.17NR[40]
vv-1ac6pPIC9K-vv-lac6227.63
Canoderma lucidum TR6glacTR6 Pichia pastoris GS115pPIC9K-glacTR60.3432685.8NR[41]
Coprinus comatusLac1 Pichia pastoris KM71HpPICZαB-lac10.5144550Dyes decolorization[42]
Cyathus bulleriLcc Pichia pastoris X33pPICZαBlcc-50.4727 200NR[43]
Monilinia fructigenaMfLcc Pichia pastoris GS115pYM8025NR48NRDegradation of phenolic compounds[32]
Botrytis acladaBaLac Pichia pastoris X33pGAPZA-BaLac0.17653 300NR[44]
Ganoderma lucidumGlLCCI Pichia pastoris GS115pYM 79091.072NRDecolorization of methylorange[45]
Polyporus grammocephalusLac-T16 Pichia pastoris GS115pPIC3.5K-TR16lac0.3264320·8NR[46]
TR16pPIC9K-TR16lacNRNRNR
Pleurotus ostreatusrPOX3 Saccharomyces cerevisiae W30pSAL4-POX3NA4830NR[30]
3-1A0.64875
Kluyveromyces lactispYG132-POX3NA7212
Pleurotus eryngiiEry3Free Saccharomyces cerevisiae cellspY-Ery30.57288NR[47]
Immobilized Saccharomyces cerevisiae cells0.5120139
Pleurotus ostreatusPOXA3 Kluyveromyces lactisA3L1.0336-40820NR[48]
Ganoderma lucidumGLlac1 Pichia pastorispPIC-RCL1NR168NRAntioxidative properties[49]
Trametes sp. 420rLacD Pichia pastoris GS115pPIC9K-LacD0.3NR83 000Decolorization of dyes[50]
Trametes sp. 420rLacC Pichia pastoris GS115pPIC9K-LacC0.321616 200Dye decolorization[51]
ABTS: 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported; NA: not added.

目前为止,还没有昆虫来源的漆酶在酵母中成功异源表达的相关报道。笔者尝试在毕赤酵母中表达来自黄翅大白蚁Macrotermes barneyi唾液腺-前肠的漆酶基因,尽管使用了不同菌株作为宿主并优化了培养条件,但并未得到重组蛋白。可能是由于密码子的偏好性、糖基化位点以及二硫键数目较多等原因而使昆虫来源的漆酶难以在毕赤酵母中正常表达。

1.3 真核漆酶在丝状真菌中的表达

目前为止,漆酶已经在曲霉属Aspergillus sp.[52-57]、里氏木霉Trichoderma reesei [58-60]和灰白青霉Penicillium canescens [61]中表达 (表 3)。大多数情况下丝状真菌用来表达来源于白腐真菌的漆酶。例如栓菌属Trametes sp.的漆酶在里氏木霉[59]和曲霉[57-62]中成功表达。除了真菌漆酶,也有细菌来源的 (天蓝色链霉菌 Streptomyces coelicolor ) 漆酶在米曲霉中成功表达,并解析了它的三维结构[63]。由于在丝状真菌中表达重组漆酶时有一个相对较高的蛋白产量,因此有利于研究漆酶的分子量[52, 64-65]、反应机制[66]和三维结构[63-67]。丝状真菌产生的重组漆酶在纸浆脱色[68-69]、合成染料的生物转化[54]以及植物修复[62]中广泛应用。另外丝状真菌表达的重组漆酶还用于食品工业黄曲霉毒素的消除、环境中酚类化合物的检测中。

表 3 部分丝状真菌中表达的重组漆酶的特性 Table 3 Properties of some recombinant laccases expressed in filamentous fungi
Laccase originExpression hostsSubstratesMaximum enzyme activity (U/L)Optimum temperature (℃)Optimum pHReferences
Trametes villosa Aspergillus oryzaeSyringaldazineNRNR5.0-5.5[52]
ABTS8 250NR2.7
Ceriporiopsis subvermispora Aspergillus nidulansABTS230NRNR[53]
Aspergillus niger
Phanerochaete flavido-alba Aspergillus nigerABTS2 500NR3.0[54]
Trichoderma reesei i Trichoderma reeseiABTS46 800NRNR[58]
GuaiacolNR5.0-7.0
Trametes sp. AH28-2 Trichoderma reeseiABTS3 620503.0[59]
GuaiacolNR504.2
Pleurotus ostreatus Trichoderma reeseiABTS237 134503.0[60]
Trametes hirsute Penicillium canescensNR3 000NRNR[61]
ABTS: 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported.
1.4 真核漆酶在植物中的表达

植物主要用来表达来自不同植物或真菌的漆酶。主要的植物表达宿主有拟南芥Arabidopsis thaliana、番茄Lycopersicon esculentum、水稻Oryza sativa、烟草Nicotiana tabacum、甘蔗Saccharum officinarum、玉米Zea mays等。在番茄中过量表达来自马铃薯的多酚氧化酶,表达植株对丁香假单胞菌Pseudomonas syringae的抵抗能力明显增强[70]。有些情况下,构建表达漆酶的转基因植物是为了建立植物修复系统,来降解双酚、三氯苯酚、五氯苯酚及其他酚类化合物[72]。在甘蔗中表达漆酶,有助于阐明漆酶在木质化过程中的作用[73]

1.5 真核漆酶在昆虫细胞中的表达

目前发现的动物来源的漆酶主要集中于昆虫漆酶,在甲壳类以及棘皮动物中也有发现。根据昆虫漆酶的功能和表达部位的不同,将分布于昆虫唾液腺、马氏管和中肠组织中的漆酶称为漆酶1 (laccase 1),此类漆酶可能参与食物的脱毒作用[4];而将表达于表皮、卵壳等组织的漆酶称为漆酶2 (laccase 2),此类漆酶与昆虫角质层的黑化作用有关[73]

Dittmer等[74]利用昆虫杆状病毒表达系统,成功表达了烟草天蛾Manduca sexta来源的多铜氧化酶漆酶2。该研究表明在昆虫杆状病毒表达系统中表达的重组昆虫漆酶可以代替内源的酶来测定生化性质,帮助我们更广泛地了解烟草天蛾来源的漆酶以及其他昆虫来源漆酶的酶学性质和功能。Coy等[75]通过RACE的方法获得了美洲散白蚁Reticulitermes flavipes唾液腺-前肠中的两个漆酶基因 (RfLacARfLacB),因为该酶在微生物缺乏的唾液腺和前肠中活性最高,并且进化上比较特别,推测该酶由白蚁自身产生。将这两个基因在昆虫杆状病毒表达系统中表达,纯化后测定酶活,发现这两个酶对木质素的单体芥子酸及其他几种酚氧化物具有较强的活力,而对黑色素前体没有或有很低的酶活力,这就为白蚁唾液腺和前肠产生的漆酶在木质纤维素降解中起作用提供了重要的证据。然而,我们前期实验尝试将黄翅大白蚁M. barneyi中肠来源的漆酶基因在昆虫杆状病毒系统中表达,但是并没有成功表达。昆虫杆状病毒表达系统的使用,使得对昆虫漆酶的研究成为可能。但是该表达系统与细菌和真菌表达系统相比,操作较为繁琐,价格和成本也比较高。

2 漆酶表达量及酶活性能的提高

如前所述,漆酶应用方面存在表达困难、热稳定性和pH耐受性较差和酶活力较低等问题。目前提高漆酶表达量的方法主要有两个方面:构建重组基因和控制培养条件及产酶条件。在提高酶活力和热稳定性方面,主要的方法包括定点突变、随机突变和DNA Shuffling等基因工程的方法。由于酵母是目前表达漆酶应用最广泛的宿主,因此更多的研究集中于漆酶在酵母细胞中的表达优化,下面对此进行介绍。

2.1 启动子的选择

在表达漆酶时,首先要选择一个合适的强启动子。在酿酒酵母中表达漆酶时,使用强启动子半乳糖激酶 (GAL1或GAL10) 启动子,利用半乳糖进行诱导,可以使漆酶有一个较高水平的表达[64]。选择铜结合蛋白 (CUP1) 启动子,在培养基中加入CuSO4至终浓度0.4-0.5 mmol/L,不仅可以诱导重组蛋白大量表达,也为漆酶提供了适量的铜离子。尽管这些诱导型启动子的使用,使得漆酶的表达量较高,但是由于诱导物的成本较高,并不适用于工业生产。从生产成本方面考虑,强的组成型启动子更具优势。在酿酒酵母中应用于表达漆酶的组成型启动子主要有乙醇脱氢酶 (ADH1) 启动子、磷酸甘油酸激酶 (PGK1) 启动子、磷酸丙糖异构酶 (TPI1) 启动子、翻译延伸因子-1 (TEF1) 启动子以及甘油醛-3-磷酸脱氢酶 (GPD1) 启动子。在毕赤酵母中表达漆酶时,最常用的是甲醇诱导型的乙醇氧化酶 (AOX1) 启动子[43]。有研究显示当用0.5%的甲醇诱导时,比用2.0%甲醇诱导时表达水平要高出5倍,从2.0 U/mL提高到11.5 U/mL[76]。类似的,在Pichia methalonica中使用甲醇诱导型启动子 (AUG1) 来表达漆酶时,最适合的产漆酶的甲醇浓度为0.8%,同样低于1%[77]。由于甲醇有毒并且成本较高,因此人们也在努力寻求不需甲醇诱导的启动子。在毕赤酵母中利用组成型的GAP启动子,成功表达了来自于木质层孔菌Fomes lignosus [78]、灵芝Ganoderma lucidum [79]、担子菌类[80]、糙皮侧耳Pleurotus ostreatus [79]以及云芝Trametes versicolor [81]来源的漆酶基因。

2.2 信号肽的选择

通常真核生物中的漆酶均分泌到胞外,这是由于在N-末端带有信号肽,促使蛋白分泌,分泌到胞外的蛋白利于纯化和应用。在酵母细胞中异源表达漆酶时,除了利用漆酶自身的信号肽之外,也可使用酵母的信号肽。通常应用比较多的是来源于酿酒酵母的信号肽序列α-factor以及它的修改序列[80]。另外,蔗糖酶基因 (SUC2)、胞外碱性蛋白酶基因 (XPR2)、木聚糖酶 (Xylanase) 基因的信号肽也可以用于漆酶基因在酵母中的高效表达[16]。值得注意的是,有时使用漆酶自身的信号肽比使用α-factor信号肽序列得到的重组蛋白活力要高,而有时情况则相反。例如,在毕赤酵母中表达来自于云芝T. versicolor的漆酶基因laccase Ⅳ时,使用α-factor信号肽,致使分泌的重组蛋白N-端残留了一个四肽,使酶比活相对于使用自身信号肽时下降了25%,从0.88 U/mg下降为0.68 U/mg[82]。而在P. methalonica中表达来自云芝的漆酶基因Lcc1,使用α-factor信号肽时的活力 (3.17 U/mL) 是自身信号肽 (1.87 U/mL) 的1.7倍[83]。类似的,在毕赤酵母中表达来自Physisporinus rivulosus的漆酶时,使用α-factor信号肽时的酶活是使用自身信号肽的1.6倍,分别为4.9 μkat/μg和3.14 μkat/μg [84]。以上这些结果的不同说明对于不同的漆酶和不同的宿主,需要对信号肽进行特定的优化来增加表达量和酶活力。

2.3 漆酶基因改造

很多研究报道,合成酵母密码子偏好的漆酶基因,比用原始的漆酶序列获得的重组蛋白更多。通过密码子优化的方式成功表达的漆酶有来源于灰盖鬼伞Coprinus cinereus的漆酶基因LCC5I [85]、灵芝G. lucidum的漆酶基因LCCI [45-79]、桃褐腐病菌M. fructigena的漆酶基因LCC2 [32]和糙皮侧耳P. ostreatus的漆酶基因POXA1b [79]

改造漆酶编码基因,除了可以提高表达量,还可以提高漆酶的稳定性、改变最适pH、改变K mK cat以及提高底物亲和力。改造基因使用的主要技术包括随机突变、定向突变以及DNA Shuffling等。Bulter等[86]通过10轮的分子进化使得来自于嗜热毁丝霉Myceliophthora thermophila的漆酶在酿酒酵母中的异源表达量提高了8倍,达到18 mg/L,总酶活提高了170倍,K cat值提高22倍 (从 (80±2.5) min-1提高到 (1 740±34) min-1)。Festa等[87]将糙皮侧耳P. ostreatus来源的漆酶POXA1b在毕赤酵母中表达,通过易错PCR和DNA Shuffling的方法使重组漆酶活性从 (183±1) U/mg提高到 (454±2) U/mg,在60 ℃、pH 7.0条件下的半衰期t 1/2从2.2 h提高至3.1 h。

另一种使得漆酶成功表达的方法是在漆酶基因N-端或C-端加入氨基酸标签来融合表达。例如Gu等[31]通过在来源于毛头鬼伞C. comatus的两个新型漆酶同工酶的N-末端加入10个氨基酸组成的标签,使得这两个漆酶同工酶成功地在毕赤酵母中异源表达,可能是增加这10个氨基酸标签后,有利于蛋白的分泌表达。

3 展望

毫无疑问,漆酶在工业和生物技术中都有着广泛的应用前景,是一种环境友好型的木质素降解酶。在自然界中,白腐真菌是漆酶主要的生产者,但野生型真菌漆酶的产量低、培养周期长,限制了漆酶的大规模应用。随着分子生物学技术和基因工程技术的发展,已经有一些不同来源的漆酶在真核和原核表达系统中实现异源表达。但是,目前真核漆酶的异源表达情况还不理想,还未达到工业化水平。酵母细胞具有真核中的翻译后修饰能力、培养基廉价、遗传操作简单和可以分泌表达等优势,因此用作漆酶异源表达的宿主是非常有前景的。未来将加强分子生物学和分子遗传学方面的研究,更加全面详细地了解漆酶的结构和功能,进而在漆酶的基因水平上加以改造,逐步实现真正的高效异源表达,达到工业化水平。

参考文献
[1] Thurston CF. The structure and function of fungal laccases. Microbiology, 1994, 140(1): 19–26. DOI: 10.1099/13500872-140-1-19
[2] Bao WL, O'Malley DM, Whetten R, et al. A laccase associated with lignification in loblolly pine xylem. Science, 1993, 260(5108): 672–674. DOI: 10.1126/science.260.5108.672
[3] Driks A. Bacillus subtilis spore coat. Microbiol Mol Biol Rev, 1999, 63(1): 1–20.
[4] Dittmer NT, Suderman RJ, Jiang HB, et al. Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. Insect Biochem Mol Biol, 2004, 34(1): 29–41. DOI: 10.1016/j.ibmb.2003.08.003
[5] Hullo MF, Moszer I, Danchin A, et al. CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol, 2001, 183(18): 5426–5430. DOI: 10.1128/JB.183.18.5426-5430.2001
[6] Grass G, Rensing C. CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochem Biophys Res Commun, 2001, 286(5): 902–908. DOI: 10.1006/bbrc.2001.5474
[7] Claus H. Laccases and their occurrence in prokaryotes. Arch Microbiol, 2003, 179(3): 145–150. DOI: 10.1007/s00203-002-0510-7
[8] Si J, Li W, Cui BK, et al. Advances of research on characteristic, molecular biology and applications of laccase from fungi. Biotechnol Bull, 2011(2): 48–55. (in Chinese).
司静, 李伟, 崔宝凯, 等. 真菌漆酶性质、分子生物学及其应用研究进展. 生物技术通报, 2011(2): 48-55.
[9] Ten Have R, Teunissen PJM. Oxidative mechanisms involved in lignin degradation by white-rot fungi. Chem Rev, 2001, 101(11): 3397–3413. DOI: 10.1021/cr000115l
[10] Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev, 2006, 30(2): 215–242. DOI: 10.1111/j.1574-4976.2005.00010.x
[11] Wang GD, Li QJ, Luo B, et al. Ex planta phytoremediation of trichlorophenol and phenolic allelochemicals via an engineered secretory laccase. Nat Biotechnol, 2004, 22(7): 893–897. DOI: 10.1038/nbt982
[12] Mayer AM, Staples RC. Laccase: new functions for an old enzyme. Phytochemistry, 2002, 60(6): 551–565. DOI: 10.1016/S0031-9422(02)00171-1
[13] Yu ML, Ni JF. Research advances in the insect laccase. Chin J Bioproc Eng, 2014, 12(1): 80–85. (in Chinese).
于孟兰, 倪金凤. 昆虫漆酶的研究进展. 生物加工过程, 2014, 12(1): 80-85.
[14] Giardina P, Faraco V, Pezzella C, et al. Laccases: a never-ending story. Cell Mol Life Sci, 2010, 67(3): 369–385. DOI: 10.1007/s00018-009-0169-1
[15] Liu ZC, Wang GG. Fungal laccases: structure-based function and mechanism. Acta Biophys Sinica, 2013, 29(9): 629–645. (in Chinese).
刘忠川, 王刚刚. 真菌漆酶结构与功能研究进展. 生物物理学报, 2013, 29(9): 629-645.
[16] Antošová Z, Sychrová H. Yeast hosts for the production of recombinant laccases: a review. Mol Biotechnol, 2016, 58(2): 93–116. DOI: 10.1007/s12033-015-9910-1
[17] Piscitelli A, Pezzella C, Giardina P, et al. Heterologous laccase production and its role in industrial applications. Bioeng Bugs, 2010, 1(4): 254–264. DOI: 10.4161/bbug.1.4.11438
[18] Ausec L, Črnigoj M, Šnajder M, et al. Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp... Appl Microbiol Biotechnol, 2015, 99(23): 9987–9999. DOI: 10.1007/s00253-015-6843-3
[19] Zhou W, Guan ZB, Cai YJ, et al. Progress in the Bacillus laccase. Microbiol China, 2015, 42(7): 1372–1383. (in Chinese).
周稳, 管政兵, 蔡宇杰, 等. 芽胞杆菌漆酶的研究进展. 微生物学通报, 2015, 42(7): 1372-1383.
[20] Ma YY, Jia HH, Wei P. Progress in study and application of bacterial laccase. Biotechnol Bull, 2013(2): 41–48. (in Chinese).
马莹莹, 贾红华, 韦萍. 细菌漆酶的研究及应用进展. 生物技术通报, 2013(2): 41-48.
[21] Yang JQ. Heterologous expression and modification of laccase from Panus rudis [D]. Beijing: Academy of Military Medical Science, 2005 (in Chinese).
杨建强. 革耳 (Panus rudis) 漆酶的异源表达和酶学性质改良[D]. 北京: 中国人民解放军军事医学科学院, 2005.
[22] Salony, Garg N, Baranwal R, et al. Laccase of Cyathus bulleri : structural, catalytic characterization and expression in Escherichia coli. Biochim Biophys Acta Proteins Proteomics, 2008, 1784(2): 259–268. DOI: 10.1016/j.bbapap.2007.11.006
[23] Nicolini C, Bruzzese D, Cambria MT, et al. Recombinant laccase: I. Enzyme cloning and characterization. J Cell Biochem, 2013, 114(3): 599–605. DOI: 10.1002/jcb.v114.3
[24] Pan CY. Cloning and expression of genes encoding laccases in Termitomyces albuminosus, Culex pipiens pallens and Musca domestica [D]. Hangzhou: Zhejiang University, 2009 (in Chinese).
潘程远. 鸡枞菌与淡色库蚊及家蝇中漆酶编码基因的克隆与表达[D]. 杭州: 浙江大学, 2009.
[25] Xue D. Cloning laccase gene from edible fungl and established the system of transformation lactic acid bacteria by electroporation [D]. Hohhot: Inner Mongolia University, 2014 (in Chinese).
薛丹. 食用菌中漆酶基因的克隆及乳酸菌电激转化体系的建立[D]. 呼和浩特: 内蒙古大学, 2014.
[26] Rodgers CJ, Blanford CF, Giddens SR, et al. Designer laccases: a vogue for high-potential fungal enzymes?. Trends Biotechnol, 2010, 28(2): 63–72. DOI: 10.1016/j.tibtech.2009.11.001
[27] Maestre-Reyna M, Liu WC, Jeng WY, et al. Structural and functional roles of glycosylation in fungal laccase from Lentinus sp... PLoS ONE, 2015, 10(4): e0120601. DOI: 10.1371/journal.pone.0120601
[28] Jadhav JP, Parshetti GK, Kalme SD, et al. Decolourization of azo dye methyl red by Saccharomyces cerevisiae MTCC 463. Chemosphere, 2007, 68(2): 394–400. DOI: 10.1016/j.chemosphere.2006.12.087
[29] Kalyani D, Tiwari MK, Li JL, et al. A highly efficient recombinant laccase from the yeast Yarrowia lipolytica and its application in the hydrolysis of biomass. PLoS ONE, 2015, 10(3): e0120156. DOI: 10.1371/journal.pone.0120156
[30] Pezzella C, Autore F, Giardina P, et al. The Pleurotus ostreatus laccase multi-gene family: isolation and heterologous expression of new family members. Curr Genet, 2009, 55(1): 45–57. DOI: 10.1007/s00294-008-0221-y
[31] Gu CJ, Zheng F, Long LK, et al. Engineering the expression and characterization of two novel laccase isoenzymes from Coprinus comatus in Pichia pastoris by fusing an additional ten amino acids tag at N-terminus. PLoS ONE, 2014, 9(4): e93912. DOI: 10.1371/journal.pone.0093912
[32] Bao WH, Peng RH, Zhang Z, et al. Expression, characterization and 2, 4, 6-trichlorophenol degradation of laccase from Monilinia fructigena. Mol Biol Rep, 2012, 39(4): 3871–3877. DOI: 10.1007/s11033-011-1166-7
[33] Wang HW, Zhu H, Liang XF, et al. Molecular cloning and expression of a novel laccase showing thermo-and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants. Biotechnol Lett, 2014, 36(1): 167–173. DOI: 10.1007/s10529-013-1347-7
[34] Wang B, Yan Y, Tian YS, et al. Heterologous expression and characterisation of a laccase from Colletotrichum lagenarium and decolourisation of different synthetic dyes. World J Microbiol Biotechnol, 2016, 32(3): 40. DOI: 10.1007/s11274-015-1999-7
[35] Liu HP, Tong CF, Du B, et al. Expression and characterization of LacMP, a novel fungal laccase of Moniliophthora perniciosa FA553. Biotechnol Lett, 2015, 37(9): 1829–1835. DOI: 10.1007/s10529-015-1865-6
[36] Lu YP, Wu GM, Lian LD, et al. Cloning and expression analysis of Vvlcc3, a novel and functional laccase gene possibly involved in stipe elongation. Int J Mol Sci, 2015, 16(12): 28498–28509. DOI: 10.3390/ijms161226111
[37] Yang J, Ng TB, Lin J, et al. A novel laccase from basidiomycete Cerrena sp.: cloning, heterologous expression, and characterization. Int J Biol Macromol, 2015, 77: 344–349. DOI: 10.1016/j.ijbiomac.2015.03.028
[38] Feng BZ, Li PQ. Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici. Braz J Microbiol, 2014, 45(1): 351–358. DOI: 10.1590/S1517-83822014005000021
[39] Tian YS, Xu H, Peng RH, et al. Heterologous expression and characterization of laccase 2 from Coprinopsis cinerea capable of decolourizing different recalcitrant dyes. Biotechnol Biotechnol Equip, 2014, 28(2): 248–258. DOI: 10.1080/13102818.2014.913402
[40] Wu L, Yang JH, Chen MJ, et al. Cloning and heterologous expression of laccase genes vv-lac 1 and vv-lac 6 from Volvaria volvacea. Acta Microbiol Sin, 2014, 54(7): 828–835. (in Chinese).
吴林, 阳经慧, 陈明杰, 等. 草菇漆酶基因vv-lac 1和vv-lac 6的克隆及异源表达. 微生物学报, 2014, 54(7): 828-835.
[41] You LF, Liu ZM, Lin JF, et al. Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris. J Basic Microbiol, 2014, 54(Suppl 1): S134–S141.
[42] Bao SY, Teng Z, Ding SJ. Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus. Mol Biol Rep, 2013, 40(2): 1927–1936. DOI: 10.1007/s11033-012-2249-9
[43] Garg N, Bieler N, Kenzom T, et al. Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase. BMC Biotechnol, 2012, 12: 75. DOI: 10.1186/1472-6750-12-75
[44] Kittl R, Gonaus C, Pillei C, et al. Constitutive expression of Botrytis aclada laccase in Pichia pastoris. Bioengineered, 2012, 3(4): 232–235. DOI: 10.4161/bioe.20037
[45] Sun J, Peng RH, Xiong AS, et al. Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris. Mol Biol Rep, 2012, 39(4): 3807–3814. DOI: 10.1007/s11033-011-1158-7
[46] Huang SJ, Liu ZM, Huang XL, et al. Molecular cloning and characterization of a novel laccase gene from a white-rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris. Lett Appl Microbiol, 2011, 52(3): 290–297. DOI: 10.1111/lam.2011.52.issue-3
[47] Bleve G, Lezzi C, Mita G, et al. Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells. Appl Microbiol Biotechnol, 2008, 79(5): 731–741. DOI: 10.1007/s00253-008-1479-1
[48] Faraco V, Ercole C, Festa G, et al. Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis. Appl Microbiol Biotechnol, 2008, 77(6): 1329–1335. DOI: 10.1007/s00253-007-1265-5
[49] Joo SS, Ryu IW, Park JK, et al. Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties. Mol Cells, 2008, 25(1): 112–118.
[50] Hong YZ, Zhou HM, Tu XM, et al. Cloning of a laccase gene from a novel basidiomycete Trametes sp. 420 and its heterologous expression in Pichia pastoris. Curr Microbiol, 2007, 54(4): 260–265. DOI: 10.1007/s00284-006-0068-8
[51] Li JF, Hong YZ, Xiao YZ. Cloning and heterologous expression of the gene of laccase C from Trametes sp. 420 and potential of recombinant laccase in dye decolorization. Acta Microbiol Sin, 2007, 47(1): 54–58. (in Chinese).
李剑凤, 洪宇植, 肖亚中. 栓菌420漆酶C基因的克隆、高效表达及重组酶的染料脱色潜能. 微生物学报, 2007, 47(1): 54-58.
[52] Yaver DS, Xu F, Golightly EJ, et al. Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl Environ Microbiol, 1996, 62(3): 834–841.
[53] Larrondo LF, Avila M, Salas L, et al. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology, 2003, 149(5): 1177–1182. DOI: 10.1099/mic.0.26147-0
[54] Benghazi L, Record E, Suárez A, et al. Production of the Phanerochaete flavido-alba laccase in Aspergillus niger for synthetic dyes decolorization and biotransformation. World J Microbiol Biotechnol, 2014, 30(1): 201–211. DOI: 10.1007/s11274-013-1440-z
[55] Ramos JAT, Barends S, Verhaert RMD, et al. The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes. Microb Cell Fact, 2011, 10: 78. DOI: 10.1186/1475-2859-10-78
[56] Tamayo-Ramos JA, Barends S, de Lange D, et al. Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system. Biotechnol Bioeng, 2013, 110(2): 543–551. DOI: 10.1002/bit.24723
[57] Wang YC, Xue W, Sims AH, et al. Isolation of four pepsin-like protease genes from Aspergillus niger and analysis of the effect of disruptions on heterologous laccase expression. Fungal Genet Biol, 2008, 45(1): 17–27. DOI: 10.1016/j.fgb.2007.09.012
[58] Kiiskinen LL, Kruus K, Bailey M, et al. Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology, 2004, 150(9): 3065–3074. DOI: 10.1099/mic.0.27147-0
[59] Zhang JW, Qu YB, Xiao P, et al. Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2. J Biosci Bioeng, 2012, 113(6): 697–703. DOI: 10.1016/j.jbiosc.2012.01.016
[60] Dong XR, Qin LN, Tao Y, et al. Overexpression and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei. Acta Microbiol Sin, 2012, 52(7): 850–856. (in Chinese).
董欣睿, 秦丽娜, 陶勇, 等. 糙皮侧耳 (Pleurotus ostreatus) 漆酶POXA1在里氏木霉中的高效表达及酶学性质. 微生物学报, 2012, 52(7): 850-856.
[61] Abianova AR, Chulkin AM, Vavilova EA, et al. A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens. Prikl Biokhim Mikrobiol, 2010, 46(3): 342–347.
[62] Fujihiro S, Higuchi R, Hisamatsu S, et al. Metabolism of hydroxylated PCB congeners by cloned laccase isoforms. Appl Microbiol Biotechnol, 2009, 82(5): 853–860. DOI: 10.1007/s00253-008-1798-2
[63] Skálová T, Dohnálek J, østergaard LH, et al. The structure of the small laccase from Streptomyces coelicolor reveals a link between laccases and nitrite reductases. J Mol Biol, 2009, 385(4): 1165–1178. DOI: 10.1016/j.jmb.2008.11.024
[64] Andberg M, Hakulinen N, Auer S, et al. Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure. FEBS J, 2009, 276(21): 6285–6300. DOI: 10.1111/j.1742-4658.2009.07336.x
[65] Berka RM, Schneider P, Golightly EJ, et al. Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol, 1997, 63(8): 3151–3157.
[66] Bukh C, Lund M, Bjerrum MJ. Kinetic studies on the reaction between Trametes villosa laccase and dioxygen. J Inorg Biochem, 2006, 100(9): 1547–1557. DOI: 10.1016/j.jinorgbio.2006.05.007
[67] Hakulinen N, Kruus K, Koivula A, et al. A crystallographic and spectroscopic study on the effect of X-ray radiation on the crystal structure of Melanocarpus albomyces laccase. Biochem Biophys Res Commun, 2006, 350(4): 929–934. DOI: 10.1016/j.bbrc.2006.09.144
[68] Sigoillot C, Camarero S, Vidal T, et al. Comparison of different fungal enzymes for bleaching high-quality paper pulps. J Biotechnol, 2005, 115(4): 333–343. DOI: 10.1016/j.jbiotec.2004.09.006
[69] Ravalason H, Herpoël-Gimbert I, Record E, et al. Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching. J Biotechnol, 2009, 142(3/4): 220–226.
[70] Li L, Steffens JC. Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta, 2002, 215(2): 239–247. DOI: 10.1007/s00425-002-0750-4
[71] Chiaiese P, Palomba F, Tatino F, et al. Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater. Enzyme Microb Technol, 2011, 49(6/7): 540–546.
[72] Cesarino I, Araújo P, Mayer JLS, et al. Expression of SofLAC, a new laccase in sugarcane, restores lignin content but not S׃G ratio of Arabidopsis lac17 mutant. J Exp Bot, 2013, 64(6): 1769–1781. DOI: 10.1093/jxb/ert045
[73] Arakane Y, Muthukrishnan S, Beeman RW, et al. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proc Natl Acad Sci USA, 2005, 102(32): 11337–11342. DOI: 10.1073/pnas.0504982102
[74] Dittmer NT, Gorman MJ, Kanost MR. Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol, 2009, 39(9): 596–606. DOI: 10.1016/j.ibmb.2009.06.006
[75] Coy MR, Salem TZ, Denton JS, et al. Phenol-oxidizing laccases from the termite gut. Insect Biochem Mol Biol, 2010, 40(10): 723–732. DOI: 10.1016/j.ibmb.2010.07.004
[76] Hong F, Meinander NQ, JÖnsson LJ. Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnol Bioeng, 2002, 79(4): 438–449. DOI: 10.1002/bit.v79:4
[77] Guo M, Lu FP, Du LX, et al. Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica. Appl Microbiol Biotechnol, 2006, 71(6): 848–852. DOI: 10.1007/s00253-005-0210-8
[78] Liu W, Chao Y, Liu S, et al. Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris. Appl Microbiol Biotechnol, 2003, 63(2): 174–181. DOI: 10.1007/s00253-003-1398-0
[79] Rivera-Hoyos CM, Morales-álvarez ED, Poveda-Cuevas SA, et al. Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris. PLoS ONE, 2015, 10(1): e0116524. DOI: 10.1371/journal.pone.0116524
[80] Mate DM, Gonzalez-Perez D, Kittl R, et al. Functional expression of a blood tolerant laccase in Pichia pastoris. BMC Biotechnol, 2013, 13: 38. DOI: 10.1186/1472-6750-13-38
[81] Bohlin C, JÖnsson LJ, Roth R, et al. Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger. Appl Biochem Biotechnol, 2006, 129(1/2/3): 195–214.
[82] Brown MA, Zhao ZW, Mauk AG. Expression and characterization of a recombinant multi-copper oxidase: laccase Ⅳ from Trametes versicolor. Inorganica Chim Acta, 2002, 331(1): 232–238. DOI: 10.1016/S0020-1693(01)00814-3
[83] Guo M, Lu FP, Pu J, et al. Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica. Appl Microbiol Biotechnol, 2005, 69(2): 178–183. DOI: 10.1007/s00253-005-1985-3
[84] Hildén K, M?kel? MR, Lundell T, et al. Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus. Appl Microbiol Biotechnol, 2013, 97(4): 1589–1599. DOI: 10.1007/s00253-012-4011-6
[85] Lin YQ, Zhang Z, Tian YS, et al. Purification and characterization of a novel laccase from Coprinus cinereus and decolorization of different chemically dyes. Mol Biol Rep, 2013, 40(2): 1487–1494. DOI: 10.1007/s11033-012-2191-x
[86] Bulter T, Alcalde M, Sieber V, et al. Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol, 2003, 69(2): 987–995. DOI: 10.1128/AEM.69.2.987-995.2003
[87] Festa G, Autore F, Fraternali F, et al. Development of new laccases by directed evolution: functional and computational analyses. Proteins Struct Funct Bioinform, 2008, 72(1): 25–34. DOI: 10.1002/prot.21889