白腐菌在两种固体基质上的漆酶产量及同工酶活性比较研究

一株新分离的白腐菌Z1可在两种农业废弃物(麸皮和稻草秸杆)基质上生长并分泌漆酶,其中在麸皮基质上的漆酶产量比在稻草秸杆基质上的漆酶产量高出10倍左右,同时活性电泳显示Z1菌在麸皮基质上可产生3条漆酶同工酶,比在稻草秸杆基质上多1条,其活性也比在稻草秸杆基质中的漆酶活性高。     

胞外漆酶同工酶谱及其在食用菌育种中的应用I．黑木耳及光帽鳞伞不同菌株的鉴别

利用聚丙烯酰胺凝胶等电聚焦圆盘电泳,分析比较了黑木耳(Auricularia auricula) 16个菌株和光帽鳞伞(Pfioliota nameko) 9个菌株的胞外漆酶同工酶酶谱。经多批次重复, 得到了所有菌株的恒定酶谱。依照这些酶谱及各酶带等电点(pT)值的不同,确立了它们的漆酶标志位点相似系谱。16株黑木耳被区分成4种类型9个组。9株光帽鳞伞被分成两种类 型3个组,但各组内之菌株间仍有异同。试验结果表明,把漆酶同工酶谱应用于菌株鉴别将是一种有意义的方法。     

胞外漆酶同工酶谱及其在食用菌育种中的应用——Ⅰ.黑木耳及光帽鳞伞不同菌株的鉴别

利用聚丙烯酰胺凝胶等电聚焦圆盘电泳,分析比较了黑木耳(Auricularia auricula)16个菌株和光帽鳞伞(Pholiota nameko)9个菌株的胞外漆酶同工酶酶谱。经多批次重复,得到了所有菌株的恒定酶谱。依照这些酶谱及各酶带等电点(pI)值的不同,确立了它们的漆酶标志位点相似系谱。16株黑木耳被区分成4种类型9个组。9株光帽鳞伞被分成两种类型3个组,但各组内之菌株间仍有异同。试验结果表明,把漆酶同工酶谱应用于菌株鉴别将是一种有意义的方法。     

几种担子菌胞外漆酶同工酶谱的研究

从猴头(Hericium erinaceus)、亚侧耳(Hohenbuehelia serotina)、毛柄金钱菌(Co-llybia velutipes)、多脂鳞伞(Pholiota adiposa)、香菇(Lentinus edodes)、黑木耳(Auriculariaauricula)、草菇(Volvaria volpacea)二孢蘑菇(Agaricus bisporus)和紫孢侧耳(Pleurotussapidus)、侧耳(Pleurotus ostreatus)风尾菇(P.sajor-caju)、金顶侧耳(P.citrinopileatus)、鲍鱼菇(P.cystidiosus)、佛罗里达侧耳(P.florida)、爪哇漏斗状侧耳(P.sajor-caju var.javanicus)商品化菌种的培养滤液里所得到的胞外漆酶(LC),采用聚丙烯酰胺凝胶等电聚焦盘状电泳方法,分析比较了它们的胞外漆酶同工酶。经多批次比较试验,获得了侧耳属七个种和八个不同属间各自恒定的漆酶同工酶谱。在这些酶谱中,分别包含了 A 组(pH3—5)和 B 组(pH5—8)的谱带。而且,所有菌种间漆酶带的等电点值(pI)各有异同。没有测定菌种间各漆酶带含量差别。讨论了漆酶同工酶谱用于分类研究的可能性。     

漆树8个品种和5个野生居群HPLC指纹图谱分析

采用HPLC指纹图谱技术分析了陕西漆树8个品种和5个野生居群的树皮提取物化学成分,并以提取物中6个化学成分含量为变量进行聚类分析.结果表明:(1)通过对2、3、4和5号主要特征峰分析,漆树8个品种和5个野生居群的13个样品平均相似度为0.974;方差和变异系数分析显示,各主要特征峰含量、漆酚总量之间存在差异,且变异系数最大达61.49%.(2)聚类分析结果显示,依据漆酚总量等6个变量完全可将漆树8个品种的8个样品分开,表明单烯漆酚为区分8个样品的第一关键因子,未知化合物和二烯漆酚为第二、第三关键因子.同时,采自商南的2个漆树品种样品和采自平利的6个品种样品分别聚为两类,表明其聚类结果与漆树地理分布存在密切关系.     

鬼针草属六种植物的同工酶电泳实验研究

目的:建立鬼针草6个品种过氧化物酶同工酶(POD)电泳指纹图谱,探讨鬼针草种内变异规律.方法:聚丙烯酰胺凝胶电泳分析.结果:鬼针草6个品种过氧化物酶同工酶PAGE指纹图谱差异显著.结论:聚丙烯酰胺凝胶同工酶电泳图谱可用于鉴别鬼针草6个种质.     

二棱大麦和杂种的β-淀粉酶同工酶研究

用水和木瓜蛋白酶提取的两种大麦β-淀粉酶同工酶在薄层等电聚焦电泳中能分辨出30条酶带,它们的pI在4.4—6.5之间,可以分成3个区(Ⅰ、Ⅱ、Ⅲ区)。水提取的游离态β-淀粉酶同工酶主要集中在Ⅰ区。而用木瓜蛋白酶提取的总β-淀粉酶同工酶主要分布在Ⅱ、Ⅲ区,Ⅰ区较少,它的分布区域与游离态酶的活性有关。37个二棱大麦品种的β-淀粉酶活性差异较大,但根据同工酶的电泳图谱可以分成两种类型,即Ⅰ型和Ⅱ型,两者在酶带数和分布上都有差异。 同一类型的不同品种之间杂交后,酶活性出现明显的杂种优势,但其同工酶的电泳图谱不发生改变。 对β-淀粉酶同工酶电泳类型的多型性及高β-淀粉酶活性在育种上的应用作了简要讨论。     

箭舌豌豆根系抗坏血酸及相关酶对镉胁迫的响应

以箭舌豌豆(Vicia sativa L.)品种L3(耐镉)和ZM(镉敏感)为材料,研究了不同程度镉胁迫下箭舌豌豆幼苗根系抗坏血酸(AsA)含量、脱氢抗坏血酸还原酶(DHAR)同工酶活性、抗坏血酸过氧化物酶(APX)同工酶活性以及APX基因表达的变化。结果显示:(1)2个箭舌豌豆品种根系AsA和脱氢抗坏血酸(DHA)含量在镉胁迫下显著升高;AsA/DHA比值在镉耐性品种L3中显著升高,在敏感品种ZM中显著下降;相同镉处理浓度下,L3根系AsA含量和AsA/DHA比值显著大于ZM。(2)2个品种根系DHAR的活性电泳共显示4条同工酶条带,它们的活性均随镉处理浓度的升高而升高;其中DHAR1只在L3显示,DHAR4只在ZM显示;相同镉处理浓度下,品种L3的DHAR的总活性大于品种ZM。(3)2个品种根系APX的活性电泳共显示11条同工酶条带,其中的APX1、2、4仅在敏感品种ZM中受镉胁迫诱导,APX 8在耐性品种L3中受到比敏感品种ZM更显著的诱导;克隆得到1个箭舌豌豆APX基因,荧光定量RT-PCR结果显示该基因的转录在L3和ZM根系均受镉处理诱导。研究表明,镉胁迫下2个箭舌豌豆品种根系AsA含量,AsA代谢相关酶DHAR和APX的活性以及APX的转录水平均显著升高;镉耐性品种L3较敏感品种ZM能更有效地促进AsA循环,维持更高的AsA水平,从而更有效地缓解镉胁迫诱导产生的氧化胁迫,这可能是L3较ZM具有更高镉耐性的重要机制之一。     

几种同工酶与猕猴桃品种对溃疡病抗性关系的研究

应用聚丙烯酰胺凝胶电泳、同工酶分析技术分别研究了猕猴桃植株体内过氧化物酶(POD)、多酚氧化酶(PPO)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、酯酶(EST)同工酶谱带的变化,结果表明:自然感染溃疡病前后,此6种同工酶谱带特征在不同抗感品种中表现出一定的差异.未感染溃疡病菌前,抗(感)品系枝条、叶片POD同工酶均有2条酶带,PPO同工酶有3条酶带,但感病品种酶带颜色深且粗,而抗病品种酶带颜色浅且细,叶片酶带颜色深于枝条;SOD、CAT同工酶谱带均为1条,Rf值分别为0.38、0.28,感性品种较抗耐品种谱带亮度高活性强;自然发病后,抗(感)品系POD、PPO同工酶谱带数都增加,分别为4、3条和5、4条,且抗病品种新酶带出现较感病品种早且酶带粗颜色深活性强,感病品系虽也有新酶带出现,但酶带少活性弱,抗病品系枝条、叶片POD、PPO同工酶新谱带的Rf值分别为0.63、0.67和0.85、0.87;抗感病品种SOD、CAT同工酶都被诱导产生了1条新的同工酶谱带,Rf分别为0.32和0.27,新酶带现色时间迟,且酶带颜色浅活性弱,但抗耐品种较感性品种谱带亮且活性强;EST同工酶于自然发病前后变化不大,与抗病性关系不很明显.     

真菌漆酶及其应用

漆酶是一种含铜多酚氧化酶,在白腐菌中普遍存在,少数低等真菌和植物中也产生,多为分泌型糖蛋白。至少20种漆酶得到了分离和纯化。该酶是一种氨基酸残基在500个左右的单体酶,一般都为酸性蛋白,含有4个铜离子,形成3个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的漆酶基因被克隆,许多来源的基因都是以家族形式存在于染色体上的。已研究的漆酶基因中都含有10个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊序列的存在与否决定了该酶的表达形式-诱导型或组成型,诱导型菌株的调控序列中含有一段受酚类化合物作用的序列,而不含有该序列的酶基因则都是组成型表达的。漆酶在S.cerevisiae、Trichodermareesei、A.oryzaeTATAamylase和Pichiapasti等异源表达系统中有成功表达的报道。漆酶的应用集中在以下几方面:漆酶参与的有机合成;生物检测;有毒化合物的消除;工业废水处理;纸浆的生物漂白;等等。     

Purification, crystallisation and X-ray diffraction study of fully functional laccases from two ligninolytic fungi

Laccase isozymes from the white-rot basidiomycete fungi Trametes versicolor and Pycnoporus cinnabarinus were purified to apparent iso-electric homogeneity and crystallised. T. versicolor laccase crystallises in two crystal forms, both with the orthorhombic space group P2(1)2(1)2(1), which diffract to 1.9 and 2.95 A resolution, respectively. The crystals of P. cinnabarinus laccase belong to the monoclinic space group C2 and diffract to at least 2.2 A resolution. All the laccase crystals are suitable for X-ray structure determination and contain a full complement of copper ions.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

Lignin-modifying enzymes of flavodon flavus, a basidiomycete isolated from a coastal marine environment

A basidiomycetous fungus Flavodon flavus (Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of 14C-labeled synthetic lignin to 14CO2 in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase. F. flavus showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by F. flavus and points to the bioremediation potential of this organism in terrestrial as well as marine environments.     

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.     

Novel laccases of Ganoderma sp. KU-Alk4, regulated by different glucose concentration in alkaline media

Physiological regulation of laccase production from Ganoderma sp. KU-Alk4, isolated in Thailand, was controlled by the initial glucose concentration in liquid culture. Different laccase isozymes were produced using different starting concentrations of glucose. With 1% glucose, two isozymes, KULac 1 and 2 were produced, while with 4% glucose, three different isozymes, KULac 3, 4 and 5, were produced. The KULacs differed in their molecular mass, ranging from 53 to 112 kDa. KULac 2 was a new laccase that had a different N-terminal amino acid sequence from other laccases previously reported. All the isozymes had optimum pH at 3.5 and were stable over the wide range of pH, 3.0–10.0, especially in alkaline pH. It is noteworthy that the activities of the four KULacs with 2,6-dimethoxyphenol were extremely high up to 90°C. They retained 100% of their activities at 60°C for 1 h.     

Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.     

Accumulation of a Natural Substrate of Laccase in Gills of Pleurotus florida During Sporulation

During sporulation, laccase activity of Pleurotus florida decreased to a minimum level in spite of increase in the number of isozymes. An endogenous laccase substrate was detected especially in the gill structure of the sporophore, which competitively inhibited oxidation of guaiacol by the enzyme during in vitro assay. Appearance of the laccase substrate in the gill structure may be linked with the sporulation phenomenon. Received: 21 January 2000 / Accepted: 2 May 2000     

Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2

The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71?454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5-7.5. The K(m) values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 μM. The corresponding V(max) are 504.0, 1910.0, 117.4 and 159.0 μM min(-1) mg(-1). In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.