天然介体在产漆酶真菌氧化蒽和芘中的重要作用(英文)

【目的】研究氧化还原介体在产漆酶真菌氧化蒽和芘的作用。【方法】通过非变性电泳和酶活力分析。【结果】发现血红密孔菌Z-1和木蹄层孔菌Z-5只产漆酶,其最大酶产量分别为11.90 U/mL和4.83 U/mL,不产木质素过氧化酶和锰过氧化物酶。木蹄层孔菌Z-5的胞外液尽管具有较低的漆酶活性,但是氧化了74.3%的蒽和12.4%的芘,高于血红密孔菌Z-1对蒽和芘的氧化率,提示天然介体可能存在于真菌胞外液中并且影响了漆酶对多环芳烃的氧化。实验进一步表明,木蹄层孔菌Z-5灭活和不灭活的超滤液以及灭活的胞外液对纯漆酶氧化多环芳烃的促进作用均大于血红密孔菌Z-1,说明木蹄层孔菌Z-5的天然介体比血红密孔菌Z-1能够更为有效地促进多环芳烃氧化。【结论】氧化还原结体在产漆酶真菌降解底物过程中发挥了重要作用,这也解释了木蹄层孔菌Z-5胞外液尽管漆酶活性不高,但是具有较大多环芳烃氧化率的原因。     

一株产漆酶土壤真菌F-5的分离及土壤修复潜力

漆酶因可氧化许多种有机污染物,在土壤污染修复方面的应用潜力受到广泛重视。筛选具有较高漆酶活性的土壤真菌,可以为污染土壤修复提供生物资源。通过培养基中愈创木酚颜色反应,从土壤中筛选获得1株真菌菌株F-5。18S rRNA基因序列显示该菌株属于巨座壳科(Family Magnaporthaceae)。单因素试验和正交试验结果显示,蔗糖和蛋白胨分别是最有利于该菌产漆酶的碳源和氮源。在适当培养条件下,真菌F-5培养液酶活性可达4033U/L,表现出该菌具有较强的产漆酶能力。在多环芳烃(PAHs)污染土壤的生物修复中,真菌F-5可使土壤中苯并(a)芘、二苯并(a,h)蒽等高环、高毒性多环芳烃降解,并使土壤多环芳烃毒性当量大幅降低。因此,真菌F-5适合修复PAHs污染土壤。     

三种白腐菌生物学特性与木质纤维素酶基因遗传多样性

以采自东北林业大学帽儿山实验林场的3种白腐真菌——木蹄层孔菌(Fomes fomentarius)、鲍姆鲍姆木层孔菌(Phellinus baumiibaumii)和火木层孔菌(Phellinus igniarius)为材料,用菌落直径测量法比较3种白腐菌在马铃署葡萄糖固体培养基上的生长速度,采用菌丝体干重法比较其在马铃署葡萄糖液体培养基中的生物量变化。结果显示:马铃薯葡萄糖固体培养基上3种白腐菌均为快速生长类型,其生长速度木蹄层孔菌火木层孔菌鲍姆鲍姆木层孔菌;马铃署葡萄糖液体培养基中生物量增长速度木蹄层孔菌鲍姆鲍姆木层孔菌火木层孔菌。用比色法测量其木质纤维素酶活性,结果显示:木蹄层孔菌产锰过氧化物酶和漆酶量较高,鲍姆鲍姆木层孔菌和火木层孔菌产木质素过氧化物酶量较高;木蹄层孔菌、鲍姆鲍姆木层孔菌和火木层孔菌3种白腐菌的3种主要木质素酶(锰过氧化物酶、漆酶和木质素过氧化物酶)的表达量,种间差异显著(F=3.75*、5.20**、3.01*),白桦木屑诱导处理与对照间差异显著(F=3.84*、4.19*、5.28*);两种主要纤维素酶(葡聚糖内切酶、葡聚糖外切酶)的表达量,种间差异不显著,受碳源影响作用显著(F=3.99*、4.04*)。筛选29对引物组合,对3种白腐菌几种主要木质纤维素酶基因进行TRAP-PCR分子标记检测,比较3菌种间遗传差异,扩增总条带数为357条,多态性条带数为255条,多态性条带的比例为71.43%,其中木质素降解酶基因总多态位点比率为73.77%,纤维素降解酶基因总多态位点比率为68.97%。3种白腐菌的木质纤维素降解酶基因在种间均存在较高的遗传差异。因此,特定基因的TRAP分子标记可以用于木腐菌的遗传变异分析。     

木腐菌氧化酶系检定及漆酶产生的研究

采用丁香醛连氮、苯胺蓝平板脱色等4种方法,定性检测了27种真菌的木素氧化酶系组成。结果表明5个菌种同时具有漆酶(Lac)、木素过氧化物酶(Lip)和锰过氧化物酶(Mnp)3种酶的活性,5个菌种同时具有Lac和Lip2种酶的活性,8个菌种具有1种酶的活性;12个菌种具有Lac活性。选择其中生长速度快、Lac活性高的5个菌种进行Lac产生的研究,发现木蹄层孔菌Fomes fomentarius诱导、静止培养条件下产生的Lac酶活峰值高达9496U/mL,远远高于其它已报道过的菌种;静止培养条件下贝形刺革菌Hymenochaete badio-ferruginea和乳白耙菌Irpexlacteus产Lac,其峰值也分别达到了652和292U/mL,均明显高于其它培养方式,说明木蹄层孔菌等3种真菌静止培养可代替振荡培养进行Lac产生、制备等相关的后续研究。木蹄层孔菌等5种真菌对刚果红等染料脱色作用研究表明,各菌种对4类染料均具有高效广谱的脱色作用。     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

一株高效降解芘的细菌分离、鉴定及其降解效果

摘要：【目的】获得高效降解高分子量多环芳烃的细菌,并研究其对多环芳烃的降解能力。【方法】利用富集培养和芘升华平板方法,从焦化厂污染土壤中分离多环芳烃降解细菌,对分离菌株通过形态特征、16S rRNA基因和gyrb基因序列相似性分析进行鉴定,并研究该菌对高分子量多环芳烃（HMW-PAHs）的降解效果。【结果】筛选到一株能以芘、苯并蒽、屈、苯并芘、茚并芘、苯并苝、荧恩为碳源和能源生长并降解这些底物的菌株HBS1,该菌株的16S rRNA基因和gyrb基因序列与Gordonia amicalis的相应基因的相似     

木蹄层孔菌不同居群间生长特性、木质素降解酶与SRAP标记遗传多样性

以帽儿山、长白山、凉水、本溪木蹄层孔菌(Fomes fomentarius) 4个居群为研究对象,采用菌丝长度测量法比较4个地点木蹄层孔菌菌株在PDA固体培养基上的生长速度,采用菌丝体干重法比较4个地点木蹄层孔菌菌株在PDA液体培养基中生物量的变化,结果显示木蹄层孔菌在23 ℃下生长速度显著高于28 ℃,说明木蹄层孔菌的生长对温度较敏感,23 ℃更适合木蹄层孔菌的生长。在同一温度下培养,4个地点的木蹄层孔菌生长速度无显著差异。通过比色法检测4个地点的木蹄层孔菌木质素降解相关酶(木质素过氧化物酶,锰过氧化物酶,漆酶)活性差异,结果显示同一种酶酶活性在4个地点间没有显著差异;在不同培养基上培养时,3种酶在PDA培养基上的活性均显著高于完全培养基。同时,采用序列相关扩增多态性(Sequence-related amplified polymorphism, SRAP)技术初探了木蹄层孔菌4个居群的遗传多样性和遗传分化, 结果表明木蹄层孔菌4个居群中多态位点比率最高的是本溪,其次是帽儿山和凉水,而长白山最低;AMOVA分析结果显示,居群间的遗传分化为24.74%,居群内的遗传分化为75.26%,木蹄层孔菌的遗传分化主要发生在居群内部。根据Nei's遗传距离对木蹄层孔菌4个居群进行UPGMA聚类分析,结果显示帽儿山和本溪居群最先聚类,其次聚类的是长白山居群,最后是凉水居群。     

基于吐温-80下2株功能菌协同降解多环芳烃的特性研究

【目的】研究恶臭假单胞菌B6-2和克雷伯氏菌CW-D3T构建的混合功能菌对多环芳烃的协同修复效能,并探究非离子表面活性剂吐温-80对混菌降解多环芳烃的影响,以期为芳烃化合物的生物修复提供技术参考和理论依据。【方法】通过生长曲线及平板菌落计数法反映混菌生长情况及比例,从而评估混菌降解体系的可行性;通过高效液相色谱法探究各体系以及不同吐温-80浓度下混培体系对多环芳烃的降解效能;最后通过烷烃吸附法测定细胞表面疏水性,以探究吐温-80对混合功能菌降解多环芳烃的影响机制。【结果】等比例混合的2株菌共培养生长状态优于纯培体系,对混合多环芳烃(菲、荧蒽、芘)的降解率分别为33.4%、30.1%、28.6%(7 d),相较于菌CW-D3T,分别提高了1.31倍、1.46倍、1.42倍。混培体系中加入500 mg/L的吐温-80对菲、荧蒽、芘的降解率分别为47.7%、43.2%、38.8%(7 d),相较于对照组各提高了1.55倍、1.38倍、1.31倍,而更高浓度的吐温-80无明显促进作用或轻微抑制。添加吐温-80使菌CW-D3T和混菌的表面疏水性提高,而菌B6-2表面疏水性降低。结合细菌生长量分析...     

真菌漆酶及其介体系统：来源、机理与应用

漆酶是一类含铜氧化酶,广泛分布于植物、真菌、细菌、昆虫中,它们能够高效催化芳香族和非芳香族化合物氧化降解,并最终将分子氧还原为水作为副产物。一些小分子介体能够进一步提高漆酶的降解底物范围、催化效率和稳定性。它们与漆酶构成漆酶/介体系统（laccase mediator systems, LMS）,能够更有效地降解非酚类、多环芳烃类等难降解化合物,在造纸制浆与漂白、染料脱色、环境脱毒等领域有着巨大的应用前景,成为近年来的研究热点之一。对漆酶的来源与功能、真菌漆酶结构与反应机理、介体类型与作用机理、LMS的应用进行了综述,以期为漆酶的应用研究提供参考。     

血红密孔菌(Pycnoporussanguineus)漆酶基因的克隆与序列分析

为克隆血红密孔菌 (Pycnoporussanguineus)漆酶基因 ,根据真菌漆酶氨基酸序列保守区设计了 1对简并引物 .以血红密孔菌基因组DNA为模板 ,PCR扩增出长 12 2 7bp的漆酶基因片段 .以此序列为基础 ,通过 5′及 3′RACE技术克隆出漆酶全长cDNA序列 ,序列长为 190 2bp ,其 5′端和 3′端非编码区长分别为 5 1bp和 2 97bp ,开放阅读框长 15 5 4bp ,编码 5 18个氨基酸的蛋白 .该蛋白具有 4个铜离子结合区域 ,预测其相对分子量为 5 6 313 2 ,等电点为 5 5 9,其氨基酸序列与Pycnoporuscinnabarinus漆酶 (lcc3 2 )的同源性最高 ,为 96 % .以该cDNA编码区的两端序列为引物 ,PCR扩增得到漆酶的长度为 2 15 4bp的全长DNA序列 ,序列中包括 10个内含子序列 ,长为 5 2～ 70bp     

Oxidation of Anthracene and Benzo[a]pyrene by Laccases from Trametes versicolor

The in vitro oxidation of the two polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, which have ionization potentials of <=7.45 eV, is catalyzed by laccases from Trametes versicolor. Crude laccase preparations were able to oxidize both anthracene and the potent carcinogen benzo[a]pyrene. Oxidation of benzo[a]pyrene was enhanced by the addition of the cooxidant 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), while an increased anthracene oxidizing ability was observed in the presence of the low-molecular-weight culture fluid ultrafiltrate. Two purified laccase isozymes from T. versicolor were found to have similar oxidative activities towards anthracene and benzo[a]pyrene. Oxidation of anthracene by the purified isozymes was enhanced in the presence of ABTS, while ABTS was essential for the oxidation of benzo[a]pyrene. In all cases anthraquinone was identified as the major end product of anthracene oxidation. These findings indicate that laccases may have a role in the oxidation of polycyclic aromatic hydrocarbons by white rot fungi.     

Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli

Laccases produced by white rot fungi are capable of rapidly oxidizing benzo[a]pyrene. We hypothesize that the polycyclic aromatic hydrocarbon (PAH)-degrading bacteria producing laccase can enhance the degree of benzo[a]pyrene mineralization. However, fungal laccases are glycoproteins which cannot be glycosylated in bacteria, and there is no evidence to show that bacterial laccases can oxidize benzo[a]pyrene. In this study, the in vitro oxidation of PAHs by crude preparations of the bacterial laccase, CueO, from Escherichia coli was investigated. The results revealed that the crude CueO catalyzed the oxidation of anthracene and benzo[a]pyrene in the same way as the fungal laccase from Trametes versicolor, but showed specific characteristics such as thermostability and copper dependence. In the presence of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), high amounts of anthracene and benzo[a]pyrene, 80% and 97%, respectively, were transformed under optimal conditions of 60°C, pH 5, and 5 mmol l(-1) CuCl(2) after a 24-h incubation period. Other PAHs including fluorene, acenaphthylene, phenanthrene, and benzo[a]anthracene were also oxidized by the crude CueO. These findings indicated the potential application of prokaryotic laccases in enhancing the mineralization of benzo[a]pyrene by PAH-degrading bacteria.     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

Affinity chromatography as a rapid and convenient method for purification of fungal laccases

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl- and vanillyl-controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed.     

Oxidation of polycyclic aromatic hydrocarbons by laccase of Coriolus hirsutus

The oxidation of five polycyclic aromatic hydrocarbons; anthracene, benzo()pyrene, fluoranthene, phenanthrene and pyrene was catalyzed by laccase from Coriolus hirsutus in the presence of the redox mediators, 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). In the ABTS-mediated system, benzo()pyrene was the most rapidly oxidized substrate, with anthracene being the most rapidly oxidized in the HBT-mediated system. There was no clear relationship between the ionization potential and the oxidation of the substrates. ABTS increased the oxidation of benzo()pyrene more than HBT but the oxidation of the other PAHs tested were the opposite. The mediators used in conjunction increased the oxidation of benzo()pyrene compared to using the mediators alone.     

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris

Aims:  To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes.
Methods and Results:  A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62·8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus . Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators.
Conclusions:  Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators.
Significance and Impact of the Study:  This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.     

Biotransformation of bezo[a]pyrene in bench scale reactor using laccase of Pycnoporus cinnabarinus

The biotransformation of benzo[a]pyrene by purified extracellular laccase of Pycnoporus cinnabarinus was investigated in bench scale reactors. The reaction required the presence of exogenous mediator ABTS. Most of 95% of the substrate was converted within 24 hours. The enzyme preparation oxidised the substrate mainly to benzo[a]pyrene 1,6- 3,6- and 6,12-quinones in a 2/1/1 ratio after 24 h incubation.     

Enhanced activity by poly(ethylene glycol) modification of Coriolopsis gallica laccase

We are studying the enzymatic modification of polycyclic aromatic hydrocarbons (PAHs) by the laccase from Coriolopsis gallica UAMH 8260. The enzyme was produced during growth in a stirred tank reactor to 15 units ml⁻¹, among the highest levels described for a wild-type fungus; the enzyme was the major protein produced under these conditions. After purification, it exhibited characteristics typical of a white rot fungal laccase. Fifteen azo and phenolic compounds at 1 mM concentration were tested as mediators in the laccase oxidation of anthracene. Higher anthracene oxidation was obtained with the mediator combination of ABTS and HBT, showing a correlation between the oxidation rate and the mediator concentration. Reactions with substituted phenols and anilines, conventional laccase substrates, and PAHs were compared using the native laccase and enzyme preparations chemically modified with 5000 MW-poly(ethylene glycol). Chemically modified laccase oxidized a similar range of substituted phenols as the native enzyme but with a higher catalytic efficiency. The k _cat increase by the chemical modification may be as great as 1300 times for syringaldazine oxidation. No effect was found of chemical modification on mediated PAH oxidation. Both unmodified and PEG-modified laccases increased PAH oxidation up to 1000 times in the presence of radical mediators. Thus, a change of the protein surface improves the mediator oxidation efficiency, but does not affect non-enzymatic PAH oxidation by oxidized mediators. Received 10 December 2001/ Accepted in revised form 20 July 2002     

Phylogeographic relationships in the polypore fungus Pycnoporus inferred from molecular data

The genus Pycnoporus forms a group of four species known especially for producing high redox potential laccases suitable for white biotechnology. A sample of 36 Pycnoporus strains originating from different geographical areas was studied to seek informative molecular markers for the typing of new strains in laboratory culture conditions and to analyse the phylogeographic relationships in this cosmopolitan group. ITS1-5.8S-ITS2 ribosomal DNA and partial regions of β-tubulin and laccase lac3-1 gene were sequenced. Phylogenetic trees inferred from these sequences clearly differentiated the group of Pycnoporus cinnabarinus strains from the group of Pycnoporus puniceus strains into strongly supported clades (100% bootstrap value). Molecular clustering based on lac 3-1 sequences enabled the distribution of Pycnoporus sanguineus and Pycnoporus coccineus through four distinct, well supported clades and sub-clades. A neotropical sub-clade, grouping the P. sanguineus strains from French Guiana and Venezuela, corresponded to P. sanguineus sensu stricto. A paleotropical sub-clade, clustering the strains from Madagascar, Vietnam and New Caledonia, was defined as Pycnoporus cf. sanguineus. The Australian clade corresponded to P. coccineus sensu stricto. The Eastern Asian region clade, clustering the strains from China and Japan, formed a P. coccineus-like group. Laccase gene (lac 3-1) analysis within the Pycnoporus species can highlight enzyme functional diversity associated with biogeographical origin.