漆酶来源与应用

漆酶来源与应用
万云洋1,2，杜予民2
1.中国石油大学（北京）资源与信息学院，北京（102249）
2.武汉大学资源与环境科学学院，武汉（430079）
E-mail ：yunyangwan@
摘 要：本文对漆酶来源，包括动物、微生物和植物，尤其是我国的特产资源漆树及其他植
物漆酶，酶的稳定化及固定化，生物整治、对木质素的作用以及其各方面的应用作一综述。

关键词：漆酶，漆树，生物整治，木质素，固定化
漆酶(EC1.10.3.2)，对－二酚:(双)氧氧化还原酶，又名酚酶，多酚氧化酶，漆酚氧化酶
和等，是一种含铜的糖蛋白氧化酶，是多铜氧化酶的一种[1]。

对漆酶的研究已有一百多年的
历史，是有记载以来开发最早的酶之一：1883年，日本人吉田在研究生漆液成份时发现这
种酶成份，但当时他误为淀粉酶物质(diastatic matter)，1898年，法国人Bertrand 在研究越南
产漆液的时候，首次提出了漆酶(laccase)的概念并沿用至今[2-5]；Reinhammar 等[6;7]、杜予民
等[8-12]对漆酶及漆树液全成份的分离纯化作了很好的工作；另外，熊野等[13;14]对漆酶反应机
理，黄葆同、甘景镐[15]等对中国漆酶化学的发展，Morpurgo(意大利)[16;17]，Solomon(美国)
等[18-24]对漆酶铜原子中心的研究作出了各自的贡献。

漆酶虽然是研究史中的老酶，但其各种新功能也正在被发现和挖掘。

本文结合自身工作实践，专门就漆酶来源、特别是植物漆酶来
源和其各方面的应用研究作一综述，进一步推动漆酶(尤其是植物漆酶)研究的发展。

Figure 1. Dominating distribution of lacquer trees in the world.
1. 漆酶的来源
1.1植物漆酶
由上述可知，对漆酶的研究首先就是从漆树来源开始的。

漆树(Rhus vernicifera )
种属于
75 90 105120135 15015
30
45
被子植物亚门双子叶植物纲蔷薇亚纲无患子目漆树属(Toxicodendron)漆树科(Anacardiaceae)，源产于我国，是我国的植物国宝。

从图1上可见，漆树现在世界上的分布主要在我国中西部以毛坝(东经109˚, 北纬30˚)为中心的地区，环我国东南的地区和国家(朝鲜，韩国，日本，越南，缅甸，泰国，柬埔寨，老挝等)，并集中分布在如图红线所示的三角区域(东北至日本的八户，西北至不丹的锡金，南到越南胡志明)[25]。

目前全国被鉴定的漆树良种46个，生漆的产量和出口量均占世界总量的85%左右，其中湖北恩施的毛坝漆、陕西平利的牛王漆、浙江临安的严州漆享有世界声誉。

漆树漆酶是目前从植物源－漆树－提取的活性最高的漆酶，是我国的一种特产资源，我们对目前已知产漆酶的植物源作一概括，详见表1。

1.2 微生物漆酶
微生物漆酶，包括真菌漆酶和细菌漆酶，已经被大量的研究和报道[26-29] (表4)，尤其是真菌漆酶，据估计，已经报道产漆酶真菌不下于1000种[30]。

现在各国对此研究相当多和非常的热，对于研究漆树漆酶很有借鉴作用。

目前报道的细菌漆酶来源有只有Bacillus sphaericus [31]、Azospirillum lipoferum[29;32]和地中海生海洋性细菌Alteromonas菌株[33]。

不过Alexandre[34]推断漆酶在细菌中也广泛存在，并正在被不断发现[35]。

1.3 动物漆酶
相对来讲，动物体中发现的漆酶很少。

目前有报道的有猪肾、麻蝇(Phormia cegina, Musca domestica, Lucilia sericata)、烟草天蛾(Manduca sexta)、绿头苍蝇(Calliphora vicina)、蚊子和双翅目的迁移类蝗虫，昆虫等[29;36-38]，不过对于此类漆酶还是要小心对待，因为很有可能是一些具有类似催化性能的多酚氧化酶，而不是漆酶。

Table 1. Sources of plant laccases
Sources 中文名References
Rhus vernicifera 漆树
[25;39;40]
Rhus succedanea 木蠟树(安南漆)
Melanorrhoea usitata 缅甸连加斯
Melanorrhoea laccifera 高棉漆
Manifera indica 檬果(芒果)
[39;41]
Schinus molle 加州胡椒树
Pistacia palaestina 巴勒斯坦黄连木
Pleiogynium timoriense 帖木李
Banana 香蕉[42]
Peach 梨[43]
Grape 葡萄
[39]
Lactuca virosa 毒莴苣
Digitalis purpurea 毛地黄
Coffer bean 咖啡豆
Mungbean hypocotyls 绿豆胚轴[44]
Acer pseudoplatanus 悬铃木（大槭树）[45]
Pinus taeda 火炬松[46]
Populus trichocarpa 西部香脂杨
[47]
Populus euramericana 黄杨
Liriodendron lulipifera 马栗树[48]
Aesculus parviflora 小花七叶树[45]
Camella sinesis L. 茶[49]
Nicotiana tabacum 烟草[50]
Arabidopsis thaliana 拟南芥[48;51] Podocarpaceae 罗汉松[43]
Forsythia suspensa 连翘[52-54]
1.4 漆酶同工酶
在讨论漆酶来源的时候不能不讲漆酶同工酶，是研究漆酶另一个很重要的方面，是漆酶源的补充，也促进了漆酶研究的深入发展。

同工酶研究的重要性可以从几个方面体现。

首先，可以发现一些新类型的酶。

白漆酶和黄漆酶正是通过同工酶的研究而提出来的[55-57]。

其次，可以对漆酶底物专一性进行对比考察。

第三，对系统发育树研究。

Ranocha[47]在对杨属植物研究时报道了两种分子量分别为90kDa和110kDa的同工酶，并对它们进行了生化特性，分子克隆和表达研究，认为不同植物来源以及同种植物之间的漆酶序列同源性都很不同，p I 各不相同，体现了系统发育植物树的分散性。

漆酶同工酶的报道很多[58;59]，尤其是真菌漆酶，几乎每一种被报道产漆酶的真菌，则必定含有几种同工酶[26;60;61]。

Antorini等人[62]从两种木质素分解真菌中分离纯化的几种同工酶进行X-射线衍射分析。

同工酶可以存在在同一细胞，同一组织，同一个体不同组织或者同一种属中[63-66]。

相对来讲，植物漆酶中的同工酶报道不多[47;67]，我们首次报道了中国漆树漆酶的两种同工酶[11;12]。

各种同工酶的差异可能主要体现在糖链部分。

现在同工酶的研究已经成为细胞分化及形态遗传学的重要内容[68]。

2. 酶稳定技术和固定化
酶的稳定性，指的是在操作使用的时候，酶的催化能力不随着储存，使用频率，应用环境的改变而下降的能力，仍然是目前生物技术研究的一个关键问题。

酶作为一种生物物质，要完全不改变是不可能的，但是将损失尽量降低则是有可能的。

为了改善和提高漆树漆酶的催化能力，应该借鉴和参考其他的行之有效的手段，比如上面提到的酶表面活性剂改性，蛋白质工程技术，化学改性(比如交联，共价吸附，表面修饰等)[69;70]，添加外源物质等[54;71]。

酶的固定化实际上只是酶和蛋白稳定化的一种技术，也是对酶进行改性的一种常规手段。

由于其本身的问题和对环境敏感的特点，并由于固定化漆酶在多方面的应用，使得酶固定化的研究得到了深入的发展[72]。

将漆酶固定在电极上是很好的方法，这种方法对于微量的无溶剂检测尤其适用[73-76]。

将漆酶和葡萄糖脱氢酶固定在一个Clark氧电极上，构造出一种酶传感器，可以在不到1min 之内同步区分可待因和吗啡[77]，以及可以检测肾上腺素类物质[78;79]。

将漆酶固定在可以再生利用的铜螯合载体上，可用于去除酒类生产中的酚[80]。

漆酶固定在尼龙海绵体上，可以对聚合染料聚R-478脱色90%[81]。

固定化的方法很多，表4对漆酶的固定化研究作了枚举。

由于大多数酶都是水溶性的，不溶或者难溶于有机溶剂，所以对酶进行化学物理改性是最常见的手段，固定化技术实质上也是一种改性方法。

但是这些手段往往是以牺牲酶活为代价的，近年来发展的一种非离子型表面活性剂双十二烷基N-D-葡糖酸-L-谷氨酸酯(DHAD)改性酶
的方法，如图2所示，逐渐得到大家的重视。

这可能涉及对蛋白质分子表面的改性或者修饰。

这种非离子表面活性剂最早由Okahata等人[82]报道，稍后Goto等报道了一种改进的方法[83]，我们改进后，首次用绿色方法合成的率高达96%以上的DHAD[84]。

由于这种非离子表面活性剂同时带有亲水和亲油基团，同时由于操作简单，对酶活的影响小，的率高等优点，近来颇受关注[85-93]。

图2 非离子型表面活性剂双十二烷基N-D-葡糖酸-L-谷氨酸酯(DHAD)改性酶方法
Table 2. Immobilization of fungal and Rhus laccases
comment carrier
sp. nylon-66
Cerrena unicolor Phenols Glass
beads Free and
immobilized
laccases Compared
Covalent-APTES-glutaraldehyde [94]
Syringaldazine
phenols
CPG-activated/covered,
dextran layer
Stability, K m, V max Covalent-APTES-
glutaraldehyde [69]
Coriolas hirsutus ABTS, Dye, Affi-Gel-15(agarose) 85% activity after
10 cycles
Covalent ester crossing-linking [95]
Lentinula edodes Effluent,
Phenols
Chitosan 520U/g,
storage
time, pH temp.
improved
Adsorption and subsequent with
glutaraldehyde cross-linking
[96]
ABTS,
Effluent,
Phenols
Eupergit 45%immobilized,
60% activity, temp.
Covalently-activated oxirane [97]
Pleurolus ostreatus ABTS, DMP,
Phenols,
Eupergit DMP
Continuous
elimination,
pH, temp., stability
improved
Covalently-activated oxirane [98]
Polyporus versicolor Phenols(apple
juice)
Sepharose
4H-Epi-IDA-Cu2+
39% phenols
removed
48% flavanols
removed
Adsorption [99]
Pyriculuria uryzae Phenols Microperl,
polyclar 95%
immobilized,
co-immobilized
Co-immobilization
(laccase/tyrosinase)
[100]
Phenols Polyethersulphone
membrane
40% immobilized,
kinetics reactor,
stability
Adsorption [101]
Phenols,
DMP, PDA
Natural and modified
chitosan, transitional
metals
30~70 immobilized,
properties improved
Adsoption, glutaraldehyde
cross-liking and/or chelation
[72]
Protein
coupling
p-benzoquinone-activated
agarose
27% immobilized,
150% activity
Affinity p-benzoquinone-activated
agarose
[102]
Phenols Transition metals,
hydroxide oxides
silica-ZrCl4
75% activity,
properties
Chelation or metal binding [103]
Rhus
vernicifera
Phenols Porous silica treated by 75% activity after Chelation or metal binding [104]
times,
20
ZrCl4 used
properties(pH, K m,
opt. Temp. etc.)
Phenols Urushiol resin-metal ions 10% activity,
[105]
properties
activity,
Adsorption electrostatic interaction [73]
electrodes 10%
ABTS Gold
mediator Fe(CN)63-
3. 漆酶的应用
漆酶的应用范围也是相当的广泛。

如上所述，也可以添加一些中介物质促进反应。

不过
对漆酶的各种应用，归结到一点，就是利用了它的催化氧化性能，漆酶结构和催化机理探讨
见文献[1]，此文不再详述。

本文涉及其应用的几个主要方面和一些最新进展。

3.1 生物整治[106]
目前用于生物整治或生物修复的主要是真菌类酶，尤其是白腐真菌所产漆酶的高水平
而广受关注。

3.1.1 对烯烃的作用
用白腐真菌Trametes hirsuta产的漆酶氧化烯烃，分两部进行：酶先催化氧化一级底物，
加到反应中的中介物，然后被氧化的中介物再氧化二级底物——烯烃，使其成为相应的醛或
酮。

用羟基苯并三唑作中介物所得结果最好，脂肪多聚不饱和的和芳香烯丙基醇在20℃下
处理2h则可完全得到氧化。

在45℃下进行20h，脂肪烯丙基乙醇的氧化率可达70%。

对其
它烯烃，比如烯丙基乙醚，cis-2-庚烯和环辛烯的氧化率也有一定的催化效果[107]。

3.1.2 对TCP和其它有害物质的作用
用白腐真菌Panus tigrinus和Coriolus versicolor的液体培养基对2,4,6-三氯苯酚(TCP)
异构体的毒性作了研究。

在这两种情况下，无论是原封不动的真菌培养液还是纯化了的木质
素降解酶，Mn-过氧化酶和漆酶，两种真菌的木质素降解酶体系都能够将三氯苯酚转化成2,6-
二氯-1,4-氢醌和2,6-二氯-1,4-苯醌，只不过，在P.tigrinus培养液中对2,4,6-TCP起主要作用
的是Mn过氧化酶，而在C.versicolor中，则主要是漆酶[108]。

漆酶还可以降解一种内分泌混
乱化合物双酚A[90;109-112]。

在有中介物质，比如1-羟基苯并三唑(HBT)和ABTS作自由基中介物时能够氧化咔唑，
N-乙基咔唑，芴和硫芴[113;114]。

在有HBT协助时，从双酚和分酚化合物中可以剔除一种雌
激素活性物质，4-异丙基苯酚[115]。

在pH为3，在有2mmol/L的ABTS作氧化还原中介物，
纯化漆酶能够将一种除草剂Isoxaflutole在土壤和植物中的活化形式——二酮腈转化成酸类
物质[116]。

3.1.3 对废水和染料的处理及其生态效应
漆酶还能够对工业废水进行处理，对多种工业染料进行脱色，对自然界的木块进行降
解等等[117-123]。

近来，漆酶也被用于多环芳烃、石油烃污染物的生物处理，值得关注。

Criquet等人[124]报道，漆酶在一些落叶，如栎树叶中可以非常稳定的存在，他们认为这
可能是由于落叶吸收了腐殖酸的缘故。

由此或许可以猜测漆酶在落叶的降解中具有重要的生
态功能。

总之，漆酶在生物整治中的潜力很大，还没有得到很好的体现[125]。

3.2. 对木质素的作用
对于漆酶在木质化过程中的功能有多种论述[47;54;126;127]，既涉及木质素的生物合成，也牵涉木质素的生物降解[128;129]，或者说植物漆酶合成木质素，真菌漆酶降解木质素[130]。

尽管Cullen和Kersten认为漆酶对于木质素的生物降解并非绝对必要，但是也承认漆酶能够将木质素的酚单元氧化成苯氧基[131]。

Freudenberg等[131-133]很早就研究了芥子醇在漆酶等作用下的脱氢聚合反应。

有报道认为漆树漆酶不能催化愈创木酚和松柏醇，所以并不涉及到木质素的生物合成[134;135]，但是很多植物（表1），如悬铃木[45;136]、连翘[53;54]、火炬松[46]及针叶树[137;138]等所产漆酶均发现同木质素的合成有关，能够催化松柏醇。

我们的研究也发现，漆树漆酶能够以非常慢的速度催化松柏醇[139]。

尤其是当有2, 2’-连氮-二(3-乙基苯并噻唑-6-磺酸)(ABTS)调节的时候，漆酶在催化愈创木酚和β-O-4-二聚醇时得到分子量更大的产物[140]。

而事实上，从1993年漆酶被用于去除纸浆木质素开始[141]，这方面的研究已经相当的多。

特别是白腐真菌等真菌漆酶对木质素的降解[142-144]。

虽然对于漆酶在木质化过程中的功能和作用尚缺乏结论性的证据，但是由于涉及到木质素这种复杂的成份，涉及到生物资源的深度开发，利用和环境保护等重大课题，因此研究相当活跃，意义重大，将是今后的研究热点和重点。

漆酶还被用于毛发染色等具有现实意义的应用。

已经有专利报道了在有中介物质羟基氐存在时将漆酶用于染发剂[145;146]。

总之，从目前的研究报道来说，漆酶的确是一种多才多艺的酶，还有许多功能和应用有待发现与发展。

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