寧娜，譚慧軍，孫新新，倪金鳳

?

真核生物來源漆酶的異源表達(dá)研究進(jìn)展

寧娜，譚慧軍，孫新新，倪金鳳

山東大學(xué)微生物技術(shù)國家重點(diǎn)實(shí)驗(yàn)室，山東濟(jì)南 250100

漆酶屬于多銅氧化酶家族中的一種，廣泛存在于昆蟲、植物、真菌和細(xì)菌中。由于其作用的底物范圍較廣，因此在紡織、制漿、食品以及木質(zhì)素的降解等方面有廣闊的應(yīng)用前景。但是自然界中的漆酶存在表達(dá)量和酶活低、高溫易失活等問題，限制了它的應(yīng)用。對(duì)漆酶進(jìn)行大量高效的異源表達(dá)，是解決這一問題的有效途徑。近年來，越來越多不同來源的漆酶基因被克隆，并在不同宿主中異源表達(dá)。但這些大多局限于實(shí)驗(yàn)室研究，還未達(dá)到工業(yè)化生產(chǎn)的水平。筆者對(duì)真核生物來源漆酶的異源表達(dá)研究進(jìn)展進(jìn)行綜述，重點(diǎn)介紹了真核生物來源的漆酶在不同表達(dá)系統(tǒng)中的異源表達(dá)情況以及在酵母細(xì)胞中表達(dá)漆酶時(shí)提高表達(dá)量和酶活性能的方法，以期為研究者們提供參考。

真核漆酶，異源表達(dá)，酶活力，穩(wěn)定性

漆酶 (Laccase，苯二酚：雙氧氧化還原酶；EC 1.10.3.2) 與抗壞血酸氧化酶和各種鐵氧化酶同屬于多銅氧化酶家族[1]。漆酶最早被發(fā)現(xiàn)于植物中[2]，隨著分子生物學(xué)和生物信息學(xué)技術(shù)的發(fā)展，人們陸續(xù)在細(xì)菌[3]、真菌以及昆蟲[4]中發(fā)現(xiàn)了漆酶。不同來源的漆酶具有不同的體內(nèi)功能。細(xì)菌漆酶主要與細(xì)菌色素的形成和金屬離子抵抗有關(guān)[5-6]。也有報(bào)道稱芽孢桿菌中的漆酶，在抗紫外線孢子的發(fā)育中起作用[7]。目前研究最多的是真菌漆酶，主要集中于擔(dān)子菌和子囊菌中的漆酶，這類菌株多為白腐真菌[8]。白腐真菌是目前所知道的唯一能夠利用自身氧化酶系統(tǒng)將木質(zhì)素降解為二氧化碳的微生物[9]，漆酶在此過程中起了重要作用。另外，真菌漆酶還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]。植物漆酶雖然發(fā)現(xiàn)較早，但研究卻相對(duì)較少。植物中的漆酶主要與木質(zhì)素的合成以及損傷部位的修復(fù)有關(guān)[2]。也有少量的報(bào)道證明植物中的漆酶在植物種間競爭[11]以及植物抵抗微生物的侵染[12]中起作用。昆蟲漆酶根據(jù)生理作用的不同可以分為漆酶1和漆酶2。目前昆蟲漆酶的研究多集中于漆酶2在昆蟲外骨骼的硬化、表皮色素沉積以及角質(zhì)層鞣化方面的作用，而對(duì)可能在木質(zhì)素降解或食物脫毒中起作用的漆酶1的報(bào)道較少[13]。

漆酶底物范圍廣泛，主要包括酚類化合物、芳香族化合物、脂肪胺和無機(jī)陽離子等[14]。漆酶可以利用銅離子特有的氧化還原能力，對(duì)還原性底物進(jìn)行單電子氧化，同時(shí)傳遞4個(gè)電子，將作為第二底物的氧氣還原成水[15]。基于漆酶底物范圍的廣泛性以及副產(chǎn)物只有水的環(huán)境友善性，使得漆酶在木質(zhì)素降解、造紙工業(yè)、染料脫色、食品和飲料業(yè)等方面都具有潛在的應(yīng)用價(jià)值[16]。然而，自然界中分離到的野生菌株的漆酶有產(chǎn)量和活性較低、純化困難、高溫易失活、不易操作等缺點(diǎn)，很大程度上阻礙了對(duì)漆酶生理生化性質(zhì)的基礎(chǔ)研究及其工業(yè)化應(yīng)用[10,17-18]。近年來，越來越多不同來源的漆酶基因被克隆，并在不同宿主中異源表達(dá)。但這些大都局限于實(shí)驗(yàn)室研究，還未達(dá)到工業(yè)化生產(chǎn)的水平。提高漆酶的表達(dá)量、重組蛋白的耐熱性和pH穩(wěn)定性是目前漆酶工業(yè)應(yīng)用需要面對(duì)的問題。對(duì)于細(xì)菌漆酶的研究進(jìn)展已經(jīng)有學(xué)者對(duì)其進(jìn)行了總結(jié)[19-20]，因此文中將結(jié)合筆者的研究工作，對(duì)近年來真核來源的漆酶 (植物、真菌和昆蟲來源的漆酶) 的異源表達(dá)情況進(jìn)行概述，以期為相關(guān)研究者們提供參考。

1 真核生物來源的漆酶在不同表達(dá)系統(tǒng)中的表達(dá)

目前為止，異源表達(dá)真核生物來源的漆酶 (以下簡稱真核漆酶) 所用表達(dá)宿主主要有細(xì)菌、酵母、絲狀真菌和昆蟲桿狀細(xì)胞，其中使用最為廣泛的宿主是畢赤酵母。下面根據(jù)所使用表達(dá)系統(tǒng)的不同，對(duì)真核漆酶的異源表達(dá)情況進(jìn)行介紹。

1.1 真核漆酶在細(xì)菌中的表達(dá)

由于細(xì)菌具有好操作、培養(yǎng)周期短、經(jīng)濟(jì)實(shí)惠等特點(diǎn)，經(jīng)常被用來表達(dá)漆酶。其中大腸桿菌表達(dá)系統(tǒng)是應(yīng)用最廣泛的原核表達(dá)系統(tǒng)。表1顯示了真核漆酶在細(xì)菌中的表達(dá)情況。 楊建強(qiáng)等[21]將野生革耳來源的漆酶基因在大腸桿菌中表達(dá)，得到可溶性漆酶蛋白，在可溶漆酶蛋白中添加CuSO4并在室溫下孵育復(fù)性，獲得有活性的漆酶，這是首次報(bào)道的真菌來源的漆酶基因在大腸桿菌中實(shí)現(xiàn)表達(dá)。另外來自黑蛋巢菌[22]和白腐擔(dān)子菌類木硬孔菌[23]的真菌漆酶也在大腸桿菌中成功異源表達(dá)。

然而，潘程遠(yuǎn)等[24]在大腸桿菌中表達(dá)來源于雞樅菌的漆酶cDNA () 時(shí)，得到重組漆酶蛋白，但未檢測到漆酶活性。大腸桿菌雖然操作簡便，但一般不具備對(duì)重組蛋白進(jìn)行二級(jí)結(jié)構(gòu)修飾的能力，而漆酶是一種高糖基化修飾的酶，因此在大腸桿菌中表達(dá)真核來源的漆酶時(shí)往往會(huì)得到?jīng)]有酶活性的重組蛋白。

1.2 真核漆酶在酵母細(xì)胞中的表達(dá)

酵母是最低等的真核生物。具有操作簡單、生長速率快、可進(jìn)行高密度發(fā)酵；培養(yǎng)基廉價(jià)，適用于工業(yè)生產(chǎn)；不含有病原體、熱原體和病毒包涵體；可對(duì)重組蛋白進(jìn)行蛋白酶解、形成二硫鍵和糖基化等翻譯后修飾的優(yōu)點(diǎn)。但是對(duì)在酵母中異源表達(dá)的漆酶進(jìn)行糖基化分析，發(fā)現(xiàn)酵母對(duì)漆酶的糖基化有時(shí)會(huì)影響甚至破壞漆酶的酶活性質(zhì)和酶活力[26-27]。

盡管有一些酵母菌會(huì)產(chǎn)生自己的漆酶[28-29]，但是很多不同的酵母，例如釀酒酵母、畢赤酵母、乳酸克魯維酵母、解脂耶氏酵母、隱球酵母sp.作為宿主，都成功表達(dá)了其他真菌 (擔(dān)子菌門和子囊菌門) 來源的漆酶。表2總結(jié)了2007年以來截止到目前在酵母中成功表達(dá)的部分真核漆酶。非常有趣的是，漆酶的表達(dá)水平因酵母宿主和漆酶亞型的不同而不同。例如，在乳酸克魯維酵母.和釀酒酵母中表達(dá)來自于糙皮側(cè)耳中的漆酶POX3時(shí)，釀酒酵母中表達(dá)的漆酶酶活性比在乳酸克魯維酵母中的高[30]。Gu等[31]在畢赤酵母中表達(dá)來自于毛頭鬼傘的漆酶Lac3和Lac4時(shí)，Lac4的酶活可達(dá)到 1 465 U/L，而Lac3的酶活為689 U/L。表達(dá)漆酶時(shí)在培養(yǎng)基中加入不同濃度的硫酸銅 (0.1–1.0 mmol/L)，可以促進(jìn)漆酶的表達(dá)并提高酶活力。Pezzella等[30]在釀酒酵母中表達(dá)來自于糙皮側(cè)耳中的漆酶，不加硫酸銅時(shí)酶活為30 U/L，加入0.6 mmol/L硫酸銅時(shí)酶活提高至75 U/L。另外在培養(yǎng)過程中，達(dá)到最大酶活的時(shí)間差異較大，從48–504 h不等，這種差異主要與漆酶亞型、表達(dá)載體以及培養(yǎng)規(guī)模等相關(guān)。酵母菌產(chǎn)生的重組漆酶在應(yīng)用方面的研究主要集中于染料脫色方面 (表2)，也有關(guān)于漆酶對(duì)酚類化合物的降解作用[32]以及對(duì)植物生長的促進(jìn)作用[33]的相關(guān)研究。

表1 漆酶在細(xì)菌中的表達(dá)

Table 1 The expression of laccase genes in bacteria

Laccase originExpression hostsSubstratesActiveReferences Panus rudisEscherichia coliABTSYes[21] Cyathus bulleriEscherichia coliABTSYes[22] Rigidoporus lignosusEscherichia coliSyringaldazineYes[23] Termitomyce albuminosusEscherichia coliABTSNo[24] Pleurotus eryngiiLactobacillus buchneriNRNR[25]

ABTS: 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported.

表2 漆酶在酵母細(xì)胞中的表達(dá)

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 lagenariumCILACIIPichia pastoris GS115pPIC9KNRNRNRDecolorization of dyes[34] Moniliophthora perniciosa FA553LacMPPichia pastoris X33pPICZaA-6AA-LacMP0.496232NR[35] Volvariella volvaceaVvlcc3Pichia pastoris GS115pPIC9K-Vvlcc30.3504296.83NR[36] Basidiomycete cerrena sp.Lac1Pichia pastoris X33pPICZα-Lac11.02166 300Decolorization of dyes[37] Phytophthora capsiciPCLAC2Pichia pastoris GS115pPIC9K/Pclac2NR16884 000NR[38] Coprinopsis cinerea Okayama-7#130CcLcc2Pichia pastoris GS115pYM7898NR72NRDyes decolorization[39] Coprinus comatusLac3Pichia pastorisKM71HpPICZαB-10AALac30.5336689Dyes decolorization[31] Lac4pPICZαB-10AALac41 465 Phomopsis liquidambariLACB3Schizosacchar-omyces pombepESP-3-lacB3NRNRNRGrowth promotion of plants[33] Volvaria volvaceavv-lac1Pichia pastoris GS115pPIC9K-vv-lac1NRNR333.17NR[40] vv-1ac6pPIC9K-vv-lac6227.63 Canoderma lucidum TR6glacTR6Pichia pastoris GS115pPIC9K-glacTR60.3?432685.8?NR[41] Coprinus comatusLac1Pichia pastoris KM71HpPICZαB-lac10.5144550Dyes decolorization[42] Cyathus bulleriLccPichia pastoris X33pPICZαBlcc- 50.4727 200NR[43] Monilinia fructigenaMfLccPichia pastorisGS115pYM8025NR48NRDegradation of phenolic compounds[32] Botrytis acladaBaLacPichia pastoris X33pGAPZA- BaLac0.17653 300NR[44] Ganoderma lucidumGlLCCIPichia pastoris GS115pYM 79091.072NRDecolorization of methylorange[45] Polyporus grammocephalus TR16Lac-T16Pichia pastoris GS115pPIC3.5K-TR16lac0.3264320·8NR[46] pPIC9K-TR16lacNRNRNR 續(xù)表2 Pleurotus ostreatusrPOX3Saccharomyces cerevisiaeW303-1ApSAL4- POX3NA4830NR[30] 0.64875 Kluyveromyces lactispYG132- POX3NA7212 Pleurotus eryngiiEry3Free Saccharomyces cerevisiae cellspY-Ery30.57288NR[47] Immobilized Saccharomyces cerevisiae cells0.5120139 Pleurotus ostreatusPOXA3Kluyveromyces lactisA3L1.0336–40820NR[48] Ganoderma lucidumGLlac1Pichia pastorispPIC-RCL1NR168NRAntioxidative properties[49] Trametes sp.420rLacDPichia pastoris GS115pPIC9K-LacD0.3NR83 000Decolorization of dyes[50] Trametes sp.420rLacCPichia pastoris GS115pPIC9K-LacC0.321616 200Dye decolorization[51]

ABTS: 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate); NR: not reported; NA: not added.

目前為止，還沒有昆蟲來源的漆酶在酵母中成功異源表達(dá)的相關(guān)報(bào)道。筆者嘗試在畢赤酵母中表達(dá)來自黃翅大白蟻唾液腺-前腸的漆酶基因，盡管使用了不同菌株作為宿主并優(yōu)化了培養(yǎng)條件，但并未得到重組蛋白?？赡苁怯捎诿艽a子的偏好性、糖基化位點(diǎn)以及二硫鍵數(shù)目較多等原因而使昆蟲來源的漆酶難以在畢赤酵母中正常表達(dá)。

1.3 真核漆酶在絲狀真菌中的表達(dá)

目前為止，漆酶已經(jīng)在曲霉屬sp.[52-57]、里氏木霉[58-60]和灰白青霉[61]中表達(dá) (表3)。大多數(shù)情況下絲狀真菌用來表達(dá)來源于白腐真菌的漆酶。例如栓菌屬sp. 的漆酶在里氏木霉[59]和曲霉[57-62]中成功表達(dá)。除了真菌漆酶，也有細(xì)菌來源的 (天藍(lán)色鏈霉菌) 漆酶在米曲霉中成功表達(dá)，并解析了它的三維結(jié)構(gòu)[63]。由于在絲狀真菌中表達(dá)重組漆酶時(shí)有一個(gè)相對(duì)較高的蛋白產(chǎn)量，因此有利于研究漆酶的分子量[52,64-65]、反應(yīng)機(jī)制[66]和三維結(jié)構(gòu)[63-67]。絲狀真菌產(chǎn)生的重組漆酶在紙漿脫色[68-69]、合成染料的生物轉(zhuǎn)化[54]以及植物修復(fù)[62]中廣泛應(yīng)用。另外絲狀真菌表達(dá)的重組漆酶還用于食品工業(yè)黃曲霉毒素的消除、環(huán)境中酚類化合物的檢測中。

表3 部分絲狀真菌中表達(dá)的重組漆酶的特性

Table 3 Properties of some recombinant laccases expressed in filamentous fungi

Laccase originExpression hostsSubstratesMaximum enzyme activity (U/L) Optimum temperature (℃)Optimum pHReferences Trametes villosaAspergillus oryzaeSyringaldazineNRNR5.0–5.5[52] ABTS8 250NR2.7 Ceriporiopsis subvermisporaAspergillus nidulansABTS230NRNR[53] Aspergillus niger Phanerochaete flavido-albaAspergillus nigerABTS2 500NR3.0[54] Trichoderma reeseiiTrichoderma reeseiABTS46 800NRNR[58] GuaiacolNR5.0–7.0 Trametes sp. AH28-2Trichoderma reeseiABTS3 620503.0[59] GuaiacolNR504.2 Pleurotus ostreatusTrichoderma reeseiABTS237 134503.0[60] Trametes hirsutePenicillium 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達(dá)

植物主要用來表達(dá)來自不同植物或真菌的漆酶。主要的植物表達(dá)宿主有擬南芥、番茄、水稻、煙草、甘蔗、玉米等。在番茄中過量表達(dá)來自馬鈴薯的多酚氧化酶，表達(dá)植株對(duì)丁香假單胞菌的抵抗能力明顯增強(qiáng)[70]。有些情況下，構(gòu)建表達(dá)漆酶的轉(zhuǎn)基因植物是為了建立植物修復(fù)系統(tǒng)，來降解雙酚、三氯苯酚、五氯苯酚及其他酚類化合物[72]。在甘蔗中表達(dá)漆酶，有助于闡明漆酶在木質(zhì)化過程中的作用[73]。

1.5 真核漆酶在昆蟲細(xì)胞中的表達(dá)

目前發(fā)現(xiàn)的動(dòng)物來源的漆酶主要集中于昆蟲漆酶，在甲殼類以及棘皮動(dòng)物中也有發(fā)現(xiàn)。根據(jù)昆蟲漆酶的功能和表達(dá)部位的不同，將分布于昆蟲唾液腺、馬氏管和中腸組織中的漆酶稱為漆酶1 (laccase 1)，此類漆酶可能參與食物的脫毒作用[4]；而將表達(dá)于表皮、卵殼等組織的漆酶稱為漆酶2 (laccase 2)，此類漆酶與昆蟲角質(zhì)層的黑化作用有關(guān)[73]。

Dittmer等[74]利用昆蟲桿狀病毒表達(dá)系統(tǒng)，成功表達(dá)了煙草天蛾來源的多銅氧化酶漆酶2。該研究表明在昆蟲桿狀病毒表達(dá)系統(tǒng)中表達(dá)的重組昆蟲漆酶可以代替內(nèi)源的酶來測定生化性質(zhì)，幫助我們更廣泛地了解煙草天蛾來源的漆酶以及其他昆蟲來源漆酶的酶學(xué)性質(zhì)和功能。Coy等[75]通過RACE的方法獲得了美洲散白蟻唾液腺-前腸中的兩個(gè)漆酶基因 (和)，因?yàn)樵撁冈谖⑸锶狈Φ耐僖合俸颓澳c中活性最高，并且進(jìn)化上比較特別，推測該酶由白蟻?zhàn)陨懋a(chǎn)生。將這兩個(gè)基因在昆蟲桿狀病毒表達(dá)系統(tǒng)中表達(dá)，純化后測定酶活，發(fā)現(xiàn)這兩個(gè)酶對(duì)木質(zhì)素的單體芥子酸及其他幾種酚氧化物具有較強(qiáng)的活力，而對(duì)黑色素前體沒有或有很低的酶活力，這就為白蟻唾液腺和前腸產(chǎn)生的漆酶在木質(zhì)纖維素降解中起作用提供了重要的證據(jù)。然而，我們前期實(shí)驗(yàn)嘗試將黃翅大白蟻中腸來源的漆酶基因在昆蟲桿狀病毒系統(tǒng)中表達(dá)，但是并沒有成功表達(dá)。昆蟲桿狀病毒表達(dá)系統(tǒng)的使用，使得對(duì)昆蟲漆酶的研究成為可能。但是該表達(dá)系統(tǒng)與細(xì)菌和真菌表達(dá)系統(tǒng)相比，操作較為繁瑣，價(jià)格和成本也比較高。

2 漆酶表達(dá)量及酶活性能的提高

如前所述，漆酶應(yīng)用方面存在表達(dá)困難、熱穩(wěn)定性和pH耐受性較差和酶活力較低等問題。目前提高漆酶表達(dá)量的方法主要有兩個(gè)方面：構(gòu)建重組基因和控制培養(yǎng)條件及產(chǎn)酶條件。在提高酶活力和熱穩(wěn)定性方面，主要的方法包括定點(diǎn)突變、隨機(jī)突變和DNA Shuffling等基因工程的方法。由于酵母是目前表達(dá)漆酶應(yīng)用最廣泛的宿主，因此更多的研究集中于漆酶在酵母細(xì)胞中的表達(dá)優(yōu)化，下面對(duì)此進(jìn)行介紹。

2.1 啟動(dòng)子的選擇

在表達(dá)漆酶時(shí)，首先要選擇一個(gè)合適的強(qiáng)啟動(dòng)子。在釀酒酵母中表達(dá)漆酶時(shí)，使用強(qiáng)啟動(dòng)子半乳糖激酶 (GAL1或GAL10) 啟動(dòng)子，利用半乳糖進(jìn)行誘導(dǎo)，可以使漆酶有一個(gè)較高水平的表達(dá)[64]。選擇銅結(jié)合蛋白 (CUP1) 啟動(dòng)子，在培養(yǎng)基中加入CuSO4至終濃度0.4–0.5 mmol/L，不僅可以誘導(dǎo)重組蛋白大量表達(dá)，也為漆酶提供了適量的銅離子。盡管這些誘導(dǎo)型啟動(dòng)子的使用，使得漆酶的表達(dá)量較高，但是由于誘導(dǎo)物的成本較高，并不適用于工業(yè)生產(chǎn)。從生產(chǎn)成本方面考慮，強(qiáng)的組成型啟動(dòng)子更具優(yōu)勢(shì)。在釀酒酵母中應(yīng)用于表達(dá)漆酶的組成型啟動(dòng)子主要有乙醇脫氫酶 (ADH1) 啟動(dòng)子、磷酸甘油酸激酶 (PGK1) 啟動(dòng)子、磷酸丙糖異構(gòu)酶 (TPI1) 啟動(dòng)子、翻譯延伸因子-1 (TEF1) 啟動(dòng)子以及甘油醛- 3-磷酸脫氫酶 (GPD1) 啟動(dòng)子。在畢赤酵母中表達(dá)漆酶時(shí)，最常用的是甲醇誘導(dǎo)型的乙醇氧化酶 (AOX1) 啟動(dòng)子[43]。有研究顯示當(dāng)用0.5%的甲醇誘導(dǎo)時(shí)，比用2.0%甲醇誘導(dǎo)時(shí)表達(dá)水平要高出5倍，從2.0 U/mL提高到11.5 U/mL[76]。類似的，在中使用甲醇誘導(dǎo)型啟動(dòng)子 (AUG1) 來表達(dá)漆酶時(shí)，最適合的產(chǎn)漆酶的甲醇濃度為0.8%，同樣低于1%[77]。由于甲醇有毒并且成本較高，因此人們也在努力尋求不需甲醇誘導(dǎo)的啟動(dòng)子。在畢赤酵母中利用組成型的GAP啟動(dòng)子，成功表達(dá)了來自于木質(zhì)層孔菌[78]、靈芝[79]、擔(dān)子菌類[80]、糙皮側(cè)耳[79]以及云芝[81]來源的漆酶基因。

2.2 信號(hào)肽的選擇

通常真核生物中的漆酶均分泌到胞外，這是由于在N-末端帶有信號(hào)肽，促使蛋白分泌，分泌到胞外的蛋白利于純化和應(yīng)用。在酵母細(xì)胞中異源表達(dá)漆酶時(shí)，除了利用漆酶自身的信號(hào)肽之外，也可使用酵母的信號(hào)肽。通常應(yīng)用比較多的是來源于釀酒酵母的信號(hào)肽序列α-factor以及它的修改序列[80]。另外，蔗糖酶基因 ()、胞外堿性蛋白酶基因 ()、木聚糖酶(Xylanase) 基因的信號(hào)肽也可以用于漆酶基因在酵母中的高效表達(dá)[16]。值得注意的是，有時(shí)使用漆酶自身的信號(hào)肽比使用α-factor信號(hào)肽序列得到的重組蛋白活力要高，而有時(shí)情況則相反。例如，在畢赤酵母中表達(dá)來自于云芝的漆酶基因laccase IV時(shí)，使用α-factor信號(hào)肽，致使分泌的重組蛋白N-端殘留了一個(gè)四肽，使酶比活相對(duì)于使用自身信號(hào)肽時(shí)下降了25%，從0.88 U/mg下降為0.68 U/mg[82]。而在中表達(dá)來自云芝的漆酶基因Lcc1，使用α-factor信號(hào)肽時(shí)的活力 (3.17 U/mL) 是自身信號(hào)肽 (1.87 U/mL) 的1.7倍[83]。類似的，在畢赤酵母中表達(dá)來自的漆酶時(shí)，使用α-factor信號(hào)肽時(shí)的酶活是使用自身信號(hào)肽的1.6倍，分別為4.9 μkat/μg和3.14 μkat/μg[84]。以上這些結(jié)果的不同說明對(duì)于不同的漆酶和不同的宿主，需要對(duì)信號(hào)肽進(jìn)行特定的優(yōu)化來增加表達(dá)量和酶活力。

2.3 漆酶基因改造

很多研究報(bào)道，合成酵母密碼子偏好的漆酶基因，比用原始的漆酶序列獲得的重組蛋白更多。通過密碼子優(yōu)化的方式成功表達(dá)的漆酶有來源于灰蓋鬼傘的漆酶基因[85]、靈芝的漆酶基因[45-79]、桃褐腐病菌的漆酶基因[32]和糙皮側(cè)耳的漆酶基因[79]。

改造漆酶編碼基因，除了可以提高表達(dá)量，還可以提高漆酶的穩(wěn)定性、改變最適pH、改變m和cat以及提高底物親和力。改造基因使用的主要技術(shù)包括隨機(jī)突變、定向突變以及DNA Shuffling等。Bulter等[86]通過10輪的分子進(jìn)化使得來自于嗜熱毀絲霉的漆酶在釀酒酵母中的異源表達(dá)量提高了8倍，達(dá)到18 mg/L，總酶活提高了170倍，cat值提高22倍 (從 (80±2.5) min?1提高到(1 740±34) min?1)。Festa等[87]將糙皮側(cè)耳來源的漆酶POXA1b在畢赤酵母中表達(dá)，通過易錯(cuò)PCR和DNA Shuffling的方法使重組漆酶活性從(183±1) U/mg提高到(454±2) U/mg，在60 ℃、pH 7.0條件下的半衰期1/2從2.2 h提高至3.1 h。

另一種使得漆酶成功表達(dá)的方法是在漆酶基因N-端或C-端加入氨基酸標(biāo)簽來融合表達(dá)。例如Gu等[31]通過在來源于毛頭鬼傘的兩個(gè)新型漆酶同工酶的N-末端加入10個(gè)氨基酸組成的標(biāo)簽，使得這兩個(gè)漆酶同工酶成功地在畢赤酵母中異源表達(dá)，可能是增加這10個(gè)氨基酸標(biāo)簽后，有利于蛋白的分泌表達(dá)。

3 展望

毫無疑問，漆酶在工業(yè)和生物技術(shù)中都有著廣泛的應(yīng)用前景，是一種環(huán)境友好型的木質(zhì)素降解酶。在自然界中，白腐真菌是漆酶主要的生產(chǎn)者，但野生型真菌漆酶的產(chǎn)量低、培養(yǎng)周期長，限制了漆酶的大規(guī)模應(yīng)用。隨著分子生物學(xué)技術(shù)和基因工程技術(shù)的發(fā)展，已經(jīng)有一些不同來源的漆酶在真核和原核表達(dá)系統(tǒng)中實(shí)現(xiàn)異源表達(dá)。但是，目前真核漆酶的異源表達(dá)情況還不理想，還未達(dá)到工業(yè)化水平。酵母細(xì)胞具有真核中的翻譯后修飾能力、培養(yǎng)基廉價(jià)、遺傳操作簡單和可以分泌表達(dá)等優(yōu)勢(shì)，因此用作漆酶異源表達(dá)的宿主是非常有前景的。未來將加強(qiáng)分子生物學(xué)和分子遺傳學(xué)方面的研究，更加全面詳細(xì)地了解漆酶的結(jié)構(gòu)和功能，進(jìn)而在漆酶的基因水平上加以改造，逐步實(shí)現(xiàn)真正的高效異源表達(dá)，達(dá)到工業(yè)化水平。

REFERENCES

[1] Thurston CF. The structure and function of fungal laccases. Microbiology, 1994, 140(1): 19–26.

[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.

[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,, and the malaria mosquito,. Insect Biochem Mol Biol, 2004, 34(1): 29–41.

[5] Hullo MF, Moszer I, Danchin A, et al. CotA ofis a copper-dependent laccase. J Bacteriol, 2001, 183(18): 5426–5430.

[6] Grass G, Rensing C. CueO is a multi-copper oxidase that confers copper tolerance in. Biochem Biophys Res Commun, 2001, 286(5): 902–908.

[7] Claus H. Laccases and their occurrence in prokaryotes. Arch Microbiol, 2003, 179(3): 145–150.

[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). 司靜, 李偉, 崔寶凱, 等. 真菌漆酶性質(zhì)、分子生物學(xué)及其應(yīng)用研究進(jìn)展. 生物技術(shù)通報(bào), 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.

[10] Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev, 2006, 30(2): 215–242.

[11] Wang GD, Li QJ, Luo B, et al.phytoremediation of trichlorophenol and phenolic allelochemicalsan engineered secretory laccase. Nat Biotechnol, 2004, 22(7): 893–897.

[12] Mayer AM, Staples RC. Laccase: new functions for an old enzyme. Phytochemistry, 2002, 60(6): 551–565.

[13] Yu ML, Ni JF. Research advances in the insect laccase. Chin J Bioproc Eng, 2014, 12(1): 80–85 (in Chinese).于孟蘭, 倪金鳳. 昆蟲漆酶的研究進(jìn)展. 生物加工過程, 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.

[15] Liu ZC, Wang GG. Fungal laccases: structure-based function and mechanism. Acta Biophys Sinica, 2013, 29(9): 629–645 (in Chinese). 劉忠川, 王剛剛. 真菌漆酶結(jié)構(gòu)與功能研究進(jìn)展. 生物物理學(xué)報(bào), 2013, 29(9): 629–645.

[16] Anto?ová Z, Sychrová H. Yeast hosts for the production of recombinant laccases: a review. Mol Biotechnol, 2016, 58(2): 93–116.

[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.

[18] Ausec L, ?rnigoj M, ?najder M, et al. Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity insp.. Appl Microbiol Biotechnol, 2015, 99(23): 9987–9999.

[19] Zhou W, Guan ZB, Cai YJ, et al. Progress in thelaccase. Microbiol China, 2015, 42(7): 1372–1383 (in Chinese). 周穩(wěn), 管政兵, 蔡宇杰, 等. 芽胞桿菌漆酶的研究進(jìn)展. 微生物學(xué)通報(bào), 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). 馬瑩瑩, 賈紅華, 韋萍. 細(xì)菌漆酶的研究及應(yīng)用進(jìn)展. 生物技術(shù)通報(bào), 2013, (2): 41–48.

[21] Yang JQ. Heterologous expression and modification of laccase from[D]. Beijing: Academy of Military Medical Science, 2005 (in Chinese). 楊建強(qiáng). 革耳() 漆酶的異源表達(dá)和酶學(xué)性質(zhì)改良[D]. 北京: 中國人民解放軍軍事醫(yī)學(xué)科學(xué)院, 2005.

[22] Salony, Garg N, Baranwal R, et al. Laccase of: structural, catalytic characterization and expression in. Biochim Biophys Acta Proteins Proteomics, 2008, 1784(2): 259–268.

[23] Nicolini C, Bruzzese D, Cambria MT, et al. Recombinant laccase: I. Enzyme cloning and characterization. J Cell Biochem, 2013, 114(3): 599–605.

[24] Pan CY. Cloning and expression of genes encoding laccases in,and[D]. Hangzhou: Zhejiang University, 2009 (in Chinese). 潘程遠(yuǎn). 雞樅菌與淡色庫蚊及家蠅中漆酶編碼基因的克隆與表達(dá)[D]. 杭州: 浙江大學(xué), 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電激轉(zhuǎn)化體系的建立[D]. 呼和浩特: 內(nèi)蒙古大學(xué), 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.

[27] Maestre-Reyna M, Liu WC, Jeng WY, et al. Structural and functional roles of glycosylation in fungal laccase fromsp.. PLoS ONE, 2015, 10(4): e0120601.

[28] Jadhav JP, Parshetti GK, Kalme SD, et al. Decolourization of azo dye methyl red byMTCC 463. Chemosphere, 2007, 68(2): 394–400.

[29] Kalyani D, Tiwari MK, Li JL, et al. A highly efficient recombinant laccase from the yeastand its application in the hydrolysis of biomass. PLoS ONE, 2015, 10(3): e0120156.

[30] Pezzella C, Autore F, Giardina P, et al. Thelaccase multi-gene family: isolation and heterologous expression of new family members. Curr Genet, 2009, 55(1): 45–57.

[31] Gu CJ, Zheng F, Long LK, et al. Engineering the expression and characterization of two novel laccase isoenzymes frominby fusing an additional ten amino acids tag at N-terminus. PLoS ONE, 2014, 9(4): e93912.

[32] Bao WH, Peng RH, Zhang Z, et al. Expression, characterization and 2,4,6-trichlorophenol degradation of laccase from. Mol Biol Rep, 2012, 39(4): 3871–3877.

[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 fungusand its potential for growth promotion of plants. Biotechnol Lett, 2014, 36(1): 167–173.

[34] Wang B, Yan Y, Tian YS, et al. Heterologous expression and characterisation of a laccase fromand decolourisation of different synthetic dyes. World J Microbiol Biotechnol, 2016, 32(3): 40.

[35] Liu HP, Tong CF, Du B, et al. Expression and characterization of LacMP, a novel fungal laccase ofFA553. Biotechnol Lett, 2015, 37(9): 1829–1835.

[36] Lu YP, Wu GM, Lian LD, et al. Cloning and expression analysis of, a novel and functional laccase gene possibly involved in stipe elongation. Int J Mol Sci, 2015, 16(12): 28498–28509.

[37] Yang J, Ng TB, Lin J, et al. A novel laccase from basidiomycetesp.: cloning, heterologous expression, and characterization. Int J Biol Macromol, 2015, 77: 344–349.

[38] Feng BZ, Li PQ. Cloning, characterization and expression of a novel laccase genefrom. Braz J Microbiol, 2014, 45(1): 351–358.

[39] Tian YS, Xu H, Peng RH, et al. Heterologous expression and characterization of laccase 2 fromcapable of decolourizing different recalcitrant dyes. Biotechnol Biotechnol Equip, 2014, 28(2): 248–258.

[40] Wu L, Yang JH, Chen MJ, et al. Cloning and heterologous expression of laccase genes1 and6 from. Acta Microbiol Sin, 2014, 54(7): 828–835(in Chinese).吳林, 陽經(jīng)慧, 陳明杰, 等. 草菇漆酶基因1和6的克隆及異源表達(dá). 微生物學(xué)報(bào), 2014, 54(7): 828–835.

[41] You LF, Liu ZM, Lin JF, et al. Molecular cloning of a laccase gene fromand heterologous expression in. 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. Mol Biol Rep, 2013, 40(2): 1927–1936.

[43] Garg N, Bieler N, Kenzom T, et al. Cloning, sequence analysis, expression oflaccase inand characterization of recombinant laccase. BMC Biotechnol, 2012, 12: 75.

[44] Kittl R, Gonaus C, Pillei C, et al. Constitutive expression oflaccase in. Bioengineered, 2012, 3(4): 232–235.

[45] Sun J, Peng RH, Xiong AS, et al. Secretory expression and characterization of a soluble laccase from thestrain 7071-9 in. Mol Biol Rep, 2012, 39(4): 3807–3814.

[46] Huang SJ, Liu ZM, Huang XL, et al. Molecular cloning and characterization of a novel laccase gene from a white-rot fungusTR16 and expression in. Lett Appl Microbiol, 2011, 52(3): 290–297.

[47] Bleve G, Lezzi C, Mita G, et al. Molecular cloning and heterologous expression of a laccase gene fromin free and immobilizedcells. Appl Microbiol Biotechnol, 2008, 79(5): 731–741.

[48] Faraco V, Ercole C, Festa G, et al. Heterologous expression of heterodimeric laccase fromin. Appl Microbiol Biotechnol, 2008, 77(6): 1329–1335.

[49] Joo SS, Ryu IW, Park JK, et al. Molecular cloning and expression of a laccase from, 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 basidiomycetesp. 420 and its heterologous expression in. Curr Microbiol, 2007, 54(4): 260–265.

[51] Li JF, Hong YZ, Xiao YZ. Cloning and heterologous expression of the gene of laccase C fromsp. 420 and potential of recombinant laccase in dye decolorization. Acta Microbiol Sin, 2007, 47(1): 54–58 (in Chinese).李劍鳳, 洪宇植, 肖亞中. 栓菌420漆酶C基因的克隆、高效表達(dá)及重組酶的染料脫色潛能. 微生物學(xué)報(bào), 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. Appl Environ Microbiol, 1996, 62(3): 834–841.

[53] Larrondo LF, Avila M, Salas L, et al. Heterologous expression of laccase cDNA fromyields copper-activated apoprotein and complex isoform patterns. Microbiology, 2003, 149(5): 1177–1182.

[54] Benghazi L, Record E, Suárez A, et al. Production of thelaccase infor synthetic dyes decolorization and biotransformation. World J Microbiol Biotechnol, 2014, 30(1): 201–211.

[55] Ramos JAT, Barends S, Verhaert RMD, et al. Themulticopper oxidase family: analysis and overexpression of laccase-like encoding genes. Microb Cell Fact, 2011, 10: 78.

[56] Tamayo-Ramos JA, Barends S, de Lange D, et al. Enhanced production oflaccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system. Biotechnol Bioeng, 2013, 110(2): 543–551.

[57] Wang YC, Xue W, Sims AH, et al. Isolation of four pepsin-like protease genes fromand analysis of the effect of disruptions on heterologous laccase expression. Fungal Genet Biol, 2008, 45(1): 17–27.

[58] Kiiskinen LL, Kruus K, Bailey M, et al. Expression oflaccase inand characterization of the purified enzyme. Microbiology, 2004, 150(9): 3065–3074.

[59] Zhang JW, Qu YB, Xiao P, et al. Improved biomass saccharification bythrough heterologous expression ofgene fromsp. AH28-2. J Biosci Bioeng, 2012, 113(6): 697–703.

[60] Dong XR, Qin LN, Tao Y, et al. Overexpression and characterization of a laccase gene fromin. Acta Microbiol Sin, 2012, 52(7): 850–856 (in Chinese).董欣睿, 秦麗娜, 陶勇, 等. 糙皮側(cè)耳()漆酶POXA1在里氏木霉中的高效表達(dá)及酶學(xué)性質(zhì). 微生物學(xué)報(bào), 2012, 52(7): 850–856.

[61] Abianova AR, Chulkin AM, Vavilova EA, et al. A heterologous production of thelaccase in the fungus. 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.

[63] Skálová T, Dohnálek J, ?stergaard LH, et al. The structure of the small laccase fromreveals a link between laccases and nitrite reductases. J Mol Biol, 2009, 385(4): 1165–1178.

[64] Andberg M, Hakulinen N, Auer S, et al. Essential role of the C-terminus inlaccase for enzyme production, catalytic properties and structure. FEBS J, 2009, 276(21): 6285–6300.

[65] Berka RM, Schneider P, Golightly EJ, et al. Characterization of the gene encoding an extracellular laccase ofand analysis of the recombinant enzyme expressed in. Appl Environ Microbiol, 1997, 63(8): 3151–3157.

[66] Bukh C, Lund M, Bjerrum MJ. Kinetic studies on the reaction betweenlaccase and dioxygen. J Inorg Biochem, 2006, 100(9): 1547–1557.

[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 oflaccase. Biochem Biophys Res Commun, 2006, 350(4): 929–934.

[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.

[69] Ravalason H, Herpo?l-Gimbert I, Record E, et al. Fusion of a family 1 carbohydrate binding module ofto thelaccase 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.

[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, a new laccase in sugarcane, restores lignin content but not S?G ratio ofmutant. J Exp Bot, 2013, 64(6): 1769–1781.

[73] Arakane Y, Muthukrishnan S, Beeman RW, et al.is the phenoloxidase gene required for beetle cuticle tanning. Proc Natl Acad Sci USA, 2005, 102(32): 11337–11342.

[74] Dittmer NT, Gorman MJ, Kanost MR. Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm,. Insect Biochem Mol Biol, 2009, 39(9): 596–606.

[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.

[76] Hong F, Meinander NQ, J?nsson LJ. Fermentation strategies for improved heterologous expression of laccase in. Biotechnol Bioeng, 2002, 79(4): 438–449.

[77] Guo M, Lu FP, Du LX, et al. Optimization of the expression of a laccase gene fromin. Appl Microbiol Biotechnol, 2006, 71(6): 848–852.

[78] Liu W, Chao Y, Liu S, et al. Molecular cloning and characterization of a laccase gene from the basidiomyceteand expression in. Appl Microbiol Biotechnol, 2003, 63(2): 174–181.

[79] Rivera-Hoyos CM, Morales-álvarez ED, Poveda-Cuevas SA, et al. Computational analysis and low-scale constitutive expression of laccases synthetic genesfromandfromin. PLoS ONE, 2015, 10(1): e0116524.

[80] Mate DM, Gonzalez-Perez D, Kittl R, et al. Functional expression of a blood tolerant laccase in. BMC Biotechnol, 2013, 13: 38.

[81] Bohlin C, J?nsson LJ, Roth R, et al. Heterologous expression oflaccase inand. 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. Inorganica Chim Acta, 2002, 331(1): 232–238.

[83] Guo M, Lu FP, Pu J, et al. Molecular cloning of the cDNA encoding laccase fromand heterologous expression in. Appl Microbiol Biotechnol, 2005, 69(2): 178–183.

[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. Appl Microbiol Biotechnol, 2013, 97(4): 1589–1599.

[85] Lin YQ, Zhang Z, Tian YS, et al. Purification and characterization of a novel laccase fromand decolorization of different chemically dyes. Mol Biol Rep, 2013, 40(2): 1487–1494.

[86] Bulter T, Alcalde M, Sieber V, et al. Functional expression of a fungal laccase inby directed evolution. Appl Environ Microbiol, 2003, 69(2): 987–995.

[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.

(本文責(zé)編 郝麗芳)

Advance of heterologous expression study of eukaryote-origin laccases

Na Ning, Huijun Tan, Xinxin Sun, and Jinfeng Ni

State Key Laboratory of Microbial Technology, Shandong University, Ji’nan 250100, Shandong, China

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.

eukaryote-origin laccases, heterologous expression, enzyme activity, stability

Supported by:National Basic Research and Development Program of China (973 Program) (No. 2011CB707402), National Natural Science Foundation of China (Nos. 31272370, 30870085).

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃 (973計(jì)劃) (No. 2011CB707402)，國家自然科學(xué)基金 (Nos. 31272370, 30870085) 資助。

September 21, 2016; Accepted: January 9, 2017

Jinfeng Ni. Tel: +86-531-88363323; E-mail: jinfgni@sdu.edu.cn

網(wǎng)絡(luò)出版時(shí)間：2017-01-16

http://www.cnki.net/kcms/detail/11.1998.Q.20170116.1003.001.html